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Allergic disease: the diagnosis of peanut allergy and
the role of heat shock proteins in chronic allergic inflammation
Joost Aalberse
Colofon
Cover: Landgoed Oostbroek, Utrecht
Design: Rozemarijn Klein Heerenbrink, Photography: Joost Aalberse
Print: Gildeprint, Enschede
ISBN: 978-94-6233-136-5
© 2013 by Cell Stress and Chaperones, for chapter 4
© 2013 by Allergy, for chapter 6
© 2014 by The Journal of Allergy and Clinical Immunology: in Practice, for textbox chapter 7
2011 by Plos One, for chapter 3
2012 by Frontiers in Immunology, for chapter 2
2014 by Clinical and Translational Allergy, for chapter 5
2015 by J.A. Aalberse, for chapter 1 and chapter 8
Address of correspondence
J.A. Aalberse, Wilhelmina Children’s Hospital, University Medical Center Utrecht
e-mail [email protected]
Allergic disease:
the diagnosis of peanut allergy
and
the role of heat shock proteins in
chronic allergic inflammation
(met een samenvatting in het Nederlands)
Proefschrift
Ter verkrijging van de graad van doctor aan de Universiteit Utrecht
op gezag van de rector magnificus, Prof. Dr. G.J. van der Zwaan
ingevolge het besluit van het college voor promoties
in het openbaar te verdedigen op
donderdag 26 november 2015 des middags te 4.15 uur
door
Joost Adriaan Aalberse
geboren op 22 januari 1977 te Amsterdam
Promotor:
Prof. Dr. B.J. Prakken
Copromotoren:
Dr. F. van Wijk
Dr. E.F. Knol
“Know Thyself” Alexander Pope
Uit: Pope, Alexander; An Essay on Man, in Four Epistles. Hartford: Silas Andrus, 1824.
Know then thyself, presume not God to scan;
The proper study of mankind is Man.
Placed on this isthmus of a middle state,
A being darkly wise and rudely great:
With too much knowledge for the Sceptic side,
With too much weakness for the Stoic’s pride,
He hangs between; in doubt to act or rest,
In doubt to deem himself a God or Beast,
In doubt his mind or body to prefer;
Born but to die, and reasoning but to err;
Alike in ignorance, his reason such
Whether he thinks too little or too much:
Chaos of thought and passion, all confused;
Still by himself abused, or disabused;
Created half to rise and half to fall;
Great lord of all things, yet a prey to all;
Sole judge of truth, in endless error hurled:
The glory, jest, and riddle of the world!
Contents
Chapter 1
General Introduction
11
Chapter 2
HSP; Bystander Antigen in Atopic Diseases?
31
Chapter 3
Cord Blood CD4+ T cells respond to self-heat shock
protein 60
51
Chapter 4
Recognition of self-heat shock protein 60 by T cells
from patients with atopic dermatitis
69
Chapter 5
Plasma IL-25 is elevated in a subgroup of patients
with clinical reactivity to peanut
85
Chapter 6
Moving from peanut extract to peanut components:
Towards validation of component-resolved IgE tests
95
Chapter 7
Discussion
115
Chapter 8
Lekensamenvatting (Nederlands)
131
Chapter 9
Dankwoord
138
List of Publications
143
CV
142
Chapter 1
General Introduction
J.A. Aalberse
Chapter 1
General Introduction
PART 1
A historical introduction
The term ‘allergy’ was coined by Pirquet in 1906(1;2). Symptoms of allergic diseases had been
described long before, with seasonal symptoms of hay fever and adverse responses to food(3-8).
Pirquet noticed an adverse response in some patients receiving a chickenpox vaccine. The pa-
tients developed not only a protective immune response, but also symptoms of hypersensitivity, such as skin rash, arthropathy and fever. Pirquet called this altered response ‘allergy’ de-
rived from the Greek words allos (other) and ergon (work). Allergy according to the definition
of Pirquet included hypersensitivity syndromes such as hay fever and food allergy, but also
auto immune disease and serum sickness. It was almost 60 years later, in 1963, that Gell and
Coombs made a classification for the, at that time, confusing wide variety of hypersensitivity
diseases(9). Remarkably, this classification is still being used. For this thesis I will focus on type
I hypersensitivity, which corresponds to atopic allergy. IgE, an immunoglobulin identified in
1966, is considered the cornerstone of atopy(10-12).
Atopic syndrome and allergy
The terminology of atopy and allergy can be confusing. Allergy is hypersensitivity towards
a harmless environmental allergen with an immunological response. This can be a humoral
(antibody mediated) or a cellular (cell mediated) response. The humoral response in an allergic reaction typically depends on the IgE isotype.
According to the definition of the World Allergy Organization atopy can be defined as the
tendency to become sensitized and produce IgE in response to exposure to allergens(14).
Atopy has a strong genetic link, with 50-70% of mothers (and in lesser extent fathers) passing their atopic tendency to their children(15). Individuals with atopy that develop allergic
disease can have symptoms with different tissue involvement, including the skin, the upper and lower respiratory system and the gut. These diseases can follow a typical pattern
during life, often referred to as the atopic march(16). Quickly after birth it starts with atopic
dermatitis and food allergy, and continues in some with asthma and allergic rhinitis. These
diseases (as seen in figure 1, in bold) are typically IgE mediated atopic diseases, which corresponds also to type I hypersensitivity.
Diagnosis of allergic disease
The diagnosis of IgE mediated allergic diseases is not always straightforward. Measuring of allergen-specific IgE is not sufficient for the diagnosis, as IgE can also be present in non-allergic
individuals. Moreover, it is not indicative of the organ involved and also the severity of the symptoms cannot accurately be predicted by the levels of IgE.
12
General Introduction
Figure 1 | Nomenclature of hypersensitivity.
Figure adapted from Johansson et al Allergy 2001(13) illustrating the misunderstanding that can occur
when atopy and allergic disease is discussed.
The diagnosis of allergic disease is foremost made on clinical observations: both on the history
of the patient as on the physical examination by the doctor. Important in the history is the relation of the symptoms with exposure to the possible allergic trigger, but also other experienced
atopic diseases as well as the family history related to atopic disease. The history of the patient
is however often unreliable. In food allergy it was found to be reliable only in thirty percent(17-19).
The clinical diagnosis can be supported by a skin test and laboratory findings such as IgE to
relevant allergens.
During a skin test a very small amount of allergen is put into the skin, which results in cross linking
of IgE on skin mast cells causing local swelling and redness when there is an allergic sensitization.
It is an easy and quick test and therefore often used. Another way of testing an allergic sensitization is in the blood, by measuring the level of specific IgE antibodies to an extract or a specific allergen. It is important to realize that not all individuals that are sensitized also have clinical symp-
toms, which can result in misdiagnosis. Positive predictive values with high accuracy have been
reported in literature(20), but these are arbitrary. Also it has been found that sensitization to some
allergens is more clinically relevant than sensitization to others. Until recently IgE was meas-
13
Chapter 1
ured only against an extract, but it can now also be measured against purified and recombinant
allergens. Measuring IgE only to the clinically most relevant allergens is suggested as a possible
solution to improve the correlation between the measured sensitization and the clinical response.
Diagnosis of food allergy
In addition to skin testing and IgE specific antibodies, food allergy can be diagnosed via a food
challenge(19). A double-blind placebo-controlled oral food challenge (DBPCFC) is the gold standard
for diagnosis(21-23). During a DBPCFC the patient is exposed to the presumed trigger and the clinical
response is observed in a safe clinical environment, with proper trained personnel and ideally
direct access to intensive care facilities in case of an anaphylactic response. The children have to
be prepared accurately as the psychological effect of an exposure to an allergen they have had
to avoid all life, can be dramatic. At the start of the challenge a low dose is given, followed by
incrementing doses. To ensure that it is blinded the food has to be masked (which often causes
difficulties with non-standardized recipes). Ideally the challenge is spread over two days, with a
placebo and a verum challenge day blinded for both the performing staff as well as the patient.
Standardization of the DBPCFC between various clinical centres is a current issue(21).
Figure 2 | The diagnosis of a food allergy like peanut allergy stands and falls with a well performed DBPCFC.
Often the diagnosis is only made on the history of symptoms and on specific IgE. Not all allergens are
known however and many extracts are incomplete, which sometimes results in false negative responses.
More often false positives responses are seen, as often sensitized patients do not have clinical allergic
responses. A well performed DBPCFC tests the clinical reactivity and therefore is used as the gold
standard for diagnosis of food allergy.
To conclude, it has been shown that food allergy is often misdiagnosed when only the history
of the patient and serum IgE or SPT is taken in account. DBPCFC are needed for a uniform
diagnosis of food allergy, not only for the proper diagnosis in patients, but also for proper
classification of patients in research.
Prevalence of allergic disease
The prevalence of allergic disease has increased tremendously especially in westernized
countries(24-29). As mentioned above it is important to note that allergic diseases are often
misdiagnosed, which also effects research data, with i.e. too high prevalence numbers, due to
false positive tests. Ideally, prevalence data on allergic disease should be based on both the
production of specific IgE (sensitization) as well as on a proven tissue involvement.
14
General Introduction
Presently, sensitization (IgE antibodies) to allergens in the environment is found in up to 40%
of the population(12). Allergic sensitization can cause diseases in different organs including
the lungs (asthma), the nose (atopic rhinitis), the gastro-intestinal tract gut (food allergy) and
the skin (atopic eczema). The latter two are the topic of this thesis. Thirteen percent of U.S.
children aged 17 years and under suffers from skin allergies, including atopic eczema(30). Often
co-morbidity is present with other allergic diseases, such as food allergy. Approximately 5-9%
of children in the US have food allergy, depending on the age, but also on criteria used for the
diagnosis on food allergy(30). Eight foods account for 90% of food allergic reactions in children.
These eight foods are milk, eggs, peanut, tree nuts, fish, shellfish, soy and wheat.
Peanut is the most prevalent food allergy, accounting for about 1-2% of all children in Europe(31-33)
and also has the most severe clinical reactions including anaphylaxis(34). Interestingly in recent
years it has been suggested that peanut consumption during pregnancy is associated with a
decreased risk of offspring with peanut allergy(35). In addition regular peanut consumption in
children that starts before age of 11 months reduces peanut allergy up to 7-fold (36). Peanut is
a common food, present in many foods. Unlike cow milk allergy which is regularly outgrown,
peanut allergy is persistent in approximately 80% of the children(26;37).
Plant Food Allergens
Although a large number of food proteins are consumed, only a small number is responsible
for the majority of food allergies(38). It is known that some of these food proteins have special
characteristics that can contribute to allergenicity, such as a small molecular weight, stability,
water solubility and heat- and digestion resistance(39-43). The food allergens can have proper-
ties that are functionally relevant for the plant or food, such as storage and defensive proper-
ties. These characteristics contribute to their ability to resist food processing and digestion,
which sometimes can even make them more allergenic. Lipid transfer proteins (LTPs) for ex-
ample are important in the defence of plants from different kinds of pathogens and environmental stress(44). Many fruits and nuts contain LTPs that are highly resistant to digestion(45-47).
Food processing can both increase and decrease the allergenicity of proteins(48;49).
A protein that is allergenic (i.e. allergen) can cause sensitization and subsequently induce an
allergic response to the protein during the next exposure. An allergen however does not always
do both. Some allergens only cause sensitization whereas others only induce a clinical response
by crossreactivity. Sensitization without clinical reactivity is often seen in hazelnut and peanut
sensitized subjects that are also grass or tree pollen allergic(50;51). Often this can be explained by
respectively cross-reactive carbohydrate determinants (CCD) and pathogen related 10 (PR10)
proteins. CCD induces IgE antibodies to grass, but also cross reactive IgE antibodies to hazelnut
and peanut. These cross reactive antibodies however do not cause a clinical response(52;53). Similarly Bet v 1, a pollen allergen of the PR-10 family, causes sensitization and clinical reactivity to
tree pollen, but often causes cross reactive sensitization to the peanut allergen Ara h 8 (a PR-10
homologue) without (severe) clinical reactivity. This cross reactivity can be explained by a lack
15
Chapter 1
of discrimination of IgE. The lower affinity for the cross reactive allergens might be a reason for
the absence of clinical reactivity. On the other hand some allergens can cause clinical reactivity,
but are dependent on sensitization via other allergens. These allergens are called incomplete
allergens. A nice example of an incomplete allergen is mal d 1, an apple allergen. This protein
rarely induces IgE antibodies, due to its instable form. Bet v 1 however is a very similar protein
to Mal d 1, which does induce IgE antibodies and also causes reactivity to Mal d 1(54).
Better insight in the functions, properties and cross reactivity of allergens is important for diagnosis and in treatment. Peanut is a legume (such as beans) and taxonomically distantly related
to tree nuts. Several peanut and tree nut allergens however belong to the same protein family(55).
So far 16 peanut allergens have been described. These allergens belong to different families of
seed storage proteins, pathogenesis related proteins, lipid transfer proteins, oleosins and defensins (PR)-10(56). In table 1 the characteristics of these peanut allergens are shown in more detail.
Table 1 | Peanut components
Allergen
Protein Family
Related Proteins
Ara h 1
Cupin
Gly m 8
Cor a 11
Ara h 3
Cupin
Cor a 9
Gly m 9
Ara h 2
Ara h 5
Ara h 6
Ara h 7
Ara h 8
Ara h 9
Ara h 10
Ara h 11
Ara h 12
Ara h 13
Ara h 14
Ara h 15
Ara h 16
Ara h 17
Conglutin
Profilin
Conglutin
Conglutin
PR-10
Lipid Transfer Protein
Oleosin
Oleosin
Defensin
Defensin
Oleosin
Oleosin
Lipid Transfer Protein
Lipid Transfer Protein
Cor a 14
Bet v 2
Cor a 14
Cor a 14
Bet v 1
Pru p 3
Cor a 12
Cor a 12
Gly m 2
Gly m 2
Cor a 12
Cor a 12
Pru p 3
Pru p 3
Gly m 8
Gly m 3
Gly m 8
Gly m 8
Mal d 1
Art v 3
Ses i 4
Ses i 4
Art v 1
Art v 1
Ses i 4
Ses i 4
Art v 3
Art v 3
The 16 peanut allergens so far known are outlined in table 1. Ara h 4 is not mentioned, as this is now
recognized as being similar to Ara h 3. Knowledge of the protein family helps in understanding possible
cross-reactivities and functions of the protein. Ara h: peanut allergen (Arachis hypogaea); Gly m: Soybean
allergen (Glycine max): Cor a: hazelnut allergen (Corylus avellana); Bet v: birch pollen allergen (Betula
verrucosa); Mal d: apple allergen (Malus domestica); Pru p: peach allergen (Prunus persica); Art v: mugwort pollen allergen (Artemisia Vulgaris); Ses i: sesame allergen (Sesamum indicum). It is likely that more
allergens will be discovered in coming years.
16
General Introduction
Not all allergens are present in extracts used for diagnosis, which is underlined by false-ne-
gative IgE tests(57). In numerous studies it has been shown that extracts used for IgE tests
and SPT are often not comparable(58;59). Which proteins are present in an extract depends on
the source materials and production procedures. It is suggested that the diagnosis would be
better interpretable if tests could be performed for selected, purified or recombinant allergens(60). This can be determined by molecular or component resolved diagnostics (CRD).
Mechanisms in IgE sensitization and allergy symptoms
The initial allergic response can immunologically be divided in two different phases: sensitization and elicitation. The typical allergic inflammatory response is an acute response within
two hours after exposure to the allergen. Although some food allergic patients outgrow their
allergies (as seen in cow’s milk allergy), food allergy is often persistent, with acute symptoms
upon every exposure. Acute allergic responses can result in remodelling of tissue which causes chronic inflammation independent of the trigger.
Initial allergen exposure causes an immune response that is speculated to be initiated by ep-
ithelial cell-derived cytokines such as TSLP, IL-25 and IL-33(61-66). These cytokines activate
group 2 innate lymphoid cells (ILC2) and dendritic cells. Activated ILC2 cells produce large
quantities of IL-5 and IL-13.The activated dendritic cells present the allergen via interaction
of MHC and the T cell receptor, which then induces Th2 cells. Th2 cells produce type 2 cytokines like IL-4, IL-5, IL-9 and IL-13 that result in differentiation, recruitment and activation
of eosinophils, basophils and mast cells(67;68). The production of IL-4 by Th2 cells results in
provision of B cell help in isotype switching to IgG1 and IgE. B cells continuously produce
IgE, but specific IgE is only formed after recognition of the antigen(61;69;70). IgE binds via its
Fc-part, to its high affinity receptor on the mast cells and basophils, arming them for the next
exposure. This arming phase is the sensitization phase. After re-exposure with the allergen,
elicitation of allergic symptoms can occur. Within a few minutes after the allergen has crosslinked the high affinity IgE receptors via binding to IgE on mast cells and basophils, degranulation of these cells occurs, with the release of mediators. Histamine is an important mediator,
causing vasodilatation, increased vascular permeability and pruritus. The acute allergic re-
sponse is dominated by Th2 cytokines. The number of Th1 cytokines however increases in
the chronic state of allergic diseases such as atopic dermatitis and allergic asthma, leading to
tissue remodelling(71;72). It is not yet known if tissue remodelling is also seen in food allergy(73).
In addition, TSLP, IL-25 and IL-33, more innate-type cytokines, also seem to contribute to the
chronic inflammation. Although these Th2 driving cytokines have an important regulatory
function maintaning the balance of immune homeostasis, uncontrolled production of these
cytokines can cause chronic allergic inflammation(74).
17
Chapter 1
PART 2
Immune therapy
Inflammation can be acute, persistent (repetitive acute inflammation) or chronic. The basics
for treatment in allergic disease are to control environmental factors and specific medicines
that relieve or control symptoms. The most important step in controlling environmental factors is avoiding the triggering allergen. Avoidance of allergens is however often not possible
or very difficult (i.e. pollen in rhinitis, or low amounts of peanut which can be present in many
foods). Fatalities due to allergic reactions are still reported every year(75). Therapies aimed at
relieving acute symptoms (such as anti-histamines) do not affect the persistency or chronicity
of symptoms. Corticosteroids can control symptoms of chronic inflammation, as often seen
in patients with atopic dermatitis or asthma. However the disease remains and is not cured.
Various biologicals, including cytokine blockers and anti-IgE have been introduced as therapy
in allergic disease(76;77). These have thus far been of limited success in specific patient sub-
groups only, which can be explained by the complexity of allergic diseases as well as the limited repertoire of biomarkers(78;79). Anti-IgE treatment for example is an approved treatment
for patients with moderate to severe persistent asthmatics. However, this is non-effective in
about a third of patients and only mildly effective in another third. The treatment response
cannot be explained by differences in total IgE levels(80). Better predictors for success and
improvement of this kind of therapy are being researched.
The only long-term cure for allergic disease is seen after allergen-specific immunotherapy
(SIT)(81;82). Various randomized studies have shown effective reduction of symptoms in in-
sects, pollen and dust mite allergy(83;84). Tolerance is induced by repeated and increasing doses
of the causative allergen. Immunological mechanisms include the induction of Tregs, produc-
tion of IL-10 and TGFβ as well as the production of specific antibody isotypes, such as IgG1
and IgG4. SIT often induces immune tolerance with desensitization for several years. Drawbacks of immune therapy are its antigen specificity and the so far limited number of allergens
available, as well as the number of side effects and the long duration of treatment.
In chronic allergic disease allergen targeted therapy is probably insufficient. Remodelling of
tissue, as seen in asthma, leads to chronic inflammation which can be present independent
of the original trigger(85). Antigen specific treatment will therefore not be successful in this
chronic inflammatory state.
Tolerance and immune regulation
Regulatory T cells play an important role in tolerance induction and controlling inflamma-
tion(86). FOXP3+ regulatory T cells are the best known and characterized T cells subset dedicated to suppression of unwanted immunity. FOXP3 is a transcription factor that is crucial for the
development and function of these Tregs. The significant role of FOXP3+ Tregs is illustrated by
18
General Introduction
a syndrome called IPEX (which stands for Immune-dysregulation Polyendocrinopathy Enteropathy X-linked(87)). A mutation in FOXP3 causes a defect in regulatory T cells in IPEX. Patients
with IPEX can present with auto immune diseases, atopic dermatitis and have increased levels of IgE(88;89). Although we know that FOXP3 Tregs are related with the production of IgE
and allergic symptoms the studies are limited(90-92). In general it is hypothesized that defective
Tregs promote the development of allergic disease.
Regulatory T cells are both derived from the thymus (the natural FOXP3+ Tregs (nTregs)), as induced in the periphery (iTregs). iTregs can be further divided in FOXP3+ Tregs, Tr1 cells producing
IL-10 and Th3 cells producing TGFβ(86) The distinction between the different types of Tregs is not
absolute. For example FOXP3 expressing Tregs are also capable of producing IL-10 and TGF-β.
IL-10 is an important cytokine for Tregs, but also plays an important role in the production of
IgG4(88;89;93-96), an immunoglobulin often upregulated when patients develop tolerance(97). T cell
clones derived from children with persistent cow’s milk allergy have been shown to produce
Th2 cytokines, whereas allergic control subjects that are cow’s milk tolerant produced a mixed
Th1/Th2 response, associated with significant elevated IL-10 levels(98). Children with high ex-
posure to cats that are less likely to become allergic to cats are characterized by an elevated
level of T cell derived IL-10, as well as elevated levels of allergen specific IgG and IgG4(99). Similar
observations have been made in bee keepers with repeated exposure to bee venom(100). Again
the high exposure to the allergen results in an increase in allergen specific IgG. Interestingly,
reactions to the stings disappear simultaneously with the increased IL-10 production by T cells,
while they reappear at the beginning of the next season. As repeated exposure to antigen can in-
crease IL-10 and IgG4, this concept is also used in some models of immune therapy as described
in more detail below.
The Loeys–Dietz syndrome (LDS), like IPEX, is a syndrome that is associated with allergic
diseases, high IgE and also eosinophilia(101). This syndrome however is caused by a mutation
that affects the TGFβ-receptors. TGFβ plays an important role in mediating the immunosuppressive effects of Tregs through cell-cell contact, in activating regulatory T cells, but also by
directly suppressing effector cells (102). Both IL-10 and TGFβ are also key cytokines produced
by Bregs(103), and in recent years the absence or loss of regulatory B cells (Bregs) has also been
associated with allergic disease.
Above mentioned studies suggest that FOXP3+ Tregs and the cytokines IL-10 and TGFβ play
an important regulatory role in the prevention and control of allergic responses.
Heat Shock Proteins
Heat shock proteins (HSP) are a family of stress induced proteins, ubiquitously expressed in
eukaryotic and prokaryotic cellular organisms. Heat shock, but also exposure to other kinds of
physiological and environmental stress can lead to the expression of HSP. They are evolution-
19
Chapter 1
ary highly conserved both in human, as in bacteria and are immune dominant(104;105). HSP can
both trigger innate and adaptive immune responses, and HSP-specific antibodies are already
present directly after birth(104;106-108). HSP are associated with both pro- and anti-inflammatory
responses (109;110). They serve as damage associated molecular pattern molecules (DAMP) that
can bind with several receptors, including toll like receptors (TLR) (111;112). HSP can thereby ac-
tivate the innate system and induce pro-inflammatory cytokines. Anti-inflammatory responses related to HSP are associated with adaptive immune responses(113-114).The induction and
presence of HSP specific T cells has been shown to be protective in an experimental model of
adjuvant arthritis and is associated with a favourable diagnosis in patients with juvenile idio-
pathic arthritis(104;115;116). Protective effects of various microbial HSP were seen in many other
experimental modes of chronic inflammation (109;117-119). So far little is known about the role
of HSP in human models of allergic chronic inflammation, as further reviewed in chapter 3.
Outline of this thesis
In the first part of this thesis we have investigated the immunogenic response of HSP. In chap-
ter 2 we have reviewed the role of HSP in atopic disease. In chapter 3 and chapter 4 we have
investigated the immunogenic response of HSP at birth in a disease free environment and in
patients with atopic dermatitis.
In the second part of this thesis we have investigated how we can improve the diagnosis
of peanut allergy. In chapter 5 we investigated if there was a different cytokine profile in
children with a clinical response to peanut compared to patients with only sensitization to
peanut. In chapter 6 we have discussed the role of CRD compared to IgE testing to peanut
extracts in the diagnosis of peanut allergy.
20
General Introduction
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General Introduction
29
Chapter 2
HSP: Bystander Antigen in Atopic Diseases?
Joost A Aalberse; Berent J Prakken; and Berber Kapitein
Front Immunol. 2012 31;3:139
Chapter 2
Abstract
Over the last years insight in the complex interactions between innate and adaptive immunity
in the regulation of an inflammatory response has increased enormously. This has revived the
interest in stress proteins; proteins that are expressed during cell stress. As these proteins
can attract and trigger an immunological response they can act as important mediators in this
interaction. In this respect, of special interest are proteins that may act as modulators of both
innate and adaptive immunity. Heat shock proteins (HSP) are stress proteins that have these,
and more, characteristics. More than two decades of studies on HSP has revealed that they are
part of intrinsic, “natural” mechanisms that steer inflammation. This has provoked compre-
hensive explorations of the role of HSP in various human inflammatory diseases. Most studies
have focused on classical autoimmune diseases. This has led to the development of clinical
studies with HSP that have shown promise in Phase II/III clinical trials. Remarkably, only very
little is yet known of the role of HSP in atopic diseases. In allergic disease a number of stud-
ies have investigated the possibility that allergen-specific regulatory T cell (Treg) function is
defective in individuals with allergic diseases. This raises the question whether methods can
be identified to improve the Treg repertoire. Studies from other inflammatory diseases have
suggested HSP may have such a beneficial effect on the T cell repertoire. Based on the immune
mechanisms of atopic diseases, in this review we will argue that, as in other human inflam-
matory conditions, understanding immunity to HSP is likely also relevant for atopic diseases.
Specifically, we will discuss why certain HSP such as HSP60 connect the immune response to
environmental antigens with regulation of the inflammatory response. Thus they provide a
molecular link that may eventually even help to better understand the immune pathological
basis of the hygiene hypothesis.
32
HSP: Bystander Antigen in Atopic Diseases?
Introduction
Over the last years insight in the complex interactions between innate and adaptive immunity
in the regulation of an inflammatory response has increased enormously. This has revived the
interest in stress proteins; proteins that are expressed during cell stress. As these proteins
can attract and trigger an immunological response they can act as important mediators in
this interaction. In this respect, of special interest are proteins that may act as modulators of
both innate and adaptive immunity. Heat shock proteins (HSP) are stress proteins that have
these, and more, characteristics. More than two decades of studies on HSP has revealed that
they are part of intrinsic, “natural” mechanisms that steer an inflammatory response(1;2). This
has provoked comprehensive explorations of the role of HSP in various human inflammatory
diseases. Most of these studies have focused on classical autoimmune diseases such as type
I diabetes (IDDM), rheumatoid arthritis (RA), and juvenile idiopathic arthritis (JIA)(3-5). This
has led to the development of clinical studies with HSP that have shown promise in Phase II/
III clinical trials in both IDDM and RA(6-8).
Remarkably, only very little is yet known of the role of HSP in atopic diseases. Based on the
immune mechanisms of atopic diseases, in this review we will argue that, as in other human
inflammatory conditions, understanding immunity to HSP is likely also relevant for atopic
diseases. Specifically, we will discuss why certain HSP such as HSP60 connect the immune
response to environmental antigens with regulation of the inflammatory response. Thus, they
provide a molecular link that may eventually even help to better understand the immune
pathological basis of the hygiene hypothesis: an important concept relating the increase in
the prevalence of atopic disease with exposure to microbes or microbial products early in life.
33
Chapter 2
Allergy
Atopy is the predisposition to develop allergic diseases like atopic dermatitis, food allergy,
asthma, and allergic rhinitis. It is characterized by a predominant typical T helper 2 (Th2)-like
immune response(9). Firstly, an environmental allergen, like an inhalant or food allergen in-
teracts with the innate immune system. After uptake by antigen-presenting cells, subsequent
T cell priming leads to the stimulation of type 2 cytokines such as interleukin (IL)-4, IL-5,
and IL-13. These cytokines interact with their receptors to induce a class switch toward IgE
production and to increase the number of eosinophils and mast cells. In immediate hypersen-
sitivity reactions IgE binds to its receptors and causes mast cells to degranulate. Apart from
IgE, increased levels of IgG4 are often measured in allergic individuals. IgG4 is considered as
an antibody with anti-inflammatory activity(10). The levels of IgG4 are usually elevated in se-
rum from people that are repeatedly exposed to the same antigen, like beekeepers, or subjects
receiving increased doses of antigen during specific immune therapy (SIT). In addition to this
humoral immune response, allergy is characterized by a cellular response, mostly mediated
by Th2 cells(11,12), but also by Th1 cells (like in atopic dermatitis) and regulatory T cells. The
combination of these humoral and cellular responses, both innate and adaptive, leads to an
allergic inflammatory reaction(13). Here we will discuss various aspects of the allergic immune
responses in the context of the possible role of environmental antigens such as HSP in steer-
ing this immune response.
Allergy and the Hygiene Hypothesis
The prevalence of atopic disease has increased tremendously in the second half of the twentieth century(14). Although the cause of this increase is not known though, a genetic cause
is unlikely due to the time span in which the increase took place. Therefore environmental
changes are more likely causes of this observed increase. Strachan(15) first proposed the
concept of the hygiene hypothesis. This hypothesis states that the increase in the preva-
lence of atopic disease is due to a decreased exposure to microbes or microbial products
early in life, especially in western societies. The concept was supported by the observation
of an inverse association between both atopic dermatitis and hay fever and the number of
children in a household. Although the hygiene hypothesis and its proposed immune mechanism are still under debate, in the 1990s numerous of studies have been published supporting this hypothesis. For example, these observations relate the number of siblings, the
number of infections and exposure to endotoxins on the farm, with the incidence of atopic
diseases(16-20).
Immune mechanisms of the hygiene hypothesis
What could be the immunological cause of this inverse relation between exposure to environmental antigens and the incidence of allergic disease? The explanation takes us back to
the original observations in the eighties of Mosmann and Coffman who defined two main
34
HSP: Bystander Antigen in Atopic Diseases?
subtypes of helper T lymphocytes: Th1 and Th2 cells. Th2 cells are characterized by their pro-
duction of cytokines like IL-4, IL-5, IL-9, and IL-13 and chemokines like TARC and MDC. They
are involved in mediating allergic responses and host defense against parasitic infection(21).
During fetal life and shortly after birth a predominant Th2 response is present(22). During life,
a shift to a predominant Th1 response occurs in non-allergic subjects, but this shift may be
incomplete in allergic individuals. As a consequence, an allergic individual will develop a Th2
response to an allergen leading to IgE production and typical type 2 cytokine production (IL4, IL-5, and IL-13) and ultimately to a clinical allergic reaction, whereas non-allergic subjects
develop a Th1 response leading to a protective IgG response and production of IFN-γ(23). This
is the basis for the immunological explanation of the hygiene hypothesis, as exposure to mi-
crobes and microbial products would stimulate a Th1 response. Those individuals that were
not enough exposed to these stress factors would be more prone to develop predominant Th2
responses, and thus an allergic response. Obviously this is still a simplification of the immune
pathogenesis that does not explain all observations. For example, a Th1 response in aller-
gic individuals would implicate the development of late hypersensitivity responses, whereas
healthy individuals do not respond to allergens with a clinically noticeable response(24). The
hygiene hypothesis also does not explain why not only the incidence of allergic diseases is
increasing but also that of auto immune diseases like RA and IDDM(25). These diseases are
obviously known as typical Th1-like diseases.
Triggering an allergic immune response
In order to fully assess and better appreciate the value of the hygiene hypothesis it is important to understand which cells are responsible for the initial trigger leading to the characteristic allergic immune response. First, naive T cells have to be activated, in a way that
promotes the classical Th2 like response. There is recent evidence that basophils may play a
crucial role in antigen recognition and processing. When recruited to lymph nodes they are
able to induce Th2 cell differentiation through the release of IL-4(26;27). Other cells also have
the capacity to process and present allergens, including mast cells, macrophages, eosinophils, and natural helper cells. Natural helper cells are innate cell populations that include
innate Th2 cells and are activated by IL-25 and IL-33 to secrete Th2 cytokines(28). There is
an increasing realization that Th2 cells are not the only cells involved in the pathophysiol-
ogy of allergic disease, as particularly in severe disease other cells than Th2 play a role in
aggravating and perpetuating the immune response (Table 1)(29).
35
Chapter 2
Table 1 | Different subtypes described to play a role in the pathogenesis of allergic diseases.
Cell
Main transcription
factor
Activating cytokines
Effector cytokines
Role in allergic diseases
Th1
T-Bet
IL-12
IFNy
Th2
GATA-3
IL-4, IL-25, IL-33, TSLP
IL-4, IL-5, IL-13, IL-25
Possible role in chronic
asthma or atopic dermatitis
Th17
Batf
TGFb, IL-1b IL-6, IL-21
IL-6, IL-8, IL-17; IL-22
Th9
PU-1
IL-4, TGFβ, IL-10, IL-25
IL-9, IL-10
Eosinophil production;
IgE induction
Mucus production, lung
inflammation, dermatitis
Possibly related to
steroid resistant asthma;
related to neutrophil
production
Th22
RORyT(?)
?
IL-22
Tr1
FOXP3
TGFb
Tregs
FOXP3
TGFb
IL-5, IL-10, IL-13, TGFb Regulatory function on
B cells by suppression of
allergen-specific IgE and
induction of IgG4 and IgA
IL-10
Limiting airway inflammation in mice. Neg.
association with Th2
cytokines
Diminished function
and number in allergic
individuals
First of all, whereas Th2 cytokines have a clear role in initiating an allergic response, espe-
cially Th1 cells have been implicated in more chronic severe allergic disease, both in atopic
dermatitis and allergic asthma(30-32).
Secondly, in addition to the Th2 cytokines IL-4, IL-5, and IL-13, recently also IL-9 was shown
to play an important role in asthma. At first IL-9 was discussed as a new Th2 cytokine. It
was shown that Th9 cells are dependent on both IL-4 and transforming growth factor-beta
(TGF-β) for their development. Interestingly, like Th2 cells, also Th9 cells are regulated by
IL-25, predominantly seen in lung inflammation(33-36). IL-25 (like IL-33 and thymic stromal
lymphopoietin) is a type II initiating cytokine(37). In addition to the role of IL-25 in lung inflam-
mation, it is also of importance in atopic dermatitis and food allergy(38;39). Although IL-25 (also
known as IL-17E) is a cytokine from the IL-17 family it has opposite effects of IL17A (IL-17).
Thirdly, Th17 cells (named after their main effector cytokine; IL-17) like Th1 cells may be associated with more severe asthma and mediate a more neutrophilic pattern of inflammation.
36
HSP: Bystander Antigen in Atopic Diseases?
Fourthly, it was recently shown that IL-22 (also produced by Th17 cells), is produced by a dis-
tinct set of CD4+ cells, named Th22-cells. These cells have been implicated as being protective
in a mouse model of allergic lung inflammation(40) and may play a role in the severity of atopic
dermatitis in humans(41).
Finally, a lot of attention has recently focused on a subset of T cells with the capacity to suppress other T cells, namely regulatory T cells (Treg). Two types of regulatory T cells will be
discussed below, namely IL-10 producing regulatory cells and FOXP3 expressing regulatory
cells.
Regulatory T Cells
Here we will discuss two types of regulatory T cells; FOXP3 expressing Tregs and IL-10 pro-
ducing regulatory T cells. However, it has to be emphasized that the distinction between these
two types is not absolute, as for example FOXP3 expressing Tregs are also capable of producing IL-10. Moreover, many different regulatory T cells, may even act in concert in a single
immune response.
IL-10 producing regulatory T cells
As discussed above, exposure to an allergen leads to the development of sensitization in a
susceptible individual. Upon a next encounter, a typical Th2 reaction will follow, including a
humoral (IgE and IgG4) response. In contrast, when healthy (non-allergic) individuals are ex-
posed to an allergen it was long thought that (in line with the Th1/Th2 dichotomy) instead of
a Th2 response a Th1 response would develop. However it has become clear that, in fact, this
is not the only difference between a normal and an allergic response to an allergen. Though
indeed some IFN-γ responses to allergens are present in healthy individuals, a study by Akdis
et al.(42) showed that this is a simplification of the reality. They isolated CD4 T cells specific
to several food- and aeroallergens from healthy and allergic individuals according to their
IL-4, IFN-γ, and IL-10 secretion profile. Interestingly, allergen-specific T cells that belong to
all three secretion profiles were detectable in both healthy and allergic individuals, with aller-
gen-specific IL-10-secreting T cells being the predominant subset in healthy individuals. They
regarded these IL-10 producing CD4+ T cells as T regulatory cell type 1 (Tr1 cells). Seemingly,
low Tr1 numbers and high Th2 cell numbers resulted in an allergic response, whereas in
healthy individuals a mixed Th1/Th2 response is associated with a strong IL-10 response(43).
The crucial role for IL-10 in allergy is also suggested by other studies on food and inhalant allergies. In cow’s milk allergy, T-cell clones derived from children that are persistently allergic
produced Th2 cytokines (IL-4, IL-13), whereas allergic control subjects that are cow milk tol-
erant produced a mixed Th1/Th2 response associated with markedly elevated IL-10 levels(44).
Similar observations of elevated IL-10 levels associated with less allergy symptoms were
made in inhalation allergies. Children raised in a house with a cat are less likely to become al-
lergic to cat allergen than those who only get indirect exposure. Many of these highly exposed
37
Chapter 2
children had an IgG and IgG4 response to the major cat allergen Fel d 1 without production
of specific IgE. This induction of high levels of allergen-specific IgG4 in the relative absence
of IgE has been referred to as a modified Th2 response(45). Interestingly also in the peripher-
al blood of these non-allergic children, T cell response to the allergens are characterized by
an elevated level of IL-10. Comparable indications for a role for IL-10-producing Tr1 cells in
the maintenance of tolerance to allergens can be found in individuals exposed to relatively
high doses of allergen like bee keepers and allergic patients undergoing immunotherapy. Bee
keepers with a repeated exposure to bee venom during the bee-keeping season demonstrate
a marked increase in allergen-specific IL-10 secretion from peripheral blood T cells as the
season progresses, while allergen-specific IgE is seen especially in the beginning of the sea-
son. Interestingly, reactions to stings disappear during the season, simultaneously with the
increased IL-10 production by T-cells(46). Apart from IL-10, also TGF-β, another cytokine that
can be produced by Tr1 cells, is reported to be induced by SIT, while both IL-10 and TGF-β are
associated respectively with the blocking antibodies IgG4 and IgA(23;47).
FOXP3 regulatory T cells
Not only IL-10 producing T cells but also FOXP3+ Tregs are associated with an allergic re-
sponse. The important role of the transcription factor FOXP3 for maintaining immune tolerance stems for multiple basic studies, mainly in experimental models. This importance
of FOXP3+ Tregs in human disease was underscored by the IPEX syndrome (immunod-
ysregulation, polyendocrinopathy, and enteropathy, X-linked). In IPEX patients a genetic
mutation causes the FOXP3 transcription factor to be defective. Patients with IPEX have
symptoms that fit both generalized autoimmunity and allergy(48;49). The relevance of FOX
P3 Tregs has been extensively demonstrated in mice models, showing an inverse correlation to Tregs and diseases like RA, inflammatory bowel disease, MS, and IDDM(50-52). Not
only in autoimmune diseases but also in allergic disease various studies have suggested a
possible defective suppressive function of Tregs(53;54). In addition to the bee keeper model
also the season-dependent antigen exposure during the pollen season offers a model to
study responses in the same individuals with and without exposure. Whereas in several studies it was shown that both non-allergic as allergic individuals have CD4+CD25+
Tregs, in the allergic subjects, these cells still produced IL-5 and IL-13 cytokines(55;56).
Further related experiments demonstrated that both dose and type of allergen appear to
have effects on the ability of CD4+CD25+ T cells to suppress responses. At the time of these
studies CD25b expression on CD4 T cells was used as a surrogate marker of FOXP3+ Tregs.
Later studies using FOXP3 as a direct marker seemed to confirm these data. During venom immunotherapy the increased allergen-specific IgG4 and reduced IgE correlated with
circulating FOXP3 positive Tregs(57). A recent study even suggested that Tregs function
may be impaired in patients with allergic diseases and that this function can be enhanced
by specific immunotherapy(58).
38
HSP: Bystander Antigen in Atopic Diseases?
Thus, altogether, there are ample suggestions that the presence of allergen-specific T cells
with a regulatory phenotype (either expressing FOXP3, and/or capable of producing IL-10
and TGF-β) may have a beneficial effect on allergic diseases. However in some atopic diseases, like atopic dermatitis and asthma, the specific trigger is often not known. This raises the
question whether methods can be identified to improve the Tregs repertoire, when the trig-
gering allergen is unknown. Studies from other inflammatory diseases, like JIA and RA, have
suggested that a certain group of antigens, called HSP, are present at the site of inflammation
and may have such a beneficial effect on the T cell repertoire.
Heat Shock Proteins
The Tregs repertoire is both formed in the thymus (so-called natural Tregs) and generated
in the periphery upon encounter with an antigen (induced or adaptive Tregs). Both self and
non-self antigens can induce Tregs in the periphery, although the repertoire of Tregs might be
biased toward self antigens(59).
In 1991, Cohen and Young proposed that a select group of self antigens is especially important
for the maintenance of peripheral tolerance(60). He described the presence of auto reactive im-
mune responses in a healthy individual to a limited set of self molecules, formed by auto reac-
tive T cells and antibodies, which he called the immunological homunculus. The self antigens
of this “homunculus” are all evolutionary highly conserved between the self and the non-self
homolog of these proteins. According to Cohen, the immune system utilizes these self anti-
gens to form an immunological picture of self which is crucial for the balance of the immune
system. One of these homunculus’ self proteins is human HSP60(2). HSP are indeed evolution-
arily highly conserved proteins and either present constitutively, functioning as chaperones,
or induced upon cell stress caused by, for instance, heat, oxidative stress, and hypoxia(61). Sev-
eral HSP have been identified and, according to their size, organized into six families: HSP100,
HSP90, HSP70, HSP60, HSP40, and HSP10. In this review we focus on the immune responses
of HSP60 in atopic disease. It has to be stressed that as only very little data are available right
now on HSP and allergic disease, and thus we need to deduce the role of HSP in atopic disorders mostly on what is known about HSP reactivity on other diseases.
Immunity to HSP and inflammation
Humoral and cellular immune responses to HSP60 have been detected both in patients with
an inflammatory disease, such as autoimmune and allergic diseases, as well as in healthy
subjects. After these initial discoveries the perception was that HSP’s may be involved in the
development of autoimmunity through antigenis mimicry: an immune response toward a microbial HSP could lead to a cross-reactive response to a self-HSP and thus cause autoimmun-
ity. However, after further studies it quickly became clear that the immune responses toward
self HSP60 were more involved in the regulation and not in the induction of autoimmunity.
Indeed, a wealth of data obtained in the last decade both in experimental models and from
observations in human diseases point to a regulatory role of immunity to self HSP60(2).
39
Chapter 2
The presence of self-HSP-reactive cells was first shown in animal studies by immunization of rats
with mammalian HSP60(62). Though immunization could induce self-HSP-specific antibodies and T
cells, a self-HSP-reactive immune repertoire was also shown to be present in the absence of immunization. The cause for this could be previous contact with homologous bacterial HSP, for example
in the mucosa of the gastro intestinal tract. The first report that immune responses to self HSP60
may have a regulatory role in inflammatory diseases was in mycobacteria-induced adjuvant ar-
thritis. In this model the induction of a self HSP60, cross-reactive T cells response was responsible
for the observed protection of HSP60 peptide immune therapy. After these initial findings, protective effects of various (conserved) microbial HSP were seen in many other experimental disease
models, including arthritis, atherosclerosis, allergic encephalomyelitis, and allergic asthma(63-66).
In vitro experiments show that immune responses modulated by HSP can result in the induc-
tion of various cytokines like IFNγ and TNFα, as well as IL-10. HSP are strong immune modulators and are able to influence the impact and direction of immune responses(67). Moreover,
HSP60 has the capacity to steer both innate and humoral immunity.
In human diseases, antibody responses to HSP60 were previously described in skin lesions of
patients with Behçets disease, whereas HSP60 has been described as a target for T-cells in au-
toimmune diseases such as IDDM and JIA(8;68;69). Data in the experimental models have pointed
out that the protective effect of HSP60 was independent of an antigenic relationship (antigen
mimicry) between HSP60 and the disease-inducing antigen. Instead it seemed that HSP60
could confer protection through so-called bystander suppression(70;71). Inflammation causes
local damage and cell stress leading to upregulation of stress proteins such as HSP60. Next,
these bystander (self) antigens can become the target of subsequent, possibly suppressive
immune responses. So, it is possible that immune responses to HSP could be involved in the
control of human chronic inflammatory diseases that have distinct, although as-yet-unknown,
initiating auto-antigens, or even allergens.
HSP responses in early life
Apparently, responses to HSP60 are important for maintaining peripheral tolerance in the
adult immune system. It is unknown when during live these specific T cells arise. It would
seem reasonable to expect that they are primed after birth upon encounter with homologous
microbial HSP in the gut. However, there are indications that self HSP60 reactivity is present
at birth. In a study by Ramage et al. it was shown that cord blood cells proliferate in response
to an in vitro challenge with the self HSP60(72). This supported the hypothesis that this reactivity is part of the normal naive immune repertoire. A more recent study by Merbl et al. put
these findings also in a different perspective(73). They found that normal cord blood contains
IgM and IgA auto-antibodies directed against a relatively uniform set of auto-antigens, such
as auto-antigens related to immune regulation such as HSP60. This obviously benign autoim-
mune self reactivity, present at birth, may have a dual function. On the one hand it may provide
40
the basis for autoimmunity in later life, while on the other hand the “inborn” autoimmunity
HSP: Bystander Antigen in Atopic Diseases?
to regulatory self antigens such as HSP60 may actually serve to protect against autoimmune
disease. Intrigued by these studies, we questioned whether CD4+ T cells specific for HSP60
are already detectable at birth before exposure to the microbial flora, and if so, to what type
of immune response these “inborn” autoreactive CD4+ T cells have upon exposure to the self
antigen HSP60. We found that HSP60 specific T cells are indeed present at birth. Moreover,
stimulation of CBMC with HSP60 leads to CD4+ T cell proliferation and cytokine production,
and induces T cells with an in vitro regulatory phenotype that are functionally suppressive(74).
HSP60 and allergy
Thus, HSP60 is a self-protein, recognized already at birth in healthy subjects, but also in individuals with chronic inflammatory diseases. Thereby a correlation between self-HSP reactivity and diminished disease activity is seen both in experimental as in human autoimmunity.
This has already led to the successful exploration of HSP60 immune therapy in human dis-
ease, firstly in IDDM. It is not known yet if the levels and responses to HSP60 are different in
individuals developing chronic inflammatory disease later in life.
For once, priming of HSP60 T cell responsiveness takes places primarily in the gut through
contact with microbial HSP60(2). Thus, priming of HSP60 immunity early in life through con-
tact with microbiota leads to more self HSP60 mediated immune regulation. This could be
one of possibly multiple mechanisms that explain why individuals exposed to more microbial
triggers early in life, could be less prone to develop immune mediated diseases. Thus it may
very well fit the concept of the hygiene hypothesis.
For that reason we set out to study the role of HSP60 immunity in human atopic diseases.
Obviously the first pre-requisite for a potential role of self HSP60 in dermatitis is the (pref-
erably increased) expression of HSP60 at the site of inflammation. Indeed we were able to
show that self HSP60 is increased expressed in the skin of patients with atopic dermatitis(75).
Moreover, in vitro stimulation with self HSP60 induced FOXP3 positive T cells, as well as T
cells producing IL-10 and IFN-γ.
It has to be emphasized that this increased expression by no means is very specific for atopic
diseases(76). Still it could be highly relevant for atopic disease as the characterization of an
antigen present at the site of inflammation, without being the disease-causing antigen has
therapeutic possibilities through, as previously mentioned, “antigen driven bystander suppression.” Bystander suppression as a mechanism is especially important in human autoimmune diseases, because often the immunizing trigger is unknown.
Theoretically, in allergic diseases this could also be an interesting option for intervention.
Although often at least one of the allergens triggering the disease is known, mono-sensitiza-
tion is rare which would imply multi-antigen immune therapy. Immune therapy with a single
bystander antigen might undermine this issue.
41
Chapter 2
Novel Immune Therapeutic Possibilities in Allergic Disease
Various novel immune therapeutic approaches to diminish allergic symptoms have been or
are being studied. Of these new interventions, blocking of effector cytokines (such as IL-4
and IL-5) and IgE is the most important. As IL-5 is important for the priming and survival of
eosinophils and these cells play an important role in the pathophysiology of allergic disease,
blocking IL-5 seemed logical. However only a highly selected patient group with severe asth-
ma, shown as sputum eosinophilia, had profit from this therapy(77). Also blocking IL-4 and IL-
13 has not yet been shown to be of clinical benefit in asthma(78). Thus, so far, the experiences
with blocking antibodies to effector cytokines suggest that either the cytokines cannot be
fully blocked or that not one of these cytokines is solely responsible for the allergic response.
As mentioned above in recent years Th2 induction cytokines have been described among
which IL-25. IL-25 is strongly elevated in a subgroup of peanut allergic subjects(38). Moreover,
in mouse models blocking IL-25 has a positive effect on bronchial hyper-reactivity(79). Apart
from blocking cytokines that are important in the pathophysiology of allergic disease, in the
last decade various studies in asthmatic patients have been performed in which an IgE-blocker was used(80). Although the first results look promising, the effect is not present in all patients, underlining again the complexity of chronic allergic diseases.
Antigen SIT, in which an allergic individual is exposed to increasing doses of the allergen trig-
gering disease, has been used for over a century. It is one of the earliest and most effective
forms of human immune therapy. Although for long the mechanisms behind this clearly ef-
fective immune intervention were not fully understood, we now know that IgG4, IL-10, and
Tregs probably play an important role. A disadvantage of this approach in which the eliciting
protein is used, is the chance of a severe anaphylactic reaction, as has been described following immune therapy in peanut allergic subjects(81).
To tackle this issue, recent work has focused on allergy peptide therapy, showing good results. Mice studies showed that after immunization with Der p 2, the house dust mite allergen,
downregulation of T cell and antibody responses was seen to Der p 2. Similar results were
seen using Fel d 1 (cat allergen) and Bet v 1 (birch allergen) peptide therapy. As most patients
are not sensitized to just one allergen, it is now studied if combination immune therapy is also
effective. Another approach is the use of a peptide of an antigen present at the site of inflam-
mation, without being the triggering antigen. This is the concept of the previous mentioned
antigen bystander suppression model(70;71).
Based on the development of immune therapies using HSP60 for other human inflammatory
diseases and encouraged by recent data showing that HSP60 expression is increased in the
skin in patients with atopic dermatitis, it is tempting to suggest that HSP60 may also be a
potential candidate for bystander therapy in allergic diseases.
42
HSP: Bystander Antigen in Atopic Diseases?
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HSP: Bystander Antigen in Atopic Diseases?
49
Chapter 3
Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)
Joost A Aalberse; Berber Kapitein; Sytze de Roock; Mark R Klein;
Wilco de Jager; Ruurd van der Zee; Maarten O Hoekstra;
Femke van Wijk; and Berent J Prakken
Plos One 2011; 6(9):e21119
Chapter 3
Abstract
To prevent harmful autoimmunity most immune responses to self proteins are controlled by
central and peripheral tolerance. T cells specific for a limited set of self-proteins such as human heat shock protein 60 (HSP60) may contribute to peripheral tolerance. It is not known
whether HSP60-specific T cells are present at birth and thus may play a role in neonatal tol-
erance. We studied whether self-HSP60 reactive T cells are present in cord blood, and if so,
what phenotype these cells have.
Cord blood mononuclear cells (CBMC) of healthy, full term neonates (n = 21), were cultured
with HSP60 and Tetanus Toxoid (TT) to study antigen specific proliferation, cytokine secretion and up-regulation of surface markers. The functional capacity of HSP60-induced T cells
was determined with in vitro suppression assays. Stimulation of CBMC with HSP60 led to
CD4+ T cell proliferation and the production of various cytokines, most notably IL-10, Inter-
feron-gamma, and IL-6. HSP60-induced T cells expressed FOXP3 and suppressed effector T
cell responses in vitro.
Self-reactive HSP60 specific T cells are already present at birth. Upon stimulation with self-
HSP60 these cells proliferate, produce cytokines and express FOXP3. These cells function as
suppressor cells in vitro and thus they may be involved in the regulation of neonatal immune
responses.
52
Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)
Introduction
The immune system possesses a range of safekeeping mechanisms that prevent the induction
of a deleterious response to self-antigens. First through negative thymic selection (central
tolerance), by which T cells expressing a T cell receptor with a high affinity for self proteins
are eliminated(1). Secondly, self reactive T cells that do escape central tolerance and develop
into mature T-cells are kept under control through peripheral tolerance(2). One of the most
prominent mechanisms of peripheral tolerance is mediated by regulatory T cells. Regulatory
T cells (Tregs) suppress effector T cell responses in vitro and can down regulate an ongoing
immune response in vivo(3). They appear essential for the homeostasis of the immune system
as deficiencies in Tregs numbers and/or function lead to inflammatory diseases both in mice
and in humans(4-6) and are already present at the start of life(7;8). Tregs are either directly de-
rived from the thymus or generated in the periphery. Although the antigen specificity of Tregs
is still largely unknown, it is assumed that they can act through recognition of self-antigens
in the periphery(9). This fits the premise that the immune response to certain self-antigens
is important for the regulation of immune responses(10). Cohen was the first to propose that
the response to a vested group of self-antigens is important for maintaining peripheral tol-
erance(11). He described the response against this limited set of self molecules in a healthy
individual, formed by auto reactive T cells and antibodies as the immunological homunculus. This ‘homunculus’ of self-antigens shares various properties, among which evolutionary
conservation between the self and the non-self homologue of these proteins. One of these
self proteins is human heat shock protein 60 (HSP60)(12). HSP60 is an evolutionary highly
conserved cellular protein. Also, it is a stress protein and acts as a danger signal for the immune system: the expression of HSP60 is strongly up regulated following cellular stress, e.g.
because of tissue damage during inflammation(13;14). HSP60 can activate both adaptive and
innate immune responses(15;16) which underscores that HSP60 has the potential to be a regu-
lator in an inflammatory response. This is further supported by the observation that in both
animal and human in vitro studies HSP60-reactive T cells have a strong immune modulatory
capacity(17-19). In experimental models these HSP60-specific T cells can effectively suppress in
vivo a variety of experimental autoimmune diseases. These findings have lead to the set up of
several clinical trials using HSP peptides, e.g. in rheumatoid arthritis (RA) and diabetes type
I, with promising results(20-22).
Thus, apparently, responses to HSP60 are important for maintaining peripheral tolerance in
the adult immune system. It is unknown when during live these specific T cells arise. It would
seem reasonable to expect that they are primed after birth upon encounter with homologous
microbial HSP in the gut(12). However, there are indications that self-HSP60 reactivity is present at birth. As early as 1991 it was shown that cord blood cells proliferate in response to an
in vitro challenge with the self antigen HSP60(23;24) and hypothesized that this reactivity is
part of the normal naïve immune repertoire. A more recent study by Merbl et al(25) put these
53
Chapter 3
findings in a different perspective. They found that normal cord blood contains IgM and IgA
autoantibodies directed against a relatively uniform set of autoantigens, such as autoantigens
that are associated with autoimmune disease (such as double stranded DNA) and autoantigens related to immune regulation such as HSP60. The authors of this study suggested that
this obviously benign autoimmune self reactivity, present already at birth, may have a dual
function. On the one hand it may provide the basis for autoimmunity in later life, while on the
other hand the “inborn” autoimmunity to regulatory self antigens such as HSP60 may actu-
ally serve to protect against autoimmune disease. Intrigued by these studies, we questioned
whether CD4+ T cells specific for HSP60 are already detectable at birth before exposure to the
microbial flora, and if so, to what type of immune response these “inborn” autoreactive CD4+
T cells have upon exposure to the self antigen HSP60. We found that HSP60 specific T cells are
indeed present at birth. Moreover, stimulation of CBMC with HSP60 leads to CD4+ T cell pro-
liferation and cytokine production, and induces T cells with an in vitro regulatory phenotype
that are functionally suppressive.
54
Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)
Results
HSP60 induces CD4+T cell proliferation in cord blood
To detect possible HSP60-specific T cells in cord blood, we first set out to study whether in vitro activation of CBMC with (human) HSP60 induces T cell proliferation. First, using thymidine
incorporation we found that five of ten (50%) cord blood samples showed a clear prolifera-
tive response to HSP60 (median Stimulation Index (SI): 2.0; Inter Quartile Range (IQR): 2.5).
Though we used a very pure batch of human HSP60 for these experiments with undetectable
LPS levels (see methods) it is still conceivable that contamination of extremely low levels may
have influenced these assays. We therefore wanted to confirm these preliminary proliferation
data using two peptide epitopes from HSP60. We have shown before that these (panDR bind-
ing-) epitopes can induce T cell proliferation in a majority of patients with JIA and RA(25). Sixty
percent of the cord blood samples (three of five) responded to either one or both peptides
tested (SI > 1.7). These responses were also significantly different (p=0.038) compared to the
proliferative response of TT, which was used as a negative control, assuming that the fetus has
not formed a prior response to TT. No significant proliferation was seen in response to tetanus
toxoid (TT) compared to background. (Figure 1a).
Figure 1 | Human HSP60 (HSP) induces cell proliferation in cord blood
A) Protein-induced proliferation of cord blood mononuclear cells (CBMC). CBMC were cultured for 96
hours in the presence of Human HSP60, both the full protein as well as HSP60-derived peptide and
Tetanus Toxoid (TT). Box whisker plots are shown and significant differences in responses are indicated
with * (p < 0.05). Background (mean +/- SD): = 1395 +/- 1446 CPM. Data (n=10) were obtained from
4 independent experiments. B) Protein-induced proliferation of cord blood mononuclear cells (CBMC)
cultured in the presence of CFSE. CBMC were cultured for 144 hours and afterwards stained for CD4.
HSP60 proliferation is represented in lighter grey. Percentage indicates number of divided CD4+ T cells.
Data are representative for 3 independent experiments. C) Protein-induced proliferation of cord blood
mononuclear cells (CBMC) cultured in the presence of CFSE. Box whisker plots are shown. Data (n=3) are
obtained from 3 independent experiments.
55
Chapter 3
Though this assay showed T cell proliferation to HSP60 we next wanted to confirm this with CFSE
staining, which allowed us to determine which cells were dividing upon HSP60 stimulation. We
found that indeed CD4+ T cells proliferated specifically after HSP60 stimulation in all samples (Figure 1b and 1c). Thus, HSP60 induced proliferation of cord blood derived CD4+ T cells.
To identify if these responses of CBMC and CD4 T cells could be the result of HSP60 acting as
a ligand for TLR2 or TLR4, as previously suggested, we also performed these proliferation
assays after incubation with a TLR2 and/or a TLR4 blocking antibody, or IgG isotype control
(Supplemental figure 1). No differences in HSP60 induced proliferation were found in the
presence of TLR blocking antibodies suggesting that the observed proliferation of CBMC in
response to HSP60 is TLR2 and TLR4 independent.
Cytokine production in cord blood stimulated cells
We next wanted to know whether stimulation with HSP60 also led to cytokine production
by CBMC. Therefore, cytokine levels in culture supernatants after 96 hours of stimulation
with HSP60, TT or medium alone were measured with a multiplex immune assay. We found
that, compared to stimulation with medium, HSP60-induced production of IL-10 (p = 0.009),
IL-5 (p=0,013) IL-6 (p=0.003), IL-13 (p=0.003), TNF-alpha (TNFα) (p=0.003) and IFN-gam-
ma (IFNγ) (p=0.005) were significantly increased. TT induced a significant increase of IL-6
(p=0.045) compared to stimulation with medium (Figure 2a).
To establish which cells are producing IL-10 and IFNγ, we next measured intracellular cytokine
production by FACS, again after stimulation in vitro with medium, HSP60 and TT. We found an
increased intracellular expression of mainly IL-10, and also IFNγ, by CD4+ T cells (Figure 2b
and 2c), after stimulation with HSP60 only (and not with TT). Thus, stimulation of CBMC with
HSP60 leads to proliferation and cytokine production (IL-10 and IFNγ) of CD4+ T cells.
HSP60 -specific induction of regulatory T cells
There are indications that HSP60, and/or peptides derived from HSP60 may induce or enhance the regulatory T cell population. We therefore wanted to know whether HSP60 may
also play a role in T cell regulation at this crucial period at, or even before birth. We therefore
stimulated cells again with TT, HSP60 or medium and next stained the cells for expression of
CD4, CD30, CD45RO (memory T cells) and FOXP3. FOXP3 is a transcription factor that is char-
acteristic for Tregs. CD30 is an activation marker that has been suggested to be a marker for
Th2 cells, whereas we recently found that CD30 is upregulated on antigen-induced Tregs(26).
As can be seen in figure 3a, following stimulation with HSP60 we found a significant increase
of CD4+CD30+ and CD4+CD45RO+ as well as an increase of CD4+FOXP3+cells, which is not seen
after stimulation with control antigen TT. The FOXP3+ expression was found in about 50%
of the CD30+ and 25% of the memory T cells and not in CD45RO- T cells (Figure 3b and 3c).
56
Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)
HSP60 induced regulatory T cells are functionally suppressive
We next questioned whether these induced Tregs were functional and thus have in vitro sup-
pressive capacity. Therefore we performed CFSE dilution - suppression assays, in which we
added HSP60 induced Tregs (CD4+CD25+CD127- T cells) and CFSE-labeled effector cells in dif-
ferent ratios. The HSP60-induced Tregs were able to functionally suppress the effector T cells.
Compared to the proliferating CD4+ T cells we saw a decreasing percentage of proliferating
cells (figure 4a). After adding HSP60-induced Tregs to the effector T cells in Treg:Teff ratios
of 1:2; and 1:1 the percentage of suppression of proliferating CD4+ T cells increased from
respectively 41.3 to 56.6%. This indicates that HSP60 induced CD4+CD25+CD127- T cells are
functional suppressor cells in vitro.
Figure 2 | Cytokine production induced by human HSP60 (HSP) in cord blood cells
A | Cells were cultured with human HSP60 or Tetanus Toxoid (TT) for 4 days. Cytokine levels in supernatant of the cultures were measured using a multiplex immunoassay. Levels of cytokines are presented in a
histogram plot. HSP stimulated cells in white, and TT stimulated cells in light grey are compared to only
medium cultured cells in black. The supernatants (n=7) were obtained in 3 independent experiments.
Cytokine production was measured in one assay. B | Percentage IL-10+ and IFNγ+ CD4+ T cells following
stimulation with human HSP60. Box whisker plots are shown and significant differences in responses are
indicated with * (p < 0.05). Data (n=4) were obtained from 2 independent experiments. C) A representative FACS-analysis of two independent experiments (n=4) showing intracellular IFNγ and IL-10 staining
of CD4+ T cells following stimulation with human HSP60.
57
Chapter 3
Figure 3 | Human HSP60 (HSP) induces CD30 and the expression of FOXP3 in cord blood
CD4+ T cells after stimulation for 6 days. A FACS analysis shows a significant higher level of CD4+CD45RA+,
CD4+CD45RO+ and CD4+CD30+ T cells as well as CD4+FOXP3+ T cells following stimulation with HSP60.
Box-Whisker plots are shown and significant differences in responses are indicated as * (p < 0.05). Data
(n=7) were obtained from 3 independent experiments. B FACS analysis of FOXP3 expression within the
CD4+CD30+ and CD4+CD45RO+ populations following HSP60 stimulation. Data (n=7) were obtained from
3 independent experiments. C) FACS analysis representing data (n=7) from 3 independent experiments
of CD4+ T cells expressing FOXP3 after stimulation with human HSP60, showing that most FOXP3 positive
CD4 T cells also express CD45RO and half of them express CD30.
58
Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)
Figure 4 | Human HSP60 (HSP)-induced CD4+CD25+ cord blood cells suppress proliferation of T
effector cells
Different amounts (0, 10.000 or 20.000) of sorted HSP60-induced regulatory T cells were cultured with
20.000 CFSE labeled effector T cells for 5 days in the presence of plate-bound anti-CD3. A | A CFSE dilution profile of effector T cells alone (open histogram) or in the presence of HSP60-induced Tregs at
a ratio of 1:1 (closed histogram). This figure represents data (n=3) from 2 independent experiments.
B | Percentages suppression measured by CFSE dilution. Data (n=3) were obtained from 2 independent
experiments.
59
Chapter 3
Discussion
With this study we show that in cord blood HSP60 induces CD4+ T cells that proliferate and
produce various cytokines, most notably IL-10 and IFNγ. Moreover such HSP60-induced T
cells express CD30, CD45RO and FOXP3, and act as suppressor T cells in vitro. These findings
underscore the universality and thus presumed importance of the “inborn” recognition of self
proteins, like HSP60, early in life.
A healthy immune system is able to initiate as well as regulate inflammation, while maintaining
tolerance for self-antigens. Immune responses to certain self proteins are hypothesized to be important for a healthy immune system(27). These “immunological homunculus” proteins, among
which stress proteins like HSP60, may serve as immune biomarkers to the body educating the
immune system on the state of present inflammation. Our results would fit this hypothesis, as
it suggests that an immune response to self HSP60 is part of an “inborn” mechanism present
already at birth that may help to regulate inflammation. It can be speculated that human HSP60
could be regarded as a recall antigen in the fetus, as self proteins are probably continuously
present in the developing fetus due to apoptosis of developing tissues. This may contribute to
induction of a (tolerogenic) response to self proteins like HSP60 before birth and may explain
why HSP60 in cord blood elicits such evident responses as seen in our study.
Interestingly, HSP60 has previously been identified as a potent regulator of inflammation in the adult
immune system. In the end of the 80’s human HSP60 was found to be recognized by the immune system during inflammation. It was then shown that HSP60-specific T cells can dampen experimental
autoimmune disorders, while in humans HSP60 is recognized by healthy individuals free of autoimmune disease or infection(28). The capacity of HSP60 to induce both innate and adaptive immunity
indicates that HSP60 has a double function: responsiveness to HSP60 can be related to inflammation, but also to a healthy immune system. Next it was shown in human studies that HSP60-spe-
cific immune responses in patients with juvenile arthritis (JIA) and RA are associated with a better
prognosis and milder symptoms, again suggesting a role in regulating inflammation (29). In line with
these findings we now show that HSP60-specific T cells are present at birth which underscores the
hypothesis that auto-reactive T cells are a principal feature of a healthy immune system.
In addition to the finding that in cord blood HSP60 can induce FOXP3+ regulatory T cells, that
are functionally suppressive, we have shown that HSP60 leads to the induction of IL-10 and
IFNγ, suggesting the upregulation of Tr1 cells. We have also shown that HSP60 leads to the in-
duction of Th2 cytokines (IL-5 and IL-13) confirming that an immune response in cord blood
is primarily a Th2 response. Another interesting finding is the huge increase of IL-6, as well as
an increase in TNFα. IL-6 has been reported as a Th2 inducer and an important anti-inflam-
matory cytokine, aiding in the protection of the fetus in utero(30). Both TNFα and IL-6 however
have also been shown to be upregulated by TLR agonists in cord blood. Our experiments
60
with HSP60 peptides and our experiments in which we block TLR2 and TLR4 indicate that
Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)
stimulating with HSP60 induces an antigen specific response. In conclusion, functional HSP60
specific CD4+ cells are already present at birth before priming of the immune cells with the
microbial flora in the gut. This mechanism may be important for the maintenance of prenatal
and neonatal immune tolerance.
Materials and Methods
Collection of cord blood
Cord blood samples (n=21) were collected from healthy subjects at the department of gynecology and obstetrics. Inclusion criteria were an uncomplicated pregnancy, full-term infant
and normal vaginal delivery. No further information about the mother, pregnancy or the de-
livery was obtained. Written informed consent was obtained. This study was approved by the
Medical Ethics Committee, UMC Utrecht (NL16498.041.07).
Isolation of CBMC
Cord blood mononuclear cells (CBMC) were isolated from heparinized venous blood by Ficoll
(Pharmacia, Uppsala, Sweden) density gradient centrifugation. The cells were then frozen
in liquid nitrogen and thawed prior the experiment. The culture was performed in RPMI-
1640 supplemented with 2mM glutamine, 100u/ml of penicillin/ streptomycin (Gibco BRL,
Gaithersburg, MD) and 10% (v/v) ABpos heat-inactivated (60 min at 56° Celsius) human serum (Sanquin, Amsterdam, the Netherlands). For measurement of the proliferative activity,
2x105 cells in 200 ml per well were cultured in triplicate in round bottomed 96-wells plates
(Nunc, Roskilde, Denmark) for 96 hours at 37° Celsius in 5% CO2 with 100% relative humidity. Cells were cultured in the absence or presence of 10 mg/ ml human HSP60, both the full
protein as two peptides p2 (human HSP60 peptide 280-292) and p4 (human HSP60 peptide
242-256)(20). The highest response to either peptide was scored. The LPS-content of HSP60
was <0.1 EU /mg protein. Tetanus-toxoid (TT; 1.5 mg/ ml, RIVM, Bilthoven, the Netherlands)
was used as a negative control, assuming that the fetus has not formed a prior response to TT.
For the final 16 hours of culture, 1mCi/ well [3H] thymidine (ICN Biomedicals, Amsterdam,
the Netherlands) was added to each well. Cells were harvested according to standard procedures and incorporated radioactivity was measured by a liquid scintillation counter and ex-
pressed as counts per minute (cpm). The magnitude of the proliferative response is expressed
as stimulation index, which is the mean cpm of cells cultured with antigen divided by the
mean cpm of cells cultured without the antigen.
TLR-blocking assay.
Isolated CBMC were cultured for 96 hours in the presence or absence of 10 µg/ml HSP60 and
in the presence or absence of anti TLR4 blocking antibody (2.5 µg/ml, clone HTA125, Serotech), anti-TLR2 blocking antibody (2.5 µg/ml, clone TL2.1, Serotech), both, or IgG2a isotype
control (2.5 µg/ml, Serotech). Proliferation was measured as described above.
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Chapter 3
CFSE - assay
To characterize which cells proliferate to HSP60, CFSE-assays were performed. For this pur-
pose 3 mM CFSE was added to 1x106 CBMC for ten minutes and washed twice with 10% FCS.
CBMC were resuspended in human AB 20% and stimulated with HSP60 for 6 days. Cells were
then stained with anti-CD3, -CD4 and -CD14 and analyzed by FACS Calibur (Becton Dickinson
Biosciences, San Jose, CA, USA), as described below.
Multiplexed particle-based Immuno assay
Cytokine levels were measured after activation of the lymphocytes in vitro as described above.
After 96 hours the supernatants of the cell cultures were stored at -80°C. Cytokines levels of
IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IFNg and TNFa were measured with the Bio-Plex system
and analysed with the Bio-Plex Manager software version 4.0 (Bio-rad Laboratories, Hercules
CA, USA) which uses the Luminex xMap technology(31). The cytokine production is presented
in a color profile to visualise the different cytokines produced.
FACS staining
CBMC were stimulated for 6 days with HSP60 and TT and then washed twice in PBS con-
taining 2% FCS (PBS-FCS) and blocked with the appropriate normal serum (30 min at 4°C).
Subsequently, the cells were incubated in 50 µl of FACS buffer containing four appropriately diluted PE-, FITC-, PerCP-, or allophycocyanin-labeled mAbs against human CD4 (clone
SK3) (Becton Dickinson Biosciences), CD30 (clone BER-H8) (Becton Dickinson Biosciences),
CD45RA (clone JS-83) (eBioscience, San Diego, CA, USA), CD45RO (clone 4CHL-1) (eBioscience, San Diego, CA, USA) and FOXP3 (PCH101) (eBioscience, San Diego, CA, USA). Stained
mononuclear cells were diluted in FACS fluid and run on a FACS Calibur (Becton Dickinson
Biosciences). CellQuest software (Becton Dickinson Biosciences) was used for analysis.
Cytokine analysis by lymphocyte intracellular staining and Flowcytometry
CBMC were stimulated as described above. During the last 4 hours of culture, Golgistop (Becton
Dickinson Biosciences) was added (final concentration of 2 mM). The cells were harvested and
stained for intracellular cytokine and FOXP3 analysis as described previously. For the staining, a
predetermined optimal concentration of PE-conjugated anti-IL-10 (clone JES3-19F1), FITC-conju-
gated anti-IFNg (clone 4S.B3) (Becton Dickinson Biosciences) and APC-conjugated FOXP3 (eBioscience, San Diego, CA, USA) was added to the cells. Cells were analyzed as described above.
Suppression Assay
To examine the functionality of induced regulatory T cells, CBMC were thawed and CD25 positive cells were depleted. The CD25 negative cells were stimulated with HSP60 for 6 days. Ef-
fector T cells (thawed CBMC of the same donor) were stained with CFSE (3mM). With a FACS
sorter added different numbers of CD4+ CD25+CD127- (regulatory) T cells were added to the
20.000 effector T cells, depending on availability of cells. In every experiment we included at
least the Teff:Treg ratios 1:0, 2:1 and 1:1. The different ratios were incubated for 120 hours
62
Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)
in the presence of plate-bound anti-CD3 (1mg/ml) and with the FACS diva the percentages of
proliferation and suppression were measured.
Statistical Analysis
Basic descriptive statistics were used to describe the patient population. A non-parametric
test (Mann Whitney U test, 2 sided) and where appropriate a parametric test (T-test, 2 sid-
ed) was applied to determine significant differences between the stimuli and the controls regarding proliferative responses, cytokine production and expression of surface markers and
intracellular markers. A p-value < 0.05 was considered significant (SPSS Statistical Program,
version 15.0; SPSS Inc, Chicago, Ill.)
Figure S1 | Human HSP60 (HSP60) induced cell proliferation of CBMC, with or without anti-TLR2
and/or anti-TLR4 blocking antibodies, or IgG isotype control.
Shown are mean stimulation index (relative to medium conditions) +/- SEM. Data (n=6) are obtained from
2 independent experiments. NS= non-significant
Acknowledgements
We would like to thank Prof M. Heringa of the department of Obstetrics and Gynaecology for
his contribution in collecting cord blood samples.
63
Chapter 3
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Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)
67
Chapter 4
Recognition of self-heat shock protein 60 by T cells
from patients with atopic dermatitis
Berber Kapitein; Joost A. Aalberse; Mark R. Klein; Wilco de Jager;
Maarten O. Hoekstra; Edward F. Knol; and Berent J. Prakken
Cell Stress and Chaperones 2013;18:87-95
Chapter 4
Abstract
Heat shock protein 60 (HSP60) is a highly conserved stress protein and target of self-reactive
T cells in various inflammatory diseases. Not much is known about a possible role in atopic
disease. As atopic diseases are considered to be the result of a disturbance in the balance
between T helper cells type 2 and regulatory T cells, it is of interest to know whether HSP60
acts as a bystander antigen in atopic disease. Our aim was to investigate whether HSP60 is
involved in the chronicity of inflammation of atopic dermatitis (AD). We studied the expres-
sion of HSP60 in skin tissue of adults with AD by immunohistochemistry. Peripheral blood
mononuclear cells (PBMC) of children with AD were cultured with HSP60 and proliferative
responses, cytokine secretion, surface markers, and functional assays were compared to re-
sponses of PBMC of healthy controls (HC). HSP60 was detected more in lesional skin of AD
patients compared to nonlesional skin. Furthermore, PBMC of children with AD proliferated
more strongly in response to HSP60 compared to HC. HSP60-reactive T cells of atopic children
produced high levels of IFNγ and low levels of IL-10. In vitro activation with HSP60 leads
to the induction of CD4+CD25bright T cells expressing FOXP3 in both HC as well as in atopic
children. However, despite their regulatory phenotype, HSP60-induced CD4+CD25bright CD127-
FOXP3+ T cells of AD patients were incapable of suppressing effector T cells in vitro. HSP60 is
recognized by proinflammatory (IFNγ high, IL-10 low) T cells in atopic patients and is more
present in lesional AD skin. This suggests that HSP60-specific T cell responses contribute to
local inflammation in AD.
70
Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis
Introduction
Atopic dermatitis (AD) is a chronic skin condition in patients with atopic disease. Atopic dis-
eases are inflammatory disorders caused by the induction of a TH2 response by an allergen.
Increasing evidence demonstrates that allergy is not a mere result of an over-reactive TH2
response, but that immune regulatory cells such as natural killer T cells and regulatory T cells
(Tregs) play a role in the regulation of this balance(1-4). These cell populations can suppress
effector T cell responses, both by cell–cell contact and by the secretion of regulatory cytokines
as IL-10 and TGF-β. In active atopic diseases, several studies show that the suppressive function of Tregs is diminished(5-7). Thus, Tregs form an attractive target for therapy in allergic
diseases. Studies in various experimental models of autoimmunity have revealed that Tregs
can be targeted in both an antigen-specific and an antigen-nonspecific fashion (8). Both venues
of immune therapy are now currently being explored in various autoimmune diseases.
Specific targeting of Tregs with antigens has the advantage that only a subpopulation of T cells
is manipulated which lowers the risk of adverse effects. However, the main dilemma of anti-
gen-specific therapy in humans is to find an antigen which is suitable. In some inflammatory
diseases, dominant disease-provoking antigens or allergens are well-known and these can
be used for antigen-specific therapy. Indeed, the proof of principle of such an approach was
underscored by Alexander et al. who showed that peptides of Fel d 1 (the major cat allergen)
can induce antigen-specific tolerance in cat allergic patients(9). In AD, as in many other chronic
inflammatory diseases, a single disease-triggering antigen is not known. For such a disease,
an antigen-specific approach is still feasible if an antigen can be identified that fulfils at least
two conditions. First, the antigen needs to be recognized by T cells from patients and, sec-
ond, the antigen must be available at the site of inflammation, preferably in a disease-specific
fashion. Heat shock proteins (HSP) could fit this profile. HSP are immunodominant antigens
and a common target of T cell recognition in various inflammatory diseases(10-13). Especially
HSP60 seems to be an important regulating antigen in human inflammatory diseases, as it
is capable of enhancing the regulatory function of human Tregs and thereby dampening the
inflammatory process(14;15).
Thus, as HSP60 can promote induction of (adaptive) Tregs, it is conceivable that it could play a
role not only in classical TH1 diseases but also TH2 diseases. Indeed, in asthma, a much stud-
ied TH2 disease, upregulation of several HSP has been shown not only in macrophages and
circulating CD4+ T cells but also in epithelial cells(16-18) but it is unknown what the functional
consequences could be. We hypothesized that upregulation and subsequent T cell immune
recognition of self-HSP60 could as well take place in a typical TH2 disease such as AD. Therefore, our aim was to investigate whether HSP60 is a target for T cells in AD and thus could play
a role in the chronicity of local inflammation.
71
Chapter 4
Methods
Patients and control subjects
Fifty-two atopic children and 30 healthy controls (HC) were included in this study. Eligible pa-
tients were children suffering from physician-diagnosed AD as defined by Hanifin and Rajka(19).
To assure underlying atopy, all children needed to have a positive radioallergosorbent test
for at least one of the three common food allergens, egg, cow’s milk, or peanut, with a history
of a clinical allergic reaction after ingestion of the protein (either parental history (Sampson
score ≥ 3) or by food challenge). Patient characteristics are given in Table 1. HC were children
who underwent a urological or orthopedic surgical procedure. None of them had a history of
allergy or a recent infection nor a first-degree relative with a history of allergy or asthma. Both
written and oral information about the study was given to the parents and written informed
consent from the parents was obtained. The study has been approved by the Medical Ethics
Committee of the University Medical Centre, Utrecht, The Netherlands. Patient samples were
used for different assays and measurements based on the availability of T cells and serum.
Table 1 | Patient characteristics
Atopic Patient (n=55)
Healthy Controls (n=30)
Male (%)
71%
78%
Eczema at presentation
51 (93%)
0
Age, mean (range)
Food allergy
Asthma
Skin prick test positive
Elevated IgE
Positive food challenge
8.8 (1.5-17.5)
55 (100%)
24 (44%)
47 (87%)
55 (100%)
31 (56%)
8.6 (1.2-17.3)
0
0
0
0
0
Immunohistochemistry
Biopsy specimens (3 mm) were taken from four adult volunteers suffering from moderate to
severe AD (for ethical reasons, only adult volunteers were used) under local anesthesia (Xylo-
caine) and snap-frozen in liquid nitrogen. The lesional biopsy was taken from an active lesion,
whereas the nonlesional biopsy was taken from healthy-looking skin. Approval was given by
the Medical Ethics Committee of the University Medical Centre, Utrecht, The Netherlands.
Subsequently the biopsies were embedded in Tissuetek® (Sakura, Torrance, CA, USA) and
stored at −80 °C until further handling. The antibody used as marker for immunohistochemical staining of the frozen sections was antihuman HSP60 (LK2, kind gift from P. van Kooten,
Department of Veterinary Medicine, University of Utrecht, The Netherlands) and mouse anti72
Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis
human CD3 (cat#347340, used 1:50; Becton Dickinson (BD) Biosciences, San Jose, CA, USA).
Single staining for HSP60 and CD3 combined with a biotinylated horse antimouse IgG (Vector
Laboratories, Inc., Burlingame, CA, USA) was performed as described previously. Stainings
were performed on sequential slides to compare HSP60 and CD3 staining in comparable locations. Skin sections were examined by light microscopy.
Proliferation and direct culture assays with PBMC
Peripheral blood mononuclear cells (PBMC) were isolated and cultured as described pre-
viously(20). For direct cultures, cells were cultured for 7 days in the absence or presence of
10 μg/ml low endotoxin human HSP60 (0.3 pg/μg protein; Faculty of Veterinary Medicine,
University of Utrecht, Utrecht, The Netherlands). Concanavalin A (2.5 μg/ml; Calbiochem, La
Jolla, CA USA) and tetanus toxoid (1.5 μg/ml; RIVM, Bilthoven, The Netherlands) were used as
positive controls. A mouse class II restricted peptide (Ova) was used as an irrelevant control.
For the proliferation assays, cells were cultured for 96 h only. For the final 16 h of culture, 1
μCi/well [3H]thymidine (ICN Biomedicals, Amsterdam, The Netherlands) was added to each
well. Cells were harvested according to standard procedures and incorporated radioactivity
was measured by a liquid scintillation counter and expressed as counts per minute. The mag-
nitude of the proliferative response is expressed as the stimulation index (SI), which is the
mean counts per minute of cells cultured with antigen divided by the mean counts per minute of cells cultured without antigen. As additional control on the effect of possible lipopolysaccharide (LPS) contamination of HSP60, proliferation assays were performed in a control
group of patients after treatment of HSP60 (10 μg/ml) or LPS (10 μg/ml; Sigma-Aldrich Corp,
St. Louis, MO, USA), with 10 μg/ml polymyxin B (Bio-Rad Laboratories, Hercules CA, USA) for
1 h at room temperature or heat inactivation at 95 °C for 30 min.
Multiplexed particle-based immunoassay
Cytokine levels were measured after the activation of lymphocytes in vitro as described above.
After 96 h, the supernatants of the cell cultures were stored at −80 °C until analysis. Cytokines
levels of IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, and IFNγ were measured with the Bio-Plex
system and analyzed with the Bio-Plex Manager software version 6.0 (Bio-Rad) which uses
the Luminex xMap technology(21). The peptide-specific cytokine production is calculated as
the cytokine production of cells cultured with peptide subtracted with the cytokine production of cells cultured without peptide.
Lymphocyte cell surface markers
At day 7 from the direct cell culture assays as described above, PBMC were harvested and
prepared for fluorescence-activated cell sorting (FACS) staining as described previously(22).
The cells were incubated in 50 μl FACS buffer containing three appropriately diluted phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)-, or Cy-Chrome-labeled monoclonal anti-
bodies against human CD4 (clone RPA-T4), CD25 (clones: activated protein C [APC], M-A251;
PE, 2A3), CD30 (clone: Ber-H83), and CD69 (clone: FN50). Stained mononuclear cells were
73
Chapter 4
diluted in FACS fluid and run on a FACSCalibur (BD Biosciences). CellQuest software (BD Biosciences) was used for analysis.
Cytokine analysis by lymphocyte intracellular staining and flow cytometry
Direct T cell lines were generated as described above. During the last 4 h of culture, GolgiStop (BD
Biosciences) was added (final concentration of 2 μM). The cells were harvested and stained for intracellular cytokine and FOXP3 analysis as described previously. For the staining, a predetermined
optimal concentration of PE-conjugated anti-IL-10 (clone JES3-19F1), anti-IL-4 (clone MP4-
25D2), FITC-conjugated anti-IFNγ (clone 4S.B3; BD Biosciences), and APC-conjugated FOXP3
(Ebioscience, San Diego, CA, USA) was added to the cells. Cells were analyzed as described above.
Functional assays
To test the induced CD4+CD25+FOXP3+ Tregs on suppressive function, PBMC were cultured
for 96 h as described above in the presence of HSP60. CD4+CD25+CD127− T cells (which were
used to quantify FOXP3+ T cells in the functional assay) and CD4+CD25− effector T cells from
the same donor were directly sorted by FACS (FACS Aria, BD Biosciences) into plate-bound
anti-CD3-coated wells (OKT-3, 1 μg/ml) in different ratios (1:0, 1:0.5, 1:1, and 1:2). T cell-de-
pleted PBMC from the same donor were irradiated (3,500 Rad) and were used as antigen-presenting cells (1:10). The cells were incubated at 37 ° C for 96 h, with the last 16 h of incubation
in the presence of [3H]thymidine (1 μCi/well). The suppressive activity was determined by
calculating the percentage difference in proliferative response (mean [3H]thymidine incorporation (counts per minute) of triplicate wells) between CD4+CD25− T cells cultured alone and
CD4+CD25− T cells cultured in the presence of induced CD4+CD25+CD127− T cells.
Statistical analysis
Basic descriptive statistics were used to describe the patient population. A nonparametric test
(Mann–Whitney U test, two-sided) was applied to determine significant differences between
the patient and control groups regarding proliferative responses, cytokine production, and
expression of cell surface markers. Where mentioned, values are noted as the mean ± standard error of the mean (SEM). A p value <0.05 was considered significant (SSPS Statistical
Program, version 15.0.; SPSS Inc, Chicago, IL, USA).
Results
Increased presence of HSP60 in skin biopsies of atopic dermatitis
Since HSP are upregulated in inflammatory disorders, we questioned whether the presence
of HSP60 is increased in the lesional AD skin(23). To demonstrate this, skin biopsies from both
nonlesional and lesional skin sites from AD patients were stained for HSP60 and CD3. The
results of a representative donor are shown in Fig 1. The presence of HSP60 was seen in both
74
the healthy skin and the AD skin, but the pattern of HSP60 presence was strikingly different.
Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis
The dermis and epidermis of atopic skin was characterized by large cell infiltrates, with the
presence of HSP60, which was not seen to that extent in the healthy skin. Staining for CD3 in
sequential slides was indicative for the presence of HSP60 in CD3+ cells in the dermis. Howev-
er, since it was not a double staining, we cannot exclude that, next to T cells, HSP60 is also lo-
calized in other cells or extracellularly in dermal tissue, in close proximity to CD3 cells. These
data show that HSP60 is highly present in inflamed tissue of patients with AD.
Figure 1 | Human HSP60 is expressed in the skin.
A HSP60 staining in an active lesion of a patient with AD. B The same skin of the patient with AD, colored with anti-CD3. Both are representative pictures of skin biopsies of an active skin lesion of a patient
with AD. Compared to the nonlesional skin (c and d), the thickened stratum corneum and epidermis are
typical for AD. In the dermis, infiltration of cells is seen. HSP60 is mostly seen in these infiltrates and
in the lower layers of the epidermis (a), whereas coloring grows less dense in the upper areas of the
epidermis. The cell infiltrates express CD3 and the HSP60 expression in the dermis seems confined to
these cells. c and d Nonlesional skin of a patient with AD. Compared to the lesional skin, the nonlesional
skin shows less HSP60 (C) and CD3 (D). ×200 (scale bar050 μm)
Human HSP60-specific proliferation
Next, we wanted to see whether this increased recognition of HSP60 is also seen in peripheral T cell of children with AD. To address this question, proliferation of PBMC was deter-
mined in response to HSP60. Proliferative responses of 28 patients were compared with
those of 18 HC. The SI of the PBMC was significantly higher in the patient group compared
75
Chapter 4
to the HC group (p = 0.004). This indicates that PBMC from children with AD proliferate
more to HSP60 compared to PBMC from HC (Fig. 2a), whereas no significant difference was
found on a control mitogen (Supplementary Fig. 1).
Figure 2 | Human HSP60 is recognized by PBMC of atopic infants and induce cytokine secretion.
A Protein-induced T cell proliferation in atopic children and HC. PBMC were cultured for 96 h in the presence of human HSP60. Boxes show IQR for each group of data, horizontal lines show the median. *p<0.05,
significant differences in responses. Light grey boxes HC, dark grey boxes atopic children. SI is indicated
on a logarithmic scale. B Protein-induced cytokine secretion of IL-10, IFNγ, and IL-6 in atopic children
and HC. Boxes show the mean levels of cytokine with SEM. White boxes medium, black boxes stimulation
with HSP60. Cytokine levels are indicated on a logarithmic scale. C FACS analysis of CD4+ CD14− T cells
from an atopic donor stimulated with HSP60 for 4 days and stained for IFNγ. Histogram shows the medium level (black) and IFNγ level (white) after stimulation with HSP60.
Human HSP60-specific cytokine induction
Given the increased T cell proliferation to HSP60 by the T cells of atopic children, we wanted to
assess the quality of T cell activation. First, cytokine production by HSP60-induced PBMC was
measured in culture medium of 26 patients and 22 HC. When compared to the medium, stimulation with HSP60 induced a significantly higher level of IL-10 in the HC group (mean ± SEM,
4.4 ± 1.8 pg/ml; p = 0.012), while in the patient group, compared to the medium, production of
IL-10 was lower after stimulation with HSP60 (mean ± SEM, −14.77 ± 17.91 pg/ml; ns). A different
pattern was seen for IFNγ. Following in vitro stimulation with HSP60, PBMC from both patients
(mean ± SEM, 198.1 ± 140.1) and HC (mean ± SEM, 5.58 ± 4.46) produced higher amounts of IFNγ.
Taken together, these results demonstrate that stimulating PBMC from atopic patients with HSP60
76
Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis
leads to the induction of IFNγ but not IL-10 (Fig. 2). Of the other cytokines tested (IL-4, IL-5, IL-12,
and IL-13), only IL-6 showed an increase after stimulation with HSP60 (Fig. 2b).
Although the LPS content of the HSP60 used was below the detection level and although
monocytes are not expected to survive after a 5-day culture, we wanted to verify that IFNγ
measured was indeed secreted by T cells. Therefore, after culturing the cells with or without
HSP60, cells were stained for CD4, CD14, and IFNγ. Indeed, HSP60-induced production of
IFNγ was produced by CD4+CD14− T cells, while virtually no monocytes (<1 %) were present.
Figure 2c shows a representative overlay histogram of CD4+CD14−IFNγ+cells.
Human HSP60-specific induction of CD4+CD25bright T cells expressing the transcription
factor FOXP3
We next questioned whether in atopic children in vitro activation with HSP60 could lead to
the induction of Tregs. To address this issue, we first investigated whether the FOXP3 expres-
sion in HSP60-specific CD4+ T cells was different between HC and atopic children. PBMC of
five HC and nine children with AD were cultured for 7 days in vitro with HSP60 (see Fig 3).
Stimulation with HSP60 gave a significantly higher percentage of CD4+CD25bright T cells and
CD4+FOXP3+ T cells in both the HC and the atopic patients compared to the medium only (see
Fig. 3a, 3b, and Table 2). Although at least a part of the FOXP3 induction is due to the activation of T cells, stimulation with HSP60 also seems to induce T cells with the phenotypical
characteristics of Tregs, expressing CD25 and FOXP3.
Figure 3 | Human HSP60 induces CD4+ CD25bright T cells and the expression of FOXP3
Cells were cultured in human HSP60 for 7 days. a FACS analysis shows a significantly higher level of CD4+
CD25bright T cells and CD4+ FOXP3+ T cells in both HC as atopic individuals. White boxes medium only,
grey boxes human HSP60. b FACS analysis of CD4+ T cells of both atopic individuals and HC expressing
FOXP3 after stimulation with either medium or human HSP60. Percentages indicate the percentage of
CD4+ FOXP3+ T cells in the total CD4+ T cell population measured.
77
Chapter 4
Table 2 | Populations of CD4+CD25bright and CD4+FOXP3+ T cells increase after stimulation with HSP60 HC
CD4+CD25bright
Atopic patients
CD4+CD25bright
CD4+FOXP3+
CD4+FOXP3+
Medium
Human HSP60
p value
1.69 ± 0.72
5.69 ± 1.73
5.13 ± 3.29
13.37 ± 8.65
< 0.05
< 0.05
1.67 ± 0.21
3.25 ± 1.39
4.95 ± 0.97
5.58 ± 1.78
< 0.001
< 0.05
This table shows the mean percentages +/- SEM for the different cell populations in FACS analysis after
stimulation with human HSP60.
Human HSP60-induced CD4+CD25+CD127− T cells are not suppressive in vitro
Since CD4+ T cells responding to HSP60 produce IFNγ while also sharing phenotypical charac-
teristics of Tregs, we questioned whether these HSP60-induced CD4+CD25+FOXP3+ are functional Tregs, with the capacity to suppress T effector cells. To evaluate the functionality of these
induced CD4+CD25+FOXP3 T cells, suppression assays were performed on PBMC of six children
with AD. Freshly sorted CD4+CD25− T effector cells were cocultured with CD4+CD25+CD127− T
cells obtained from PBMC precultured for 4 days with HSP60 (see Fig. 4).
Figure 4 | Human HSP60-induced CD4+ CD25+ CD127− T cells from atopic patients have no suppressive function.
Functional assays were performed as described in the “Methods” section. The figure shows the percentage difference in proliferative response between CD4+ CD25− T cells (Teff) cultured alone and CD4+
CD25− T cells cultured in the presence of induced CD4+ CD25+ CD127− T cells (Tregs) in three different
ratios. Only one patient showed suppressive function.
Despite high levels of FOXP3, the CD4+CD25+CD127− T cells from these cultures were not sup-
pressive in five of six patients. In only one patient, the cells were able to suppress effector
78
Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis
cells. In comparison, in all HC, these cells are suppressive in this classical suppression assay(24). These findings indicate that, even though HSP60, presented in an atopic environment,
induces T cells that resemble Tregs phenotypically, these cells are not capable of suppressing
effector T cells in vitro.
Discussion
In the present study, we demonstrated for the first time the increased presence of HSP60 in the
skin of patients with AD. Furthermore, we found increased proliferation of T cells from atopic
children after in vitro stimulation. Both increased levels of HSP60 at the site of inflammation
and the enhanced T cell response underline that HSP60 may act as the target of an (autoan-
tigenic) T cell response. The next question was what type of T cell response was induced by
HSP60 in AD. At first glance, it seemed that HSP-induced T cells had a regulatory phenotype as
they expressed CD25bright and FOXP3. However, despite the expression of FOXP3, HSP60-induced
CD4+CD25+CD127− cells of AD patients were not functionally suppressive in vitro. Thus, these
cells are most probably not Tregs but highly activated effector T cells. The presence of activated
but not Tregs may be in line with previous studies showing that, in active atopic diseases, the
suppressive function of Tregs is diminished(5;6). Interestingly, a recent study by Wehrens et al.
revealed that highly activated T effector cells are resistant to suppression through PKB/c-akt
hyperactivation. This might also be an explanation why these phenotypically regulatory cells fail
to suppress T effector cells in vitro and needs further investigation(25).
In atopic diseases, it has been speculated whether specific targeting of T cells could provide
a novel immunomodulatory pathway for treatment. Up till now, studies have focused on the
use of synthetic peptides which contain T cell epitopes derived from known allergens, such
as Fel d 1 (cat allergy) and Api m 1 (bee venom)(26;27). However, since atopic individuals are
mostly polysensitized, immunotherapy aimed at one allergen might have limited effect in the
majority of the patients.
Another possibility for immunomodulation is the use of bystander antigens, in which the antigen used is not the causative antigen, but an antigen which can alter the reaction by either by-
stander activation or suppression(28). Prerequisites for bystander epitopes are expression at the
site of inflammation and recognition by immune-competent cells such as T cells. For this type of
antigen-specific targeting of T cells, HSP60 is a potential candidate(13;15) as HSP60 was previously
described in skin lesions of patients with Behçet’s disease(29). HSP60 has also been described as a
target for T cells in TH1 diseases such as diabetes mellitus and juvenile idiopathic arthritis and as
a target for T cells in the inflammatory process contributing to atherosclerotic lesions(30-33).
It has to be noted, though, that HSP60 primarily acts as an intracellular (and intramitochondri-
al) chaperone. Only in particular conditions, such as cell stress, HSP60 will be released into the
79
Chapter 4
extracellular environment, where it may have “extra-chaperoning” roles, including interaction
with immune system cells(34;35). This secretion of HSP60 may originate either from lymphocytes
themselves, from other cutaneous cells like keratinocytes and fibroblasts, or from other sources
such as nerves or vessels (36). Also, next to HSP60, other proteins involved in inflammation, including proteins from other HSP families, may be involved in AD pathogenesis(37-39).
This is the first study to demonstrate that HSP60 is present at the site of inflammation and
that it induces specific proinflammatory T cell responses in children with AD. This is in line
with a previous experimental study showing that HSP60 can induce cytokines of a regulatory
and Th1 phenotype in the skin of dogs with AD(40). This finding is an important condition in
investigating novel targets for immunomodulation. Further studies are needed to investigate
these immunomodulating properties of HSP60 in atopic diseases.
Supplemental figure | Protein-induced T cell proliferation in atopic children and healthy controls.
PBMCs were cultured for 96 hours in the presence of ConA. Boxes show IQR for each group of data, horizontal lines show the median. Light grey boxes = healthy controls, dark grey boxes = atopic children. SI is
indicated on a logarithmic scale. The difference in SI between the HC and atopic children was not significant (p = 0.1) after stimulation with ConA.
80
Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis
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83
Chapter 5
Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut
Joost A Aalberse; Anders O van Thuijl; Yolanda Meijer; Wilco de
Jager; Tjitske van der Palen-Merkus; Aline B Sprikkelman; Maarten
O Hoekstra; Berent J Prakken; and Femke van Wijk.
Clin Transl Allergy. 2013 Dec 2;3(1):40
Chapter 5
Abstract
One of the IL-17 family members, IL-25, has been implicated with the initiation and amplifica-
tion of Th2 responses in animal models and has been associated with airway hyper-reactivity.
The involvement of IL-25 and also IL-17 in food allergic disease remains to be investigated.
In this study thirty children suspected of peanut allergic disease underwent a double-blind
placebo controlled food challenge (DBPCFC) and IL-25 and IL-17 plasma levels were determined before and after challenge. IL-25 was highly elevated only in subgroup of children with
a positive DBPCFC outcome. Plasma IL-25 was absent in children with a negative DBPCFC
outcome and in healthy controls.
This study shows that IL-25, an IL-17 family member, is highly elevated only in children with
a clinical response to peanut. This suggests a role for IL-25 in the pathogenesis of peanut allergy and elevated plasma IL-25 may be a sign of a severe atopic phenotype.
86
Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut
Findings
Members of the IL-17 cytokine family are emerging as key factors in immune responses(1).
The prototypic family member, IL-17A, triggers pro-inflammatory immune responses and
contributes to neutrophilia during chronic airway inflammation. IL-17E, also known as IL-25,
is the most divergent cytokine in the IL-17 family and, unlike the other members, has been
identified as a central player in the initiation and amplification of Th2 responses(1). In experi-
mental mouse models IL-25 mediates early differentiation towards a Th2 phenotype and de-
velopment of airway hyper-reactivity and allergic disease(2;3). Moreover, allergen provocation
in asthmatic patients increases expression of IL-25 and its receptor(4), suggesting that IL-25 is
implicated in both sensitization and memory responses in airway hypersensitivity. Increased
duodenal levels of Il-25 were found in peanut allergy in a mouse study(5). In human subjects
however, the involvement of IL-25 and IL-17 in food (peanut) allergy remains unknown. To
investigate if there is a difference in IL-25- and IL-17 expression in peanut allergic versus
peanut tolerant (i.e. peanut sensitized but, not clinical reactive) we determined IL-25 and
IL-17 plasma levels, as well as the Th2 cytokines IL-4, IL-5 and IL-13, in a well-defined cohort
of peanut sensitized children undergoing a double-blind placebo controlled food challenge
(DBPCFC).
Thirty children suspected of peanut allergic disease (based on either elevated specific IgE
to peanut (ImmunoCap >0.35 kU/L) or positive skin test to peanut) were referred to The
Wilhelmina Children’s Hospital, University Medical Center, Utrecht, The Netherlands for a
DBPCFC to obtain certainty about the diagnosis of peanut allergy (for patient characteristics
see Table 1). The study was approved by the local medical ethics review boards (METC, UMC
Utrecht; project no. 05/084 and METC AMC, Amsterdam; project no. 05/254) and informed
consent was obtained for all subjects. The oral challenge was performed as previously de-
scribed(6). Peripheral blood samples were collected before the start of the DBPCFC, as well as
when the challenge was finished. Plasma cytokine levels were determined by Xmap technology (Luminex Austin)(7).
87
Chapter 5
Table 1 | Characteristics peanut sensitized patients that underwent a DBPCFC
No
Sex
PeanutSPT at
Clinical
Age
Atopic
specific-IgE diag- Asthma#
response
(years)
Dermatitis$
at diagnosis nosis
to DBPCFC
1
M
10
4.0
<1
2
M
15
0.43
<1
5
M
4
5.9
NP
3
4
6
7
8
9
10
11
12
13
14
M
M
F
M
M
M
M
M
F
M
M
15* F
16* M
17
M
20
M
18
19
21
22
F
M
M
F
23* M
24
M
25* F
26
M
27* M
28
F
29* M
30
F
16
14
3
13
4
15
4
8
4
5
9
6
17
6
7
4
9
7
7
11
4
6
7
5
6
6
5
<0.35
2.0
0.6
1.1
7.3
0.6
97.0
0.8
1.1
>100
2.2
>100
56.0
>100
1.9
3.5
>100
55.0
2.1
4.0
6.0
4.9
15.5
21.0
5.3
2.2
NP
3
2
3
2
<1
2
4
3
2
4.5
1
3
3.5
4
4
NP
3
3
4
2
2
2
3
2
3
NP
2
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
GI
Resp Syst
+
*
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
*
+
+
+
+
+
+
+
+
+
+
+
+
*
+
+
+
+
+
+
+
+
-
Patient no. 1–12: non-responders to peanut challenge. Patient no. 13–30: positive responders to peanut
challenge. # Asthma diagnosed according to ATS criteria. $Atopic dermatitis diagnosed according to criteria from Hanifin and Rajka. SPT, Skin Prick Test (the number represents the wheal size ratio peanut
compared to histamine). DBPCFC, Double-Blind Placebo-Controlled Food Challenge. NP, Not performed.
*Children with elevated plasma IL-25 as determined in Figure 1. GI: Gastrointestinal symptoms including
stomach ache, nausea, vomiting and diarrhea; Resp: respiratory symptoms including rhinorrhea, stuffy
nose; Syst: systemic symptoms including shortage of breath, systemic urticaria and angioedema.
88
Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut
DBPCFC resulted in a positive (allergic) and negative (tolerant) challenge group and peanut
allergy was confirmed in 18 out of 30 patients. In the plasma samples of the negative and
positive challenge groups we found a striking difference for the type 2-related cytokine IL-
25. Plasma IL-25 was not detected in any of the children with a negative challenge response,
whereas plasma IL-25 was elevated in six children of the positive challenge group (Figure 1A).
In five out of six of these children the IL-25 concentration was even extremely elevated, with
levels ranging up to 13000 pg/ml (Figure 1A, B). IL-25 levels were similar in plasma samples
taken after challenge (data not shown). IL-17 was found in both the positive and negative
challenge groups (in 67% versus 83% of children respectively), but in contrast to IL-25, IL-17
levels were significantly lower in the positive challenge than in the negative challenge group
(p < 0.01) (Figure 1A, B). We show that in this cohort of peanut sensitized children, the pres-
ence of high levels of IL-25 in plasma is only present in children with a positive challenge and
clinical reactivity is inversely correlated with plasma IL-17. These findings seem to be rather
specific for the cohort of (peanut) allergic children since both IL-25 and IL-17 plasma levels
were below the detection limit in age-matched healthy controls (n = 20) (data not shown).
We searched to explain why plasma IL-25 was elevated in this subgroup of peanut allergic
patients. No difference was found in relation to the other Th2 cytokines. Levels of IL-4 were
below the detection limit and IL-5 and IL-13 levels were very low when positive. Within the
clinical responder group also no difference in peanut-specific IgE levels was found between
the IL-25 negative and IL-25 positive subgroups (Figure 1C).
Plasma IL-25 did not associate with total IgE levels (Figure 1D), previous exposure to peanut,
severity of food allergic symptoms, or the presence of asthma and atopic dermatitis, nor did
it correlate with organ specific symptoms during challenge (Table 1). As IL-25 is often men-
tioned to be related to asthma like symptoms we reviewed IgE data to inhalant allergens.
Plasma levels of IgE to either birch, house dust mite (hdm) and cat from 17 patients were
available. In general most children with a peanut sensitization were also sensitized to inhalation allergens, irrespective of the positive or negative challenge or IL-25 levels.
To extend our findings in peanut allergic children we next measured IL-17 and IL-25 in a
group of infants diagnosed with cow’s milk allergy (CMA) (confirmed by DBPCFC, n = 12). In
these infants IL-25 was found in 42% of the allergic patients but at very low levels (1.5-14 pg/
ml) and IL-17 levels were below the detection level (data not shown). These data indicate that
elevated IL-25 in serum is not a general phenomenon in clinical food allergy.
Peanut allergy is considered as an indication of a broad and possibly severe atopic phenotype and, unlike other food allergies (such as CMA), is infrequently outgrown8. The original
diagnosis of peanut allergy in our tested cohort was not based on an oral challenge and this
poses limitations on conclusions about the resolution of peanut allergy. However, the data do
demonstrate that plasma IL-25 was only present in children with ongoing peanut allergy and,
89
Chapter 5
Figure 1 | Cytokine profile and antibody levels of peanut sensitized patients.
A colour profile (A) and scatter plot (B) of IL-25 and IL-17 plasma levels (pg/ml) in children with a negative (n = 12) and children with a positive peanut challenge (n = 18). * p < 0.05 with a Mann–Whitney U
test. Peanut-specific IgE (C) and total IgE (D) serum levels in the negative and positive challenge groups,*
p < 0.05 with a Mann–Whitney U test. The positive challenge group is subdivided in IL-25 positive and
IL-25 negative children.
90
Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut
importantly, despite a peanut-free diet for at least 6 months. Together these data may indicate
that elevated plasma IL-25 is a sign of chronic immune activation that is not induced by the
provoking allergen itself but represents a risk factor for the development or persistence of
clinical reactivity to peanut. Recently it was suggested that IL-25 secretion is induced during
disruption of epithelial barriers. Our data are insufficient to make a conclusion on the nature
of a IL-25 subgroup, but it can be speculated that these patients have a more severe type of
food allergy. The finding that only 6 out of the 18 peanut allergic patients displayed highly
elevated IL-25 levels stresses the possibility of a clinical subgroup within the group of peanut
allergic children and warrants larger cohort studies.
IL-25 is expressed by a variety of innate immune cells and non-hematopoietic cells including
basophils, eosinophils, epithelial and endothelial cells9 and in the gut IL-25 is predominant-
ly found in epithelial cells10. Innate lymphoid cells have been described in the human that
respond to IL-25 and provide an innate source of Th2 cytokines11;12. Together, increased pro-
duction of IL-25 triggered by environmental antigens or microbes in the gut might therefore
contribute to the atopic phenotype by promoting Th2 differentiation and the maintenance of
allergen specific Th2 memory cells.
In conclusion, this study is the first to show that IL-25 is highly elevated in a subgroup of pea-
nut-allergic children and suggests a role for IL-25 in the development and/or persistence of
peanut allergy, in this subgroup. These findings warrant further studies in a larger cohort of
patients as well as in other food allergies.
91
Chapter 5
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Tamachi T, Maezawa Y, Ikeda K, Kagami S, Hatano M, Seto Y et al. IL-25 enhances allergic airway
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thymic stromal lymphopoietin or IL-25, is central to mite and peanut allergic sensitization.
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Aalberse JA, Meijer Y, Derksen N, van der Palen-Merkus T, Knol E, Aalberse RC. Moving from
peanut extract to peanut components: towards validation of component-resolved IgE tests.
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Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut
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Moving from peanut extract to peanut components: towards validation of
component resolved IgE test
J.A. Aalberse; Y. Meijer; N. Derksen; T. van der Palen-Merkus;
E. Knol; and R.C. Aalberse
Allergy 2013; 68 748-56
Chapter 6
Abstract
Replacement of peanut extracts by recombinant peanut components is an important step
in allergy serologic testing. Criteria are needed for the unbiased inclusion of patients into a
study to validate such a replacement.
Plasma samples from 64 peanut-positive children (42 reactors, 22 nonreactors in a double-blind, placebo-controlled food challenge) were used to compare IgE reactivity to six re-
combinant peanut allergens with reactivity to natural peanut proteins extracted at neutral or
low pH. We tested the hypothesis that poor extractability of Ara h 9 and other basic allergens
at neutral pH leads to under-representation of patients with such sensitization.
IgE reactivity to the components did not fully explain IgE reactivity to peanut extract in 5 of
32 reactors with IgE to peanut extract ≤100 kUA/L. IgE reactivity to components was stronger
than to the extract in 11 plasma samples, which was largely due to a low Ara h 8 reactivity of
the extract. IgE reactivity to Ara h 9 was much lower than reactivity to other basic proteins,
some of which bound IgE well in the RAST, but lost IgE reactivity upon immunoblotting.
Conventional peanut extracts are deficient in significant IgE-binding components. The inclu-
sion of patients for a validation study should be based on serology performed with improved
peanut reagents to avoid a bias against these under-represented, potentially important allergens. To judge clinical relevance of an allergen, the reagent used for inclusion of patients
needs to be efficient in detecting IgE to this component.
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Moving from peanut extract to peanut components: towards validation of component resolved IgE test
Introduction
It is widely appreciated that recombinant allergens are potentially superior reagents for IgE-
based diagnostic tests compared to extracts of natural source materials(1-8). We want to ad-
dress some issues related to the transition from extracts to recombinant components. This
article focuses on establishing a validation protocol, that is, a procedure to substantiate a
claim that a new test protocol (a combination of recombinant allergens) is at least as sensitive
as a test with a conventional allergen extract (peanut extract in our case). We selected peanut
as a suitable test case for a study of a validation protocol, because it is a complex source of
important allergens, six of which were available to us in recombinant form. In several recent
reports, it has been suggested that recombinant allergens may substantially improve the diagnostic accuracy of peanut allergy tests(3;6). These reports may give the impression that re-
combinant allergens could already replace peanut extract. We are concerned that this would
lead to a premature disappearance of tests for IgE to peanut extract. We want to argue that it
is important to have a transition period in which the results with recombinant allergens are
used to improve IgE-based serology to whole peanut extract (most likely a mixture of different extracts, spiked with purified natural and/or recombinant allergens). In the next round
of evaluation, component-based IgE tests should then be re-evaluated using the improved
allergen extract.
The clinical aspect of the relation between serology and its diagnostic consequences is not the
topic of this article. The data we present in this article are intended to serve as a technical case
study on a validation procedure. The focus is on serology, particularly on the possibility and
need of an improvement of its sensitivity. The aim is to be able to identify more than 95% of
all sensitized subjects. One topic that will be discussed is the necessity of using an improved
peanut reagent to increase the sensitivity of the IgE test needed to include patients for a dou-
ble-blind, placebo-controlled food challenge (DBPCFC). This is an important validation issue,
because the peanut reagent used for the inclusion of human subjects should contain sufficient
quantities of all potentially relevant allergens.
This study is based on an extensive serological analysis of plasma samples obtained from chil-
dren suspected to have a peanut allergy and subsequently subjected to a DBPCFC. Previous
studies have shown that a few recombinant allergens might suffice for diagnostic purposes(5).
Some studies indicated that measuring IgE to Ara h 2 (possibly used in combination with the
closely related allergens Ara h 6 and Ara h 7) might even be sufficient for diagnosis(1;4;7). How-
ever, the relevance of the two highly abundant storage proteins Ara h 1 and Ara h 3 is still a
matter of debate(5;9). The potential contribution of lipid transfer protein (LTP, Ara h 9) is also
of interest(9), not only because this class of proteins has been found to be very important in
other food allergies, but also because of a technical issue. Whole peanut extract is traditionally
prepared in saline at a pH of approximately 7, which works well for slightly acidic allergens
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Chapter 6
like Ara h 1, Ara h 2, Ara h 3, Ara h 6 and Ara h 7 (isoelectric point [IEP] 5.3–6.2). However,
this is not optimal for highly basic allergens, such as lipid transfer protein (LTP, Ara h 9, IEP
9.4), which are extracted more efficiently at low pH. In the results, we will highlight these basic allergens and discuss the potential consequences of their under-representation in peanut
extracts used for the inclusion of subjects for validation trials.
Based on the experiments reported in this article, we will discuss three questions: (i) How
often does our panel of six recombinant allergens detect less sIgE than our peanut extracts?
(ii) How often do our peanut extracts detect less sIgE than our panel of six recombinant allergens? (iii) How should we deal with IgE reactivity to highly cross-reactive peanut allergens that have a low abundance in peanut extract such as Ara h 5 (profilin), Ara h 8 (relat-
ed to Bet v 1) and Ara h 9 (LTP)? Taking Ara h 8 as a prototypic cross-reactive allergen, we
will discuss whether it would be useful to deplete Ara h 8 from the peanut extract or spike
the extract to enhance Ara h 8 activity. It is evident from published data as well from our
own results that adding Ara h 8 will increase sensitivity, but will markedly decrease speci-
ficity. This might be considered inappropriate in a diagnostic setting. However, we will ar-
gue that in the context of a validation trial of an allergen panel for serological testing, it is
necessary to maximize sensitivity, because we can only judge the clinical relevance of an
allergen if the reagent used for inclusion was efficient in detecting IgE to this component.
Patients, materials and methods
Study population
This serological study was performed on serum collected from children referred to our hospital for a DBPCFC because of a presumed food allergic reaction and/or atopic dermatitis. Children were clinically evaluated for a peanut DBPCFC based on a history of an allergic reaction
related to peanut ingestion or a positive skin prick test (SPT) (ALK-Abello, Nieuwegein, the
Netherlands(10) and/or elevated IgE to peanut (ImmunoCAP; Thermo Fisher Scientific, Uppsala, Sweden; see Supplementary Table S1).
The DBPCFC was performed in the Wilhelmina Children’s hospital. The children were chal-
lenged in seven increasing doses from 0.01 to 10 g peanut, alternated with placebo. All chil-
dren remained under observation after the final dose for at least 2 h depending on severity
of the symptoms to minimize the risk associated with these challenges. DBPCFC results were
considered positive after the occurrence of objective symptoms (urticaria other than perioral,
angio-oedema, vomiting, stridor and hypotension) or after the occurrence of three subjective
symptoms (nausea, oral allergy and abdominal pain).
Based on serum availability, 65 children who underwent a DBPCFC to peanut were initially selected for the clinical analysis of the study. However, one sample was serologically negative at
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Moving from peanut extract to peanut components: towards validation of component resolved IgE test
time of the challenge and therefore excluded from our analysis. Of the 64 remaining children,
42 were peanut allergic (i.e. Sampson score >1(11), whereas 22 were peanut sensitized but not
peanut allergic (no response or only perioral urticaria after DBPCFC).
Skin prick tests were performed on the patient’s back with a standardized prick needle using
a commercial peanut extract (ALK-Abello). The area of the skin wheal was determined by
computer scanning. SPT responses were standardized by dividing the wheal area of the pea-
nut prick by that obtained for the histamine control. In hindsight, we realized that we have
insufficient information on these peanut extracts (skin test, serology) used for inclusion.
Allergens
In this article, four different types of peanut-derived allergenic preparations have been used.
A peanut extract prepared by extraction in a saline solution at neutral pH (to which we will
refer as ‘conventional extract’) was used for the skin test and in the ImmunoCAP. In addition,
we used extracts prepared at low pH (acetic acid (HAc) pH 4.0–4.5) and at neutral pH with
polyvinylpolypyrrolidone (PVPP, a tannin-removing substance that improves the extractability of many proteins)(12). These two peanut preparations will be referred to as HAc and PVPP.
Furthermore, we isolated a fraction containing basic proteins by adsorption of the acetic acid
extract to SP-Sepharose at pH 6 in 20 mM acetic acid/10 mM MES and elution by 0.2 M NaCl in
20 mM MES, pH 6. This preparation will be referred to as BPP (basic peanut proteins). For use
in the RAST, the latter three extracts were coupled to CNBr-activated Sepharose as described
elsewhere(13).
The recombinant peanut components Ara h 1, 2, 3, 6, 8 and 9 were ImmunoCAP reagents. In
addition, we tested our sera for IgE reactivity to two recombinant proteins related to peanut
allergens: grass profilin Phl p 12 (related to Ara h 5) and peach LTP Pru p 3. Phl p 12 has
been reported to be a major cause of cross-reactivity between grass and peanut(5;12;14). Pru p
3 has been reported to be a major cause of peanut reactivity in Mediterranean countries(15).
The tests for IgE reactivity to the recombinant allergens were performed in the laboratory of
Thermo Fisher Scientific, Uppsala (Drs J. Lidholm and L. Elfström).
Serology
Quantitative IgE assays were performed either by ImmunoCAP (Thermo Fisher Scientific) or
by a Sepharose-based radioallergosorbent test (RAST) procedure as described elsewhere13.
Briefly, allergen was coupled to CNBr-activated Sepharose (GE Healthcare, Uppsala, Swe-
den). A suspension of the resulting allergosorbent (corresponding to 0.25–1 mg of Sepharose powder as supplied by the manufacturer and approximately 5 μg protein per test) was
incubated with 50 μl serum on a vertical rotator in a final volume of 0.75 ml. After washing
the suspension with saline using repeated centrifugation, Sepharose-bound IgE was detected
with 125I-labelled affinity-purified sheep anti-IgE (approximately 1 ng per test). IgE-binding
data were converted to the equivalent of international IgE units (kUA) using a chimeric IgE
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anti-Der p 2 antibody prepared at Sanquin(16). For the quantitative comparison between IgE
reactivity to whole extract and to the peanut components, we calculated a cumulative sIgE
level for the components, with the sIgE level to rPhl p 12 as a surrogate for Ara h 5. Because of
the cross-reactivity between Ara h 2 and Ara h 6, we used the highest sIgE value of these two
for the calculation. In this comparison, we used for whole extract either the reactivity of the
ImmunoCAP peanut test or the highest value of three extracts: ImmunoCAP, PVPP and HAc.
The qualitative analysis of IgE binding to peanut proteins was performed by immunoblotting
of peanut proteins separated by Novex 4–12% SDS-PAGE (nonreducing gel, unless stated otherwise) in a MES running buffer. Blotting was performed using iBlot® (Invitrogen Life Tech-
nologies, Breda, Netherlands) according to manufacturer’s instructions. The main blotting
experiment was performed by applying the two extracts (PVPP and HAc) as three doublets
on a single gel (5 μg protein/lane), transferring the whole gel to nitrocellulose and cutting the
blot into three parts containing one allergen doublet each. After incubation of each strip with
150 μl plasma and washing, IgE binding was detected using an affinity-purified 125I-labelled
sheep anti-IgE antibody as described elsewhere(13).
Results
Reactivity of the conventional peanut extract compared with the component panel
We analysed the ImmunoCAP results based on the assumption that the IgE-binding capacity
of the ImmunoCAP is sufficient to bind all allergen-specific IgE provided that the sIgE level is
not extremely high. For this upper cut-off value, we used 100 kUA/L. Of the 42 plasma samples
from patients who reacted to the DBPCFC (serum codes with a ‘p’), 10 had an sIgE level to
peanut (ImmunoCAP) that exceeded this upper cut-off value of 100 kUA/L, which made these
results unsuited for a quantitative comparison between extract and components. In Fig. 1,
the cumulative IgE binding for the remaining 32 plasma samples to components is shown in
comparison with IgE binding to the conventional peanut extract (ImmunoCAP) for 32 plasma
samples with a sIgE level to peanut extract ≤100 kUA/L. In 5 of the 32 plasma samples (16%),
the cumulative sIgE level to the components was less than 75% of the sIgE level to peanut ex-
tract. In 11 of the 32 plasma samples (34%), the cumulative sIgE level to the components was
more than 125% of the sIgE level to conventional peanut extract (Fig. 1B). As some plasma
samples reacted stronger to the PVPP and/or the HAc peanut extract than to the ImmunoCAP
peanut reagent, for numerical analysis, we used the highest value of these three tests. This obviously increased the peanut sIgE level, which resulted in a decrease in the number of plasma
samples with a lower extract sIgE level from 11 to 8 (31–25%). The number of plasma sam-
ples with a lower component sIgE level increased from 5 to 9 (29%) (Fig. 1C). With N = 32, we
need to find four deviant cases to have a more than 95% probability that in more than 5% of
100
the cases, a discrepancy between whole extract and components exists.
Moving from peanut extract to peanut components: towards validation of component resolved IgE test
Figure 1 | ImmunoCAP sIgE levels of 32 patients with a reaction upon challenge (marked 04p to 54p, ‘p’
indicates peanut allergic patients) compared to the IgE level to peanut extract and recombinant allergens.
In frame A and B, the ImmunoCap extract is shown, in frame 1c the highest level of ImmunoCAP, HAc and
PVPP. The recombinant allergens are Ara h 1, the maximum response of Ara h 2 or Ara h 6, Ara h 3, Ara h
8 as well as the sum of Ara h 9 and grass profilin, represented together as ‘other’. (A) Absolute cumulative
IgE-binding data; (B and C) cumulative IgE scores expressed as % of peanut extract, either relative to the
ImmunoCAP extract (B) or relative to the highest score of ImmunoCAP, HAc and PVPP (C).
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A
B
C
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Moving from peanut extract to peanut components: towards validation of component resolved IgE test
< Figure 2 | SDS-PAGE analysis.
A | Silver-stained SDS-PAGE (nonreduced) of two peanut extracts (PVPP and HAc) and two fractions obtained by cation-exchange chromatography from HAc (BPP5 and BPP6). Molecular masses of the marker
proteins (in lanes marked M) are indicated in kD on the left. Expected positions of peanut proteins are
indicated on the right. The prominent band at 34 kD is tentatively assumed to be a major fragment of Ara
h 3. This is based on MS data, in which an Ara h 3 fragment is found composed of an N-terminal proteolytic fragment that is disulphide-linked to the basic chain ref 16. B | Immunoblot of 11 plasma samples (p:
peanut allergic patients; n: nonpeanut allergic patients) each tested on a doublet of two peanut extracts:
PVPP (left lane of each doublet) and HAc (right lane of each doublet). The plasma samples are ranked
based on their percentage coverage by the recombinant panel compared to the highest response to three
peanut extracts (see Fig. 1C). The numbers below the serum codes indicate IgE-binding classes on a factor
3 scale starting at 0.1. A score 0 corresponds to an sIgE value <0.1 kUA/L; score 1 = 0.1–0.3, 2 = 0.3–0.9,
3 = 0.9–2.7, 4 = 2.7–8.1, 5 = 8.1–24.3, 6 = 24.3–72.9 kUA/L, 7 = 72.9–219 and 8 = >219 kUA/L. The arrow
pointing to a 34-kD component indicates a presumed major Ara h 3 fragment, whose staining is much
stronger than expected from the ImmunoCAP results in 54p and 51p relative to the undetectable staining
of Ara h 2. C Analysis of the effects of blotting pH on the transfer of protein to nitrocellulose (indicated as
‘NC’) and on IgE reactivity. Lanes on the left: BPP; lanes on the right: PVPP. Serum 54p was used for the
IgE staining.
Problem 1: reactivity of the components too low
A cumulative sIgE level to the recombinant allergen panel <75% of the sIgE level to peanut
extract might reflect a structural defect of the recombinant proteins and/or to the absence
of a relevant allergen. We investigated whether we could identify additional components by
immunoblotting. We tested the binding to two blotted peanut extracts (PVPP and HAc, 5 μg
protein/lane, see protein spectra in Fig. 2A) of 11 plasma samples, including some plasma
samples from children with a negative provocation test (indicated in the serum codes by the
letter ‘n’): 11n, 52p, 53p and 60p, which are plasma samples with too low-component reactiv-
ity; 44n and 45n, which are plasma samples with component reactivity exceeding that of the
extract; and 41p, 49p, 51p, 54p and 61p, which are plasma samples with component reactivity
that matched extract reactivity. Note the discrepancies between staining >25 kD and CAP reactivity to Ara h 1/3 and the discrepancies between staining <25 kD and CAP reactivity to Ara
h 2/6. Furthermore, note that the staining in the Ara h 1/3 area is much stronger than in the
Ara h 2/6 area, which is not reflected by CAP reactivity.
High staining intensity of Ara h 1, 2, 3 and 6 in the PVPP extract made it difficult to detect
additional IgE-binding components (Fig. 2B, lanes on the left of each doublet). We therefore
focus our analysis on the HAc extract (Fig. 2B, lanes on the right of each doublet), which has
an excellent reactivity when coupled to CNBr-activated Sepharose (Fig. 3), but a substantially
lower content of Ara h 1, 2, 3 and 6, as is illustrated by the protein stained gels (Fig. 2A) and
confirmed by the very weak IgE staining in the Ara h 1 and 3 range (>30 kD) and the absence
of IgE staining in the 15–20 kD range (using plasma samples that stained well in this range
to the blotted PVPP extract, Fig. 2B). The faint bands on the immunoblots in the Ara h 1 and
3 region are possibly due to traces of these allergens and their processing products. Upon
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removal of these contaminating allergens by cation-exchange chromatography (see Fig. 2A
for the SDS-PAGE protein spectrum of this basic peanut protein fraction (BPP)), IgE-binding
activity in the RAST dropped by 50%, but the remaining RAST activity of BPP was still substantial (Fig.3).
Figure 3 | sIgE (RAST) reactivity to PVPP (on x-axis) compared with reactivity to the HAc extract (grey
squares) and BPP fraction of HAc (black diamonds), both on the y-axis.
This low staining was not due to lack of protein transferred to the nitrocellulose. As shown in
Fig. 2C, the protein is more efficiently eluted from the gel at the recommended pH (7.2), but
protein binding to nitrocellulose is more efficient when the pH is adjusted to pH 9.5, particularly for Ara h 2/6. Note further that even after autoradiography for 4 days, IgE staining to BPP
is very weak compared to PVPP, despite the clear presence of protein on the nitrocellulose.
From these results, we conclude that BPP contains an allergen that has a high IgE-binding
activity in the RAST, but has a very low IgE-binding activity on the immunoblot. Based on the
protein spectrum, oleosins (Ara h 10, Ara h 11) are potential candidates.
Problem 2: reactivity of the extract too low
The majority of the plasma samples with a high level of IgE reactive with Ara h 8 (the Bet v 1
homologue of peanut) had a low reactivity to each extract, particularly in the conventional ex-
tract, most likely due to the low content of Ara h 8 in our peanut extracts (compare Fig. 1B,C).
LTP and profilin are peanut components that are known to be detectable by cross-reactive
antibodies induced by other allergen sources. We tested IgE reactivity to peanut LTP (Ara h 9).
Peanut profilin (Ara h 5) was not available for testing, so we instead used recombinant profi-
lin from timothy grass pollen, Phl p 12. Our data indicate that IgE to these allergens contrib104
utes less to peanut IgE reactivity than Ara h 8, at least in these Dutch peanut-reactive patients.
Moving from peanut extract to peanut components: towards validation of component resolved IgE test
It is of note that the strongest reaction to LTP (4.4 kUA/L) was found in a patient that did not
react in the DBPCFC. All plasma samples except this one reacted weaker to Ara h 9 than to Pru
p 3 (LTP of peach).
Problem 3: How should we deal with the high IgE reactivity to Ara h 8
The data relevant for this topic are presented in Fig. 4, which contains results both of patients
who reacted on DBPCFC and of nonreactors. As expected, IgE to Ara h 8 is often found in
nonreactors (14/22 = 64%). Ara h 8 is positive in 21/39 = 55% of the patients positive to
Ara h 2/6. In two cases with a positive DBPCFC, IgE to Ara h 8 was the only component with
reactivity above the cut-off of 0.35 kUA/L.
Figure 4 | sIgE (ImmunoCAP) reactivity to Ara h 8 in plasma samples with IgE to Ara h2/6 <0.35
kUA/L (left panel) or ≥0.35 kUA/L (right panel), for reactors and nonreactors in the DBPCFC.
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Chapter 6
Discussion
The most important conclusion of this study is that conventional peanut extracts are deficient
in potentially relevant allergens, which hampers a proper evaluation of the diagnostic performance of a panel of peanut components.
When discussing the way to validate the replacement of a food extract (peanut extract in our
case) by a panel of recombinant allergenic components, we have to deal with two clinical
aspects: patient inclusion and the food challenge and two immunochemical aspects: the allergen panel itself and the potency of the peanut extract needed for the validation trial.
Patient inclusion
Many of the preliminary analyses can be performed on almost any serum panel with IgE to
peanut, without the need of clinical data. In the validation phase, however, clinical data are
essential, including information on sensitization to related allergens. An important issue is
therefore the protocol used for the inclusion of patients. Selection of patients for validation of
a component-based protocol should be unbiased. A selection independent of the use of a pea-
nut extract eliminates bias introduced by possible deficiencies of the extract (to be discussed
below). Inclusion based solely on a questionnaire15;16 is a possible approach, but it may intro-
duce a bias in relation to the spectrum of peanut-related symptoms and the outcome would
be affected by the relative uncertainty of self-reported symptoms. For validation studies, it is
important to avoid a bias towards patients with severe symptoms, because these tend to have
relatively high IgE antibody levels. Broadening the scope of the questions to include the full
spectrum of symptoms has the disadvantage that more subjects need to be included, because
a larger fraction of subjects will have a negative DBPCFC.
How many patients need to be included? At the most basic level, an acceptable allergen panel
should be able to detect patients with an unambiguously diagnosed peanut allergy. A desira-
ble target would be a sensitivity of at least 95%, using a cut-off at which negative controls with
high total IgE (>2000 IU/L; such as a low IgE serum spiked with myeloma IgE or recombinant
IgE) are negative. For our peanut extracts, the 0.35 kUA/Lcut-off level is appropriate. Binomial
statistics indicate that at a 95% confidence level, a panel of at least 59 DBPCFC-positive pa-
tients is needed to establish a sensitivity of better than 95%, if all plasma samples are positive.
A panel of 93 patients is required if a negative test with a single serum is to be accommodated.
The DBPCFC
The criteria for a validation study are not necessarily the same as for a diagnostic procedure.
This is reflected in the type of symptoms needed to score a test as positive, in particular how
to deal with exclusively (peri) oral symptoms. Another issue is the top test dose. The clinical
relevance of a positive test at a 10-gram dose may be lower than one at a 10-mg dose, but the
requirement of a high dose may reflect a potent allergen with a low abundance in peanut,
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Moving from peanut extract to peanut components: towards validation of component resolved IgE test
which might be considered a relevant component in an allergen panel. A third issue is the
validation of the masking procedure used for the food challenge material. The potential instability of newly discovered allergens should be taken into consideration.
The allergen panel
As mentioned above, the panel should be positive in >95% of challenge-positive patients. An
additional criterion that should be considered is whether the panel covers all the relevant
allergens found in an optimal peanut extract (which will be specified below). One approach
is to compare the summed component levels with the level for the optimal peanut extract
(Fig.1), preferably using a serological protocol in which closely related allergens such as Ara
h 2, 6 and 7 are combined into a single test. This procedure could be improved by removing
IgE reactivity to allergens of uncertain relevance, for example, absorbing out IgE to Bet v 1 to
eliminate cross-reactive IgE responses to Ara h 8. An even more informative approach would
be to absorb out all panel-specific IgE from serum and test the depleted serum for remaining
IgE reactivity to peanut extract, on an immunoblot and/or RAST. A limitation of the immunob-
lot in this context will be discussed below. The discussion on the number of plasma samples
required and the way these plasma samples should be selected is similar to the discussion
concerning patient selection.
Insufficiency of a panel of recombinant allergens may reflect either incompleteness of the
panel or qualitative differences between the natural and recombinant allergen, particularly
regarding folding and post-translational processing(17).
The peanut extract
When we performed the studies reported in this article, we did not appreciate the critical role
of the peanut extracts, that is, the extracts used for skin testing and serology. Our data indi-
cated that our extracts were deficient in potentially relevant allergens. Only then we realized
that this might have biased our patient selection, because one of the inclusion criteria was a
positive skin test and/or peanut IgE test. In addition, the serological comparison between the
component panel and peanut extract shown in Fig. 1B is biased towards the well-established
allergens Ara h 1, 2, 3 and 6, because these allergens are abundantly represented in conven-
tional peanut extracts. When other extracts (PVPP, HAc) are included in the comparison, some
gaps in the allergen representation became more evident (Fig. 1C).
If the deficient (i.e. missing or defective) allergens are known components (e.g. Ara h 8), this
can be taken into account during the inclusion phase. However, our results with the acidic ex-
tract indicate that peanut contains at least one hitherto unidentified major allergen. Since we
found high IgE binding to the HAc extract, the (almost) negative results of the immunoblots
with the HAc preparation were surprising. For the total HAc extract, it can be hypothesized
that trace amounts of the traditional peanut allergens (Ara h 1, 2, 3, 6), too low to show well
on protein staining of the gel and on immunoblot, might still be coupled in high enough quan-
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Chapter 6
tities to CNBr-activated Sepharose to allow substantial IgE binding. However, this becomes
less likely with the more purified BPP, which is expected to have a substantially lower content
of these allergens, but still reacted almost as well in the Sepharose test (Fig. 3). One possible
explanation could be a major contribution of proteolytic fragments derived from Ara h 118 or
Ara h 319. Our preliminary data obtained for the N-terminal peptide of Ara h 1 support such an
explanation. This is unlikely to explain all discrepancies, because some of the plasma samples
that reacted well with BPP were negative for Ara h 1 and Ara h 3 (e.g. serum 04p, 34p). We
initially assumed that our blot procedure, which was performed at pH 7.2, was inefficient for
proteins with an isoelectric point well above this pH. Protein staining of the nitrocellulose
blots (Fig. 2C) indicated that this was not the explanation. These results rather suggest that
binding to nitrocellulose leads to a major loss in IgE binding. One possibility would be that
oleosins are involved. The oleosins form a protein family of basic small (10–20 kD) triglyc-
eride-binding proteins, including Ara h 10 and Ara h 11 from peanut. Their presence in BPP
would be unexpected, as they are generally assumed to be unstable in the absence of lipids
or detergents(20). It is known that oleosins form oligomers(21), which might be more stable in
aqueous buffers. Upon binding to nitrocellulose, they may unfold and thus largely lose their
IgE-binding potential.
We conclude that in a validation protocol, it is important to use an in vitro test based on an
improved peanut extract (most likely a blend of different extracts, enriched fractions and recombinant allergens) for the selection of subjects.
Ara h 8 as a prototypic problematic allergen
Many of the issues raised by the availability of recombinant allergens relate to their usefulness in the clinical sensitivity/specificity balance. The concentration of Ara h 8 in current
peanut extracts is low, so spiking the extract with recombinant protein is an obvious possi-
bility to increase sensitivity. While this would result in an unacceptably frequent detection of
birch pollen sensitized but peanut tolerant individuals (i.e. a loss of clinical specificity), it is
important to note that IgE to Ara h 8 was present in 23/42 reactors and as the only detected
sensitization in two of these 23 subjects.
Why does the clinical relevance of IgE seem to vary? The allergen inducing the IgE antibod-
ies reactive with Ara h 8 may be relevant. If the IgE is induced by the birch allergen Bet v 1,
nonsymptomatic cross-reactivity is common(22). This is presumably due to instability of Ara
h 8 during roasting and digestion. However, IgE induction via exposure to Gly m 4 (the soy
equivalent of Ara h 8) by oral consumption of a yoghurt-like soy drink(13;23) may be more likely
to result in symptomatic cross-reactivity. We do not have information on soy exposure in our
patient population, but it is unlikely to be the full explanation of the high prevalence of IgE
to Ara h 8 in the symptomatic group. This may indicate either that some birch-induced IgE
responses are composed such that they do cause symptoms or that other foods may induce
108
cross-reactive antibodies more likely to elicit an allergic reaction or that peanut itself may in-
Moving from peanut extract to peanut components: towards validation of component resolved IgE test
duce clinically relevant mast cell-activating IgE antibodies to Ara h 8. Similarly, IgE reactivity
due to cross-reactivity without clinical sensitivity has been reported for Ara h 5(5;14).
In our study population, two of 7 Ara h 8 sensitized patients without IgE to Ara h 1, 2, 3, 6 or
9 reacted to peanut in DBPCFC with more than oral symptoms. This finding suggests that it is
too simplistic to assume that all patients with such a sensitization pattern will tolerate peanut or have only oral symptoms and that it may be problematic to extrapolate results from a
population-based study(22) to patients specifically selected on the suspicion of peanut allergy.
It will be interesting to investigate the importance of Ara h 8 in geographical areas with low
exposure to birch pollen.
Including other sources of PR10 proteins (Bet v 1, Gly m 4) to the allergen panel to more
precisely define the specificity of the Ara h 8 antibodies may help to improve the specificity
of serology. However, it is unlikely that data obtained by serology will fully predict the clinical
relevance of IgE antibodies to Ara h 8. Statistical algorithms more sophisticated than ROC
analyses will be needed to achieve optimal information from serological data. Using a test
with suboptimal sensitivity for Ara h 8 may be an acceptable interim solution, but we should
be able to do better in the near future.
Conclusion
As long as the extracts used for inclusion of patients for test validation analysis are inefficient in
detecting IgE to an allergenic component, we cannot judge the clinical relevance of this allergen.
Acknowledgements
We thank Arjen Brenkman (UMC, Utrecht) for his work on characterizing the low MW compo-
nent in our peanut extract. We thank Jonas Lidholm and Lisa Elfström (Uppsala, Sweden) for
the ImmunoCAP analyses and stimulating discussions. We thank Karin Hoffmann-Sommergruber (Vienna, Austria), Arndt Petersen (Borstel, Germany), Angela Simpson (Manchester,
UK), Anna Pomes (Charlottesville, Virginia) and Steven Stapel, Laurian Zuidmeer and Jaap
Akkerdaas (Amsterdam, NL) for stimulating discussions. We thank Intersnack (Doetinchem,
NL) for generously providing raw peanuts.
109
Chapter 6
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2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
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Astier C, Morisset M, Roitel O, Codreanu F, Jacquenet S, Franck P et al. Predictive value of skin
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23.
Cabanos C, Tandang-Silvas MR, Odijk V, Brostedt P, Tanaka A, Utsumi S et al. Expression,
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Schuurman J, Perdok GJ, Lourens TE, Parren PW, Chapman MD, Aalberse RC. Production of a
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Koppelman SJ, Knol EF, Vlooswijk RA, Wensing M, Knulst AC, Hefle SL et al. Peanut allergen Ara
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111
Chapter 6
Supplementary Table
A
ID
112
B
C
D
E
F
G
chalcomavoidatopy
SPT sIgE
lenge
plaint
ance
H
I
J
K
L
Eliciting ImmunoCAP ImmunoCAP ImmunoCAP ImmunoCAP
dose
peanut
rAra h 1
rAra h 2
rAra h 3
kUA/L
kUA/L
kUA/L
grams kUA/L
01n 0
E
E
+
+
none
N10
0,10
0,10
0,10
0,10
02n 1
E
F
+
strict
N10
0,10
0,10
0,10
0,10
03p 2
ER
E
+
+
strict
1
0,10
0,10
0,10
0,10
04p 2
EA
F
+
+
strict
1
0,21
0,10
0,10
0,10
05n 0
E
E
+
+
strict
N10
0,25
0,10
0,19
0,10
06n 0
EA
E
+
strict
N10
0,26
0,10
0,10
0,10
07n 0
EAR E
+
+
relaxed N10
0,65
0,10
0,10
0,10
08n 0
EA
E
+
+
none
N10
0,67
0,10
0,72
0,10
09n 0
EAR P
+
strict
N10
0,79
0,10
0,10
0,10
10p 3
EAR P
+
+
strict
10
0,79
0,10
0,23
0,10
11n 0
ER
F
+
strict
N10
1,12
0,10
0,10
0,10
12n 0
EAR E
+
+
strict
N10
1,24
0,10
0,10
0,10
13n 0
EAR P
+
strict
N10
1,42
0,11
0,10
0,10
14p 3
EA
P
nt +
strict
1
1,72
0,10
2,12
0,10
15n 0
EAR E
+
strict
N10
2,69
0,10
0,10
0,10
16p 2
E
P
+
+
strict
10
2,82
1,37
0,72
0,10
17p 2
A
F
+
+
strict
0.3
2,97
0,89
2,54
0,10
18p 2
EAR F
+
+
strict
1
2,99
0,22
1,53
0,10
19p 2
E
F
+
+
strict
1
3,07
0,10
0,91
0,10
20n 0
EAR E
+
+
relaxed N10
3,65
0,10
0,10
0,10
21p 3
EA
F
+
+
strict
3
3,66
0,10
3,87
0,10
22p 2
EAR P
+
+
strict
0.3
3,82
0,10
2,06
0,10
23p 2
E
F
+
+
strict
0.3
4,13
0,10
1,36
0,10
24p 2
EAR P
+
+
strict
0.3
4,87
0,10
7,97
0,10
25p 2
EAR F
+
+
strict
10
5,14
0,10
3,66
0,10
26n 1
E
P
+
+
strict
N10
5,32
0,20
2,14
0,10
27p 2
EA
P
+
+
strict
10
5,45
0,10
3,55
0,10
28p 2
any? P
+
+
strict
0.3
6,37
2,59
6,97
0,10
29p 2
EA
F
+
+
strict
0.1
7,30
0,10
0,19
0,11
+
+
strict
0.3
7,43
0,30
2,67
0,10
30p 2
E
F
31n 0
EAR E
+
+
strict
N10
7,90
0,10
2,86
0,74
32p 2
ER
P
+
+
strict
1
8,30
2,18
3,57
0,10
33p 2
E
P
+
+
strict
10
9,90
0,44
11,9
0,10
34p 3
EA
F
+
+
strict
10
11,1
0,16
10,4
0,10
35p 3
EAR E
+
+
strict
10
11,8
0,16
5,90
0,21
36n 0
EA
P
+
strict
N10
12,0
0,17
0,22
0,10
37p 2
EAR F
+
+
strict
10
13,1
0,73
6,18
4,04
38n 0
ER
E
+
+
strict
N10
15,2
0,10
3,67
1,24
39n 0
E
E
+
strict
N10
17,9
0,19
0,19
0,23
40p 2
EAR E
+
+
strict
0.3
20,2
0,40
0,10
0,32
41p 4
EA
E
+
+
relaxed 0.3
21,7
4,62
7,29
0,10
42n 0
EA
F
+
strict
N10
22,8
0,22
0,56
0,40
43n 0
E
E
nt +
relaxed N10
31,0
0,10
0,10
0,28
44n 1
EA
P
+
+
strict
N10
33,0
0,76
15,2
0,92
45n 0
EA
E
+
+
strict
N10
38,7
17,1
0,11
1,34
46p 3
E
E
+
+
relaxed 0.3
46,5
0,11
13,1
11,82
47p 2
EAR E
nt +
strict
0.01
57,8
16,16
25,4
0,84
48p 2
E
P*
+
+
relaxed 3
58,0
0,10
45,9
0,11
49p 3
EAR F
+
+
strict
0.3
62,5
11,0
46,0
8,05
50p 2
EAR P
+
+
strict
1
62,9
1,11
75,2
0,10
51p 2
E
P
+
+
strict
0.1
74,7
54,4
10,9
1,72
52p 3
any? P
nt +
strict
10
80,8
35,9
7,9
9,07
53p 2
any? P
+
+
strict
3
84,4
8,16
78,2
0,18
54p 3
EAR P
+
+
strict
0.1
98,5
67,9
17,3
6,06
55p 4
EA
F
+
+
strict
1
100
46,4
54,9
6,52
56p 2
EAR F
+
+
relaxed 0.1
100
14,5
98,6
2,13
57p 2
EAR P
+
+
strict
0.3
100
69,8
83,0
28,2
58p 2
EAR P
+
+
strict
0.3
100
10,0
100
4,92
59n 0
EAR P*
+
+
relaxed N10
100
100
100
85,6
60p 3
E
P
+
+
strict
10
100
94,6
100
79,1
61p 2
EAR E
+
+
strict
1
100
95,0
100
14,7
62p 4
A
P
+
+
strict
0.3
100
100
100
94,5
100
100
100
60,1
63p 3
E
P
nt +
strict
10
64p 4
A
p
+
+
strict
0.3
100
100
100
16,5
65p 2
E
E
+
+
strict
10
100
100
100
12,7
Patient characteristics
Legend to the Columns.
A: Patient code (n=negative challenge, p=positive challenge).
B: Challenge score (Sampson score(11)).
C: Atopic history other than food allergy (E: eczema; A: asthma; R: rhinitis).
D: The reason of an allergy test (E: eczema; P: response to peanut; P*: 2 patients previously had a positive response to
a peanut challenge; F: response to other foods).
E-F:Allergy test prior to challenge (SPT; serum IgE to peanut).
Moving from peanut extract to peanut components: towards validation of component resolved IgE test
M
N
O
P
Q
R
S
T
U
ImmunoCAP ImmunoCAP ImmunoCAP ImmunoCAP ImmunoCAP Sepharose Sepharose Sepharose Sepharose
rAra h 6
rAra h 8
rAra h 9
rPru p 3
rPhl p 12
PVPP
HAc
BPP
total IgE
kUA/L
kUA/L
kUA/L
kUA/L
kUA/L
kUA/L
kUA/L
IU/L
kUA/L
0,10
0,10
0,10
0,10
0,10
0,10
0,10
0,10
51
0,10
0,10
0,10
0,10
0,10
0,10
0,17
0,10
30
0,10
0,10
0,10
0,10
0,10
1,35
nt
1470
0,10
13,48
0,10
0,10
0,10
14,4
16,6
10,2
202
0,20
0,63
0,10
0,10
0,10
0,31
0,50
0,29
37
0,30
0,10
0,10
0,10
0,10
0,12
0,43
0,26
53
0,14
38,7
0,10
0,17
0,10
2,92
0,47
0,11
1060
nt
0,10
0,10
0,19
0,10
0,64
1,09
0,53
256
0,36
1,52
0,12
0,10
0,10
1,40
nt
876
0,29
14,18
0,10
0,10
0,10
0,73
2,03
0,28
2860
0,10
0,16
0,10
0,10
0,10
6,36
3,56
0,14
116
1,09
11,75
0,10
0,83
0,10
2,17
1,31
1,26
2300
0,10
0,19
0,10
3,93
0,18
2,31
3,27
0,93
2300
1,18
1,59
0,10
0,10
0,10
1,10
2,61
1,12
250
0,23
66,0
0,10
0,10
3,03
0,59
3,18
0,10
583
0,42
0,10
0,10
0,27
0,10
1,02
0,78
0,34
183
1,39
17,4
0,10
0,10
0,10
1,82
4,02
1,01
245
2,81
2,64
0,10
0,10
0,10
2,10
3,66
1,97
558
1,43
1,16
0,10
0,10
0,11
2,19
3,34
1,62
90
0,10
28,6
0,10
0,10
3,35
3,34
nt
354
3,92
0,10
0,10
0,10
0,10
2,90
4,91
4,77
252
1,23
64,7
0,10
0,12
0,10
2,32
4,49
0,71
450
3,93
0,35
0,10
0,10
0,10
8,00
nt
nt
4,01
4,48
0,10
0,18
nt
3,97
nt
191
1,79
9,83
0,10
0,10
0,10
2,79
6,39
1,40
480
2,37
1,86
0,10
0,10
0,13
5,00
5,44
1,79
864
4,05
0,73
0,10
0,10
0,10
8,85
6,74
1,68
600
1,27
0,10
0,10
0,10
0,10
6,43
9,07
3,66
35
5,26
0,10
0,10
0,48
0,10
4,37
3,95
5,39
901
6,43
0,21
3,12
3,76
1,13
8,73
10,9
7,40
305
0,71
31,52
0,14
0,15
0,22
4,60
9,90
1,18
2920
2,99
0,25
0,10
0,10
0,10
6,92
7,61
2,02
181
4,94
0,10
0,10
0,10
0,10
63,3
68,6
5,82
30
8,54
0,10
0,10
0,10
0,10
8,49
12,3
9,85
353
4,05
31,16
0,33
5,65
0,22
16,3
15,3
4,79
3350
0,13
58,9
0,10
1,28
0,10
5,43
18,2
2,54
1600
1,93
2,66
0,12
0,12
0,14
9,79
8,73
2,11
1520
3,33
0,10
4,43
1,91
0,16
16,3
9,86
3,28
800
0,59
17,0
0,34
0,42
9,85
9,93
nt
3470
0,48
8,37
35,2
28,7
10,9
1100
12,6
0,10
0,10
12,2
0,17
0,10
0,21
0,10
17,5
21,9
16,3
165
0,40
100
0,29
0,60
0,30
11,4
30,5
16,8
4360
1,19
0,94
0,10
0,10
0,10
23,5
5,28
1,18
705
9,43
40,5
0,37
2,36
0,18
34,0
21,5
8,18
2300
0,34
71,7
0,31
5,77
0,19
62,5
51,9
7,49
3350
2,65
10,4
0,24
0,97
11,50
22,6
33,1
3,33
2300
22,1
0,10
0,10
0,10
0,10
37,9
39,5
32,5
325
40,5
4,85
0,52
9,25
0,14
68,4
nt
2340
30,2
0,10
0,10
0,11
4,86
81,2
45,1
19,4
737
48,9
2,09
0,10
0,10
0,10
52,6
65,8
47,0
719
8,07
2,10
0,10
0,44
0,10
46,7
17,3
10,9
217
7,07
0,10
0,10
0,10
0,22
51,3
17,3
18,6
511
39,3
0,15
0,10
0,16
0,64
240
168
45,7
1610
30,9
0,10
0,10
0,10
0,10
61,3
43,8
44,2
79
2,05
nt
0,20
0,79
nt
136
115
56,5
2200
49,9
9,38
0,10
0,13
0,17
86,4
114
47,1
2300
41,3
10,6
0,13
0,12
0,10
161
129
50,0
508
60,5
53,7
0,14
0,93
5,59
500
500
89,3
2136
73,5
0,11
0,15
0,17
0,26
1000
413
220
1060
85,7
5,77
0,40
2,25
34,5
1000
1000
315
2300
84,2
10,5
0,30
0,42
0,10
236
202
188
1000
97,3
0,11
0,12
0,21
0,20
1000
355
255
2040
80,7
0,10
0,10
0,10
0,10
297
272
127
808
58,3
2,91
0,10
0,10
0,10
479
1000
116
437
93,4
1,80
0,10
0,10
0,10
500
500
87,7
950
G: Exposure to peanut prior to challenge (0: strict avoidance; 1: relaxed avoidance; 2: no avoidance). 01n had no
response to an open challenge performed at home.
H: Eliciting dose in grams. N10: no response to the maximum dose of 10 grams.
I-Q: ImmunoCAP serology: IgE to peanut and single allergens components (kUA/L). nt: not tested.
R-U: Sanquin serology: total IgE (kIU/L) and peanut IgE to PVPP extract, HAc extract and BPP(kUA/L). BPP = Basic
peanut protein fraction obtained from the HAc extract. Due to limited plasma availability some Sepharoses were not
tested (nt) or tested only as a combination (PVPP + HAc).
113
Chapter 7
Discussion
Partly published in J Allergy Clin Immunol Pract 2014 635-6
Aalberse JA & Aalberse RC
Chapter 7
Food allergy: Current and future diagnosis and
implications for treatment
In this chapter I will discuss the difficulties of allergen specific diagnosis and the role of alarmins
in diagnosis and treatment.
PART 1
The clinical relevance of sensitization
In chapter 6 we have discussed the difficulties of the diagnosis with a serum IgE test. We have
shown that regularly used IgE tests for peanut sensitization are deficient of certain allergens
and have discussed the importance of an improved test to increase the sensitivity of the test.
It is both important to have a sensitive test and to know what allergens are clinically relevant.
The first is especially important for general practitioners, the latter is more important for
more specialized doctors in the hospital.
The current guideline states that an IgE test should not be performed by a GP, because of low
sensitivity and specificity. As will be discussed below, these guideline need to be reconsidered.
The diagnosis of food allergy is based upon the history of the patient, a skin prick test and/
or serum IgE tests, the latter two combined referred to as sIgE test. The sIgE test is often false
positive. In an unselected population up to 10% of people are found to have an SPT positive to
peanut(1). As only 0.5-1% is found to be truly allergic in the general population, up to 90-95%
of positively tested individuals for peanut do not have a clinical response. Even in a selected
patient group suspected for peanut allergy on history of an allergic response upon exposure,
50% is sensitized without a clinical response(2;3). As discussed below new tests show better
predicting outcomes, but adjustments of the test are clearly needed.
The gold standard for the diagnosis of peanut allergy, like other food allergies, is the double blind placebo controlled food challenge(4). This is an academic clinical diagnosis, but not
a workable diagnosis for general practitioners and smaller hospitals. Even in academic research it is often not included in food allergy diagnosis. The search for tests that can more
accurately predict a clinical response is therefore important.
As we have discussed in chapter 6, many conventional extracts are deficient in some sig-
nificant IgE binding components, which can result in a low sensitivity of the test. Also some
components are clinically irrelevant, causing a low specificity of the test.
The accuracy of a test can be described with several parameters of which sensitivity and spec-
ificity are most often used, but also positive predictive value (PPV) and negative predictive
value (NPV) can aid in interpretation of a test. These parameters often cause confusion, but
can be very helpful to understand the value of a test(5).
116
Discussion
Sensitivity relates to the test’s ability to identify a condition correctly. In other words how
often is the food specific IgE test positive when you are food allergic. Specificity relates to the
test’s ability to exclude a condition correctly. In other words how often is the IgE test found
negative if a subject is not food allergic.
These tests are independent of the population they are used for. However PPV and NPV describe the proportion of the test that is truly positive or negative, respectively, which can be
different in a general practice population and in a patient population visiting a specialized
physician in a secondary or tertiary referral clinic. The NPV is especially of importance in a GP
practice. A high NPV can rule out food allergy with high certainty. Remaining patients can then
be referred to the specialized physician who needs a high PPV lowering the need for DBPCFC.
As can be seen in figure 1 the NPV and PPV are dependent on the sensitivity and specificity of
the test, but also on the expected prevalence of the disease, which is underlined by the differ-
ence in NPV and PPV in the population of a general practice and the secondary and tertiary
referral clinic.
In the current protocol used by GPs in food allergy it is suggested to refrain from measuring
IgE because the test result has insufficient specificity and sensitivity(6). With the increased
technical knowledge on peanut allergy diagnosis as well as based on the different prevalence
numbers in a general practice compared to secondary and tertiary referral clinics, I would
suggest that the serum IgE test for peanut is included in the test protocol for GP’s to be able
to rule out peanut allergy. However when the patient tests positive for a food, it is very important that the patient is well informed on the high chances of false positive values.
Component resolved diagnostics and an ongoing need of food extracts
In recent years technical progress has been made that enables the measurement of binding
of IgE to multiple single allergens instead of crude extracts via so-called component resolved
diagnostics (CRD). As we have shown in chapter 6 the development of CRD can be of help to
achieve a better diagnosis. It has been suggested that components can replace extract based
testing. For peanut it has been shown that the measurement of solely Ara h 2 and Ara h 6 is
sufficient for diagnosis. Often inclusion criteria are however based on deficient extract, which
could bias the importance of certain allergens. We have discussed in an editorial on hazelnut
allergy that CRD is a helpful tool to improve IgE diagnosis but cannot yet replace extract-based
testing. We have argued that we can learn from component-based tests how to improve our
extract-based testing until we can definitely use only CRD (see textbox).
117
118
IgE test
+
food allergy
total
B
PT
D
NT
ND
T
86%
74%
sensitivity specificity
(Academical Center)
food allergy
+
3600
1500
+
600
4300
total
4200
5800
86%
74%
sensitivity specificity
total
5100
4900
10000
(5 times an average practice = 10000 patients)
food allergy
+
total
600
2400 3000
+
100
6900 7000
total
700
9300 10000
A/PD
D/ND
sensitivity specificity
+ A
- C
total PD
42%
pretest probability
71% PPV
88% NPV
7%
pretest probability
20% PPV
98.6% NPV
PD/T
pretest probability
IgE test
Specialist
IgE test
+
total
+
total
+
food allergy
total
2400 3065
6900 6935
9300 10000
665
35
700
95%
74%
sensitivity specificity
food allergy
+
4000
1500 5500
200
4300 4500
4200
5800 10000
95%
74%
sensitivity specificity
Figure 1c; increased sensitvity
General Practice
A/PT positive predictive value (PPV)
D/NT negative predictive value (NPV)
42%
pretest probability
73% PPV
96% NPV
7%
pretest probability
22% PPV
99.5% NPV
IgE test
Specialist
+
total
95%
95%
sensitivity specificity
food allergy
+
total
4000
300 4300
200
5500 5700
4200
5800 10000
95%
95%
sensitivity specificity
Figure 1d: increased specificity
General Practice
food allergy
+
total
665
435 1100
+
IgE test
35
8865 8900
total
700
9300 10000
42%
pretest probability
93% PPV
96% NPV
7%
pretest probability
60% PPV
99.6% NPV
and specificity. This is important for the interpretation of these tests in either a general practice or in a hospital.
The key message of this figure is that the negative and positive predictive values can be different depending on the population it is tested in, despite the same sensitivity
and specificity.
This is of
important
for the is
interpretation
of these tests
in either
a general
practice orvalues
in a hospital.
The
key message
this figure
that the negative
and
positive
predictive
can be different depending on the population it is tested in, despite the same sensitivity
Legend to figure 1
Legend to figure 1
This figure illustrates the concepts of sensitivity, specificity, positive predictive value and negative predictive value.
This figure illustrates the concepts of sensitivity, specificity, positive predictive value and negative predictive value.
Figure
1a| Formulas
| Formulas
the
2x2A=table.
A=ofnumber
of correct
positive
tests;
B= number
ofnumber
false positive
tests; tests;
C= number
ofoffalse
negative
Figure 1a
of theof
2x2
table.
number
correct positive
tests; B=
number of
false positive
tests; C=
of false negative
D= number
correct
nega- tests; D= number of correct negative tests.
number
of positive
tests; NT=
number
of negative
PD= number
of disease
NDof
number
of nopresent;
disease present.
T: total number
of tests. present. T: total number of tests.
tive
tests.PT=
PT=
number
of positive
tests;
NT=
numbertests;
of negative
tests;
PD=present;
number
disease
ND number
of no disease
Figure 1b-d | In these figures the numbers chosen are not related to a true situation but give estimates assuming a pre-test probability of 7% in general practice and 42%
Figure
1b-d | In these figures the numbers chosen are not related to a true situation but give estimates assuming a pre-test probability of 7% in general practice and 42% at the specialist.
at the specialist.
Figure 1b1b
| This
box indicates
the baseline
Figure
| This
box indicates
thesituation.
baseline situation.
Figure 1c1c
| In| the
box the box
sensitivity
is increased. Sensitivity
can be increased
by adding
missing
allergens by
withadding
better extracts
or viaallergens
CRD, but also
by decreasing
Figure
In second
the second
the sensitivity
is increased.
Sensitivity
can be
increased
missing
with
better extracts or via CRD, but also by
the cut off value for a positive test. Both interventions will cause an increase in sensitivity.
decreasing
the
cut
off
value
for
a
positive
test.
Both
interventions
will
cause
an
increase
in
sensitivity.
Figure 1d | In the final box on the right also the specificity in increased. This can be achieved by removing clinical irrelevant allergens.
Figure 1d | n the final box on the right also the specificity in increased. This can be achieved by removing clinical irrelevant allergens.
Specialist
IgE test
Figure 1b; baseline
General Practice
IgE test
Figure 1a Formulas
Chapter 7
Discussion
The point is that patients with a very high or a very low likelihood of sensitization are not informative
for deciding how useful it is to add another test to a diagnostic protocol and that it is not statistically
helpful to increase numbers by mixing groups of subjects with very different pretest likelihoods. Although subjects with a moderate pretest likelihood are most informative, subjects with a very low or a
very high likelihood can help us to decide which components might be useful in diagnostic protocols.
Tolerant subjects are informative in 2 ways. First, to establish which allergens are least likely to be
clinically relevant. Second, to establish which characteristics* make subjects likely to be food sensitized
without symptoms on oral exposure. Sera from patients who are evidently symptomatic are important
to identify the spectrum of allergenic proteins and to check whether all these components are adequately present in diagnostic extracts. If not, we need to decide whether these deficient components
(such as Cor a 1, a prominent allergen in the majority of the patients who are provocation positive) are
useful to add to our diagnostic protocol. The proposed “additional studies”** require oral provocation.
To recruit patients for provocation studies, most protocols rely on tests with extracts for inclusion.
We have discussed this potential bias elsewhere in relation to peanut components.5 Our conclusion
was that it is impossible to judge the clinical relevance of an allergic component if the extract used
for inclusion of patients is inefficient in detecting sensitization to this component (such a Cor a 1 in
hazelnut extracts). Another issue is how to deal with potential cross-reactivity, for example, between
the birch pollen allergen Bet v 1 and Cor a 1. Should patients with pollen sensitization be excluded from
these studies, and, if so, how? IgE induced by exposure to birch pollen often reacts with Cor a 1.01 from
hazel pollen and Cor a 1.04 from hazelnut.6 Because many patients who are allergic to birch tolerate
hazelnuts despite such cross-reactive antibodies, it is tempting to conclude that IgE to hazelnut with a
patient who is sensitized to birch is clinically irrelevant. However, 85% of the clinically reactive patients
in the article by Kattan et al are Cor a 1 positive***. The effect of sensitization to cross-reactive pollen
components will be an important aspect of the future studies. Similarly, the effect of sensitization to
fruit-derived lipid transfer protein (eg, peach) should be taken into account.(7,8)
7. Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification
of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled
food challenge results. J Allergy Clin Immunol 2002;109:563-70.
8. Schocker F, Lüttkopf D, Scheurer S, Petersen A, Cisteró-Bahima A, Enrique E, et al. Recombinant lipid transfer protein Cor a 8 from hazelnut: a new tool for in vitro diagnosis of potentially
severe hazelnut allergy. J Allergy Clin Immunol 2004;113:141-7
This textbox is a citation from an Editorial by Aalberse and Aalberse; A lesson from component resolved
testing: we need better extracts. Journal of Allergy and Clinical Immunology in Practice 2014 SepOct;2(5):635-6. doi: 10.1016/j.jaip.2014.07.006.
* Characteristics of allergens. Another subject dependent characteristic is IgG
** The need for additional studies was proposed by Kattan et al (J Allergy Clin Immunol Pract. 2014
Sep-Oct;2(5):633-4), in response to whom this Editorial was written.
*** In contrast to the situation in paediatric hazelnut allergy, pollen allergy is very often found in
genuine adult peanut allergy. In the latter situation, measuring IgE to Ara h 8 is not informative and
the diagnosis is much more dependent on IgE to Ara h 2 or Ara h 6.
119
Chapter 7
The IgE responses to hazelnut allergens show many similarities compared to peanut allergens,
however some aspects are clearly different. Both in peanut- and in hazelnut allergy subjects are
often also pollen allergic. Cor a1 and Ara h8 are allergens that show cross-reactivity in pollen allergic subjects with hazelnut and peanut allergy, respectively. In hazelnut allergy IgE to just Cor a1
is often seen. However in symptomatic peanut allergy IgE to only Ara h8 is uncommon(7). IgE to
Ara h2 and Ara h6 have shown to be important components in peanut allergy diagnosis(8). Future
studies will have to evaluate if also other allergens are needed for diagnosis of peanut allergy.
Predicting severity of a food allergic response
Next to the difficulties in relation to sensitization, another diagnostic problem is that when a
positive diagnosis is made, it is hard to predict severity (i.e. the threshold dose needed for a
response and local versus systemic complaints)(9). Skin prick testing and measuring serum-spe-
cific IgE are not consistent and reliable in terms of predicting the severity of reactions. Also DB-
PCFC are valuable to diagnose the food allergic response, but the dose response thresholds are
not reproducible(10). Recently it was suggested that the BAT test (basophil activation test) can
predict severity, but the data is still insufficient to draw conclusions(11). Severe allergic reactions
are more likely to occur in older patients and those with underlying asthma(12). Other data sug-
gest the role of biological mediators in allergic reactions such as platelet-activating factor. These
may provide a future additional tool in predicting those at risk of severe reactions(13).
Phenotypes and endotypes
Beyond the problem of insufficient food extracts or clinically irrelevant allergens, it is likely
that other factors also play a role in the difficulties of diagnosis. The diagnosis of asthma
and rhinitis were similarly difficult several years ago, but this has gradually changed. It has
become clear that asthma and allergic rhinitis are not single entities but syndromes(14;15). It is
plausible that food allergy is also a syndrome with different subgroups.
A phenotype can be defined as a feature or a cluster of features that can discriminate that
subgroup from the general population. An endotype is a phenotype with a pathophysiologic
character. Little is known so far on phenotypes and endotypes in food allergy. Classifications
based on phenotypes and endotypes in rhinitis, but especially in asthma have promising implications on the management(16). In the future a similar classification could result in a more
uniform diagnosis and better response to treatment also in food allergy.
Phenotypes and endotypes in asthma and rhinitis.
A group of experts proposed distinct endotypes based on seven parameters: clinical characteristics, biomarkers, airway physiology, genetics, histopathology, epidemiology and treat-
ment response(15). This has lead to practical allergy classifications (PRACTALL) in asthma and
rhinitis. Another convincing example of the success of endotypes is the IL-13 based endo-
types, which has been shown to have a clear response to inhaled corticosteroids(17). In a phase
II study it was shown that a subgroup of patients with high levels of periostin, a surrogate
120
Discussion
marker for IL-13 activity, improved significantly upon treatment with anti IL-13. Discriminat-
ing endotypes aids in diagnosis and treatment in asthma and rhinitis, but a practical allergy
classification has not yet been made for food allergy.
Phenotypes and endotypes in food allergy
It is likely that food allergy is also a syndrome, including distinct phenotypes. In the last few
years a small number of studies have described different phenotypes. It is a challenge to formulate endotypes in food allergy that may help to establish new treatment possibilities.
A large prospective cohort identified distinct phenotypes in children in their first year of life
with wheeze, eczema or food sensitization(18). The five phenotypes found included no allergic
disease, non-food sensitized eczema, egg allergy, multiple food allergies (predominantly pea-
nut) and multiple food allergies (predominantly egg). Eosinophilic oesophagitis and food pro-
tein induced enterocolitis syndrome have recently been suggested as phenotypes linked to food
allergy(19). Additional endotypes can be formulated by discriminating patients within the groups
that are proven peanut allergic or by comparing differences between peanut allergic to only
peanut sensitized individuals. Tryptase has been associated with a severe acute allergic response (anaphylaxis) with a higher basal serum level of tryptase in anaphylactic patients com-
pared to patients without anaphylaxis. We and others have compared peanut allergic to just
peanut sensitized children and compared cytokine responses. In 2012 Xie et al found clearly
higher IL-9 production after in vitro peanut stimulation in the peanut allergic group compared
to the peanut tolerant individuals(20;21). In our study in chapter 5 we have shown that IL-25,
an IL-17 family member, is highly elevated in plasma only in some children with a clinical response to peanut: a subgroup in the peanut allergic patients. This suggests a role for IL-25 in
the pathogenesis of peanut allergy and elevated plasma IL-25 may be a sign of a chronic severe
atopic phenotype(22). Overproduction of the uric acid (UA) alarmin in the local microenviron-
ment plays a critical role in the induction of peanut-allergic sensitization, likely due to its ability
to activate DCs. These findings suggest that cellular damage or tissue injury may be an essential
requisite for the development of allergic sensitization to foods(23). In chapter 2, 3 and 4 we have
discussed the potential role of another alarmin, HSP (heat shock proteins), in atopic disease (see
also part 2 in this chapter).
For the food allergic syndrome, endotypes have only just started to be formulated and large
cohorts are needed to discriminate between the endotypes. The above mentioned seven se-
lected parameters, with clinical characteristics (food sensitized, but tolerant versus clinically
reactive), biomarkers (UA, IL-25, IL-9, tryptase), mouth/gut physiology (locally activated DC’s
and mast cells), genetics, histopathology, epidemiology (including age of onset), and treat-
ment response have great potential to formulate new distinct endotypes in food allergy with
better diagnostic possibilities and improved treatment results.
121
Chapter 7
PART 2
Heat shock proteins and alarmins in allergic disease.
Immunogenic responses to heat shock proteins
This thesis shows that HSP is immunogenic both in a disease free environment as in patients with
atopic dermatitis(24;25). At the start of our research data primarily suggested that HSP is an in-
teresting protein because of its potential of inducing regulatory T cells(26). Therefore when our
research was initiated this had our prime interest. Latter research, including our own, additionally
showed that HSP not only induces regulatory T cells, but also activated, non-regulatory T cells,
depending on the environment in which the immune response takes place(27-29). In chapter 3 we
show that immunogenic regulatory responses to HSP are already present in cord blood(24). The
immune cells found in cord blood are taken from a disease free, relatively sterile environment,
directly after birth. This supports the hypothesis that responses to HSP can be a healthy immu-
nogenic response. In chapter 4 we show that immune responses to HSP are stronger in children
with atopic dermatitis than in non-atopic children(25). These responses have a pro-inflammatory
character indicating that responses towards HSP are dependent on the environment.
Alarmins in allergic inflammation
HSP are endogenous proteins that have an important chaperone function in the cell, upregulated dur-
ing stress, that are present in abundance(30). These endogenous HSP have a molecular mimicry with
microbes(31). Interestingly, HSP have an effect both on the innate as the adaptive immune system(32).
Endogenous proteins, like HSP, that are released upon tissue damage and that can activate the innate
immune system have been called damage associated molecular patterns (DAMPs) or alarmins(33-35).
Recent data have suggested that alarmins contribute to the dysregulated processes seen in chronic
inflammation. Research in mice has shown the presence of the proteins early in the inflammation,
including in a contact dermatitis model(36). In this thesis we show that HSP is found more in lesional
skin than in non-lesional skin of atopic dermatitis patients(25). Other research has shown a role for
IL-33 as an important alarmin in epithelia and lymphoid organs in allergy(37). Just recently a study
was published on the role of uric acid and its involvement in peanut allergy(23). The authors showed
that when in mice uric acid is blocked peanut sensitization could be blocked. Elevated levels of uric
acid were seen in peanut allergic children compared to healthy controls. Together these data suggest
that in allergic settings triggers of the innate immune system such as alarmins may play a role in sustaining the (partly) allergen-independent chronic inflammation that is present in the tissues. A very
interesting observation from this thesis (as discussed in chapter 5) is that a subgroup of children that
all had a positive DBPCFC to peanut without exposure to peanut for years, showed highly increased
circulating levels of IL-25. Since gut epithelial cells are a major source of IL-25 this suggest that also
in a (sub)group of peanut allergic children there may be chronic tissue inflammation involvement(22).
The role of endogenous alarmins in the initiation and maintenance of tissue inflammation in
allergic disease and their role as potential therapeutic targets is one of the interesting and
122
important avenues for future research, as partly reviewed in chapter 2(38).
Discussion
PART 3
Management of peanut allergy
Peanut allergy management
It can be concluded that there is need for improvements in the management of food allergy
by better sensitization tests, but also by identifying different phenotypes and endotypes. Im-
proved diagnosis is likely to result in more targeted therapy including appropriate diets. As
discussed below immunotherapy is the only curative therapy in allergic rhinitis and possibly
also in food allergy. Combining food allergy immunotherapy with anti IgE treatment might
reduce the risk of side effects while allowing for more rapid desensitization schemes. On the
other hand, it is plausible that alarmins can be a potential target for therapy in chronic allergic
inflammation, including food allergy.
Allergen Specific Management
Allergen specific immunotherapy is the only curative therapy, mostly applied in tree- and grass
pollen-allergic patient(39). Specific immunotherapy induces clinical tolerance by applying in-
creasing doses of the allergen(40). The effect seen with immunotherapy in venom or aero aller-
gies has different pathophysiologic mechanisms compared to immunotherapy in food allergies.
Whereas in immunotherapy for allergic rhinitis the altered T cells responses and decreased IgE
production with an increase in IgG4 clearly plays an important role, in food allergy desensitiza-
tion of mast cells and basophils seems to contribute most to the effect of immunotherapy(41-43).
A small number of trials in peanut allergy have shown a reduced risk of allergic symptoms after
therapy(44;45). The down side of immunotherapy in general is its duration, and in peanut specif-
ic immunotherapy also the high rate of adverse reactions, and waning of the protection after
stopping the active therapy(46). As in other allergens, an improved effectiveness with less side-ef-
fects is suggested via T-cell-reactive peptides and chemically modified allergens in peanut aller-
gy(47-49). Currently, peanut specific immunotherapy is not in the stage that it can be applied in
clinic. A different approach is to prevent peanut allergy. Recently it was published that peanut
allergy can be prevented by early exposure to peanut(50;51). Oral exposure to peanut early in life
was even found to diminish already existing allergic complaints. This is an exciting study, but
needs to be repeated and further evaluated to draw clear conclusions.
Immunotherapy based on endotypes
As discussed above new endotypes are designed to be used for therapy. It is conceivable that
biologicals specifically aimed at an endotype and its immunopathomechanism can be used
also in food allergy in the future(21;52). Anti-IgE is the first biological used in peanut allergy(53;54).
Both in adults and in children with high-risk peanut allergy, treatment with omalizumab (an-
ti-IgE) facilitated rapid oral desensitization and qualitatively improved the desensitization
process(55;56). As seen in asthma, it is likely that with the introduction of new biologicals such
as anti-IL-13 and anti-IL-25, new subtypes or endotypes will be demonstrated that will improve the diagnosis and treatment responses(57-60).
123
Chapter 7
Immunotherapy based on alarmins
Alarmins such as HSP, clearly play a role in (local) inflammation. They may therefore be inter-
esting targets for therapy. As discussed by Chan et al., it first needs to be defined which alarmins are important in a specific type of inflammation, and if this depends on the different phas-
es of an inflammatory response(61). A big challenge concerns the issue of balance. The same
alarmins can be both harmful and beneficial within the same disease context, depending on
the disease activity and the environment (chapters 2,3, and 4) (24;25;38). While dampening of
inflammation may be desirable in certain pathological contexts, total abolition of the host
defense is disadvantageous, leaving the patient at risk to opportunistic infections and malig-
nancies as well as impairing repair and remodelling pathways. Therefore further research is
needed to evaluate the overall effect of targeting alarmins.
In conclusion, it is to be expected that a combination of allergen specific immunotherapy, bi-
ologicals and alarmin-related therapy will be needed to tackle specific clinical manifestation
and progression of peanut allergy and other food allergies. Improved diagnostics, including
newly discovered allergens, as well as better-defined endotypes, will contribute to a better
understanding of the disease with better management of peanut allergy.
124
Discussion
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Discussion
129
Chapter 8
Lekensamenvatting
J.A. Aalberse
Chapter 8
Introductie
Dit proefschrift beschrijft de diagnose van pinda allergie en de rol van alarmins, zoals heat
shock eiwitten, bij allergische ziekten.
Allergie is een immunologische ziekte waarbij je reageert op stofjes, bijvoorbeeld in de voe-
ding of uit de lucht, waar mensen zonder die allergie niet op reageren. Zo kan je allergisch
reageren op huisstof, waardoor je bijvoorbeeld gaat niezen of reageren op de kat en daar benauwd van worden. Ook zijn er mensen die allergisch zijn voor voedselcomponenten. Iemand
met pinda allergie krijgt na het eten van pinda bijvoorbeeld uitslag, maar kan ook een heftige
reactie krijgen met shock verschijnselen, dit heet een anaphylactische shock.
De basis van een allergische ziekte lijkt zo simpel. Als je allergisch bent maak je namelijk een
antistof, IgE, aan tegen stofjes in bijvoorbeeld de lucht of in het voedsel. Deze stofjes, meestal
eiwitten, noemen we dan allergenen. Mensen die niet allergisch zijn maken geen IgE tegen
deze stofjes. Door de binding van het IgE met een allergeen ontstaat er een reactie bij bepaalde cellen in het lichaam. Door deze reactie kan iemand fysieke klachten ervaren, zoals jeuk.
Kinderen die allergisch zijn tegen pinda hebben inderdaad IgE tegen pinda. Maar helaas
wordt het dan ingewikkelder. Niet alle kinderen die IgE tegen pinda hebben in het bloed, hebben daadwerkelijk een allergische reactie tegen pinda. Mensen met IgE tegen pinda noemen
we gesensibiliseerd (gevoelig) voor pinda. Je bent echter pas echt pinda-allergisch als je een
pinda sensibilisatie hebt met een allergische reactie na blootstelling aan pinda.
De diagnose van pinda allergie wordt meestal gedaan op basis van het verhaal van de patiënt of de ouders, een huidtest en/of een bloedtest. Ten slotte is een dubbel blinde placebo
gecontroleerde provocatie test nodig. Deze provocatie test is de gouden standaard en maakt
het onderscheid tussen kinderen die alleen een gevoeligheid hebben voor pinda en kinderen
die daadwerkelijk ook een reactie hebben als ze worden bloodgesteld aan pinda. Dubbelblind
placebo gecontroleerd betekent dat noch de patiënt noch de dokter weten of de patiënt place-
bo (een hapje zonder pinda) of pinda toegediend krijgt. Zo kan je zo veel mogelijk voorkomen
dat er een bevooroordeelde observatie ontstaat bij de test.
Handig zou zijn als je die provocatietest niet nodig zou hebben. Het is namelijk best spannend
voor de patiënt en de ouders, daarnaast is het intensief en kost het veel tijd van patiënten,
verpleegkundigen, dokters en ziekenhuizen.
Helaas ontbreekt de provocatietest om deze reden nu al vaak. Veel kinderen hebben daardoor
mogelijk een onterechte diagnose van pinda allergie met alle ingrijpende gevolgen van dien.
Ook in veel wetenschappelijke artikelen is geen gebruik gemaakt van provocatie testen en
gaat het dus eigenlijk over gesensibiliseerde patiënten maar is het onbekend of ze ook daar132
werkelijk pinda allergisch zijn. Ze worden wel beschreven als pinda allergisch. Deze soms
Lekensamenvatting
onterechte conclusie geeft veel problemen bij de interpretatie van wetenschappelijke studies.
Je kunt je afvragen wat de waarde is van een studie van het aantal patiënten dat over zijn
pinda allergie heen is gegroeid als je niet met zekerheid weet of iemand ooit pinda allergisch
is geweest. Wat is het nut van een voorspellende waarde als je eigenlijk slechts sensibiliteit
voorspelt, maar geen allergische ziekte.
In onze studies beschreven in dit proefschrift hebben we wel gebruik gemaakt van provocatie
testen om de diagnose te bevestigen of te ontkrachten. In deze patiënten populatie hebben
we gekeken of we de voorspellende waarde van de bloed test die het IgE tegen allergenen
meet kunnen verbeteren of kunnen aanvullen. Voor de bloedtest met IgE zijn twee aspecten
belangrijk. Ten eerste moet het een test zijn waarin alle allergenen aanwezig zijn, waarvoor
je allergisch bent. Ten tweede is het belangrijk om te weten of al die allergenen ook daadwerkelijk klinisch relevant zijn.
Hoewel dit beide logisch klinkt is het nog niet zo vanzelfsprekend. In hoofdstuk 6 laten we
zien dat de manieren van extraheren (een methode om allergenen uit een pinda te halen) erg
verschillen, en ook verschillend succesvol zijn. Dit heeft veel te maken met de extractie buffer
(de vloeistof die gebruikt wordt om de eiwitten uit de pinda te halen). Het blijkt dat als je een
zure buffer gebruikt, zoals azijnzuur, je bijvoorbeeld meer basische allergenen kan extraheren, die je mist als je alleen water gebruikt als extractie buffer. Met meer allergenen is de kans
dat je iets mist kleiner. Helaas wordt de kans dat de diagnose goed is niet evenredig beter,
en zelfs soms slechter. Dit komt omdat sommige allergenen bij de meeste mensen niet leidt
tot allergische klachten. Deze allergenen zijn maar bij enkele patiënten klinisch relevant. De
uitdaging van de bloedtest is om aan de ene kant zoveel mogelijk eiwitten in de test te hebben
en aan de andere kant de test zo efficient mogelijk te maken.
We hebben ook gekeken wat het verschil is tussen patiënten met een IgE tegen pinda die
alleen gesensibiliseerd zijn, en de patiënten die ook echt allergische symptomen hebben na
blootstelling aan pinda. Onze onderzoeksvraag was of we aan de hand van in het bloed gemeten cytokines (bepaalde eiwitten die processen aanzetten voor de ontstekingsreactie) kunnen
voorspellen wie wel en wie niet gaat reageren op de pinda. Uit ons onderzoek, beschreven in
hoofdstuk 5, blijkt dat IL-25 alleen te vinden is bij patiënten die ook daadwerkelijk een pinda
allergische reactie vertoonden bij de provocatie. Het is echter niet te vinden bij alle patiënten
met een positieve reactie. Dit suggereert dat IL-25 mogelijk een bepaalde subgroep identificeert. Dit zou bijvoorbeeld een meer chronische groep patiënten kunnen zijn.
Il-25 is een alarmin. Een alarmin is een endogeen eiwit, dit betekent dat het wordt gemaakt
in de cel, verder komt een alarmin vrij bij schade en kan het het (aangeboren) immuunsysteem aanzetten. Meerdere alarmins lijken een rol te spelen bij allergische ziekten, zoals ook
urinezuur en IL-33.
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Chapter 8
Deze alarmins helpen niet alleen in de diagnose of bij een beter begrip van de ziekte, maar
zou ook een aanknopingspunt kunnen zijn voor therapie. Een mogelijkheid in de toekomst
zou bijvoorbeeld zijn om met behulp van antilichamen IL-25 te blokkeren om op de manier
de chronische ontsteking te onderdrukken.
Het andere deel van dit proefschrift gaat over heat shock eiwitten (HSP = heat shock proteins). Ook deze eiwitten zijn alarmins en worden in verband gebracht met therapie, bij bij-
voorbeeld auto immuunziekten. In dit proefschrift hebben we gekeken wat de rol is HSP bij
allergische ziekten.
HSP zijn endogenen eiwitten. De HSP in de cellen van de mens lijken erg op de HSP van planten en bacterieën wat er op duidt dat de eiwitten weinig zijn veranderd tijdens de evolutie.
Een belangrijke functie van HSP is dat ze chaperonen zijn: ze helpen bij het transport van
eiwitten van binnen naar buiten de cel. HSP blijken echter nog meer functies te hebben. Ze
zijn namelijk immunogeen, ze triggeren het immuunsysteem. Bij hitte (vandaar de naam heat
shock) of andere vorm van stress welke leidt tot schade aan het weefsel, worden deze eiwitten
extra aangemaakt. In hoofdstuk 2 van dit proefschrift hebben we op een rij gezet wat er tot
dan toe bekend was over de rol van HSP bij allergische ziekten.
In hoofdstuk 3 en hoofdstuk 4 hebben we laten zien dat HSP immunogeen is zowel in een
ziektevrije omgeving (direct na de geboorte) als in patiënten met eczeem. Dit ondersteunt de
hypothese dat reacties op hsp een gezonde immunogene respons kan zijn, maar dat het in
een immuun actieve omgeving ook juist een reactie kan geven die bijdraagt aan de onsteking.
De rol van endogene alarmins bij het begin van de ontsteking maar ook bij het in stand hou-
den van weefsel ontsteking bij allergische aandoeningen alsook hun rol als potentiële the-
rapeutische targets is een van de interessante en belangrijke invalshoeken voor toekomstig
allergie onderzoek.
134
Lekensamenvatting
135
Chapter 9
Dankwoord & Curriculum Vitea
Chapter 9
DANKWOORD
Promoveren bestaat uit veel stapjes. Terugkijkend zijn het voornamelijk leuke stapjes, maar
natuurlijk ook frustrerende en te kleine stapjes, of stapjes terug. Zelfs stapjes in de hele an-
dere richting. Onderweg ben ik veel mensen tegengekomen die me hebben gestimuleerd en
geholpen om te komen waar ik nu ben. Ook de mensen die voor welkome afleiding hebben
gezorgd of de humor kende van de frustratie zijn erg het bedanken waard.
In de eerste plaats: het onderzoek in dit proefschrift had nooit kunnen plaatsvinden zonder
de (net bevallen) moeders, vaders en hun kinderen die meededen aan het onderzoek. Heel
hartelijk dank.
Beste Berent,
Jij gunt je promovendi het maken van eigen stappen. Stappen out of the box, daardoor ont-
stond bijvoorbeeld het stuk over hsp in cordblood. Je luistert met een glimlach, je stuurt onopvallend en bovenal weet je altijd weer het goede gevoel boven te halen, hoe diep de dip ook
lijkt. Erg veel dank voor je aanhoudende vertrouwen en de bijzondere gesprekken.
Beste Femke,
De eerste stapjes zette ik zonder jou, ik kwam overal. Dankzij jou kreeg mijn onderzoek nog
duidelijker focus. Je geduld, je oordeelloos vermogen om telkens weer opnieuw in te gaan op
die vraag die ik toch nog net niet begreep. Inhoudelijke discussies en leuke gesprekken. Ik
vind je een bijzonder kundige onderzoeker en ben erg blij dat jij mijn co-promotor bent.
Beste Edward,
Een bijzondere verlengde stap. Ik mag promoveren bij jou, nadat mijn vader eerder bij jouw
promotieonderzoek betrokken was. Dank dat jij steeds meer betrokken bent geraakt bij mijn
onderzoek. Jouw enthousiasme is aanstekelijk. Dank voor al je tijd en inzet om mij te helpen
de promotie af te ronden. Het is gelukt!
Beste Maarten,
Zonder jou had ik de eerste stappen niet kunnen maken. We kenden elkaar al vanaf het AMC
waar ik als student meedeed aan een ander mooi allergie onderzoek van Trinette Steenhuis.
Ik ben je stappen gevolgd naar het WKZ waar ik mee mocht denken over allergie onderzoek
en heat shock eiwitten. Met je vele interesses op vele terreinen en mijn wat langere traject
sluiten we het alleen officieus samen af. Dank voor de kans die je mij gunde om dit onderzoek
op te zetten.
Beste Erica,
Wat een bijzondere secretaresse ben jij. Niet mijn secretaresse natuurlijk, maar zo liet je het
soms wel lijken. Je was er altijd. Je had altijd tijd, terwijl je het enorm druk had. Zelfs pro-ac138
tief maakte je voor mij afspraken met Berent. Je bent bijzonder in je betrokkenheid. Stijn en
Dankwoord & Curriculum Vitea
Roos vonden de ballon prachtig. Dank ook aan Heleen, Angela en Mirjam in mijn weg naar de
verdediging van mijn proefschrift.
Lieve Marloes en Yasser, lieve paranimfen
Marloes als vriendin en ex-roomie, Yasser als vriend en ex-buur, dank dat jullie mijn paranimfen willen zijn. Het geeft rust dat jullie daar straks in het zweetkamertje zitten en mij willen
vergezellen met ijsberende stappen, met jullie heerlijke grappen, net niet onder de gordel.
Dank voor jullie steun en voor het al een paar jaar vrijhouden van de agenda voor deze verdediging.
Beste kamergenoten,
De stappen tijdens een promotietraject, de frustraties en de pret, worden het best begrepen
door mede onderzoekers. Dank aan mijn lieve kamergenoten, arash, joost s, korneel, lianne,
maja, marieke, marije, marloes, nan en titia. Ik denk dan meteen aan fietsen, mooie excel programma’s, “zeg nee tegen ….”, het altijd veranderlijke weer, het dubbele proefschrift, de trap
onder de kont, de tranen van commerciele belangen, paranymfen, Lelystad, Rome, liedjes, en
lief gemene opmerkingen, maar vooral de altijd aanwezige lach en steun, door dik en dun.
Heel veel dank!
Binnenstappen in een lab is als arts niet vanzelfsprekend. De genen had ik mee, ietsepiets er-
varing, maar meer ook niet. Mark maakte het mogelijk. Moest er nou 1 of 2 ml bij. Mark weet
het. En ook bij de tiende keer legde Mark het weer heel rustig uit alsof je het voor de eerste
keer vroeg. Het lab was gebouwd op jou als steunpilaar, en daar heb ik veel aan gehad. Dank
voor de tent-lessen in het Eiffel gebied! Dank Wilco en Jenny voor jullie hulp bij de expirementen en het meten van de cytokines. Zo kwamen de publicaties dan echt.
En dan de andere prakkers en WKZ’ers: in de eerste plaats Berber. Gezamenlijke frustraties,
er tegen op boksen. We konden er wel om lachen. Tja en natuurlijk de eindeloze soap met elke
keer weer onverwachte nieuwe wendingen. Hopelijk heb je nu wat meer rust en kan je met
zijn tweeen aan de weg timmeren! Alma, Alvin, Annemarie, Annick, Astrid, Arjan, Bas, Ellen,
Eva, Evelien, Evelien, Henk, Huib, Isme, Joris, Lieke, Lise, Pleun, Ruud, Sanne, Sarah, Selma,
Sylvia, Theo en Yvonne, en erg belangrijk Sytze! Jasr, stoelendans, koffieautomaat, lunchstap-
jes door het natuurgebied achter het WKZ (inspiratie voor de omslag van dit proefschrift),
serieuze gesprekken, ezelsbruggetjes van de spar en de den. Je leert veel tijdens een promotie-traject.
En dan de stappen in de kelder van het WKZ, het militair hospitaal naar de dagbehandeling
bij Pauw, en verder door naar de operatiekamers. Dank nogmaals aan alle kinderen en ouders
die mee wilden doen aan het onderzoek, zowel de kinderen verdacht van pinda allergie, als
de kinderen die juist geen allergie hadden en bloed afstonden voor de controle samples. Daar-
naast veel dank aan de specialisten die de logistiek mogelijk maakten: Maarten Hoekstra en
139
Chapter 9
Yolanda Meijer, en ook de anesthesisten op de OK, en zeker ook alle artsen en verpleegkundigen werkzaam op Pauw, die zorg droegen voor de bloedafnames. Niet in de laatste plaats dank
aan Petra Kentie. Het onderzoek begon zonder jou, maar floreerde dankzij jou.
Een groot deel van dit onderzoek vond ook plaats in Amsterdam, met het AMC, maar voorna-
melijk op het CLB, op het lab van Rob. Het is bijzonder om zo welkom te zijn op een plek waar
ik mijn hele leven om uiteenlopende reden elke keer weer mijn stappen zet. Het monnikenwerk van het pinda pellen hebben de meeste indruk gemaakt. Veel dank aan Tjitske, Ninotska,
Astrid, Jolanda, Ellen, Ingrid, Kim, Theo, Pleun, Theresa, Henk, Meriam, Tom, Marianne, Janny,
Ronald, Bouke, Fatima, Kaoutar, Laurian, Jaap, Erica, Ed, Anders, Steven en Rob.
Natuurlijk zijn de Coffers, de 3e verdieping en de diergeneeskunde erg belangrijk geweest
voor het meedenken, meedoen en om tegen te voetballen, vooral dank aan Jorg, Jeffrey, Paul,
Nathalie, Vanessa, Ger A, en Ger R, Koos, Pirrko en Willem.
Ik heb tijdens die tien jaar ook nog stappen buiten gemaakt, alhoewel de vrienden soms het
onderspit dolven omdat ik weer dacht dat ik de promotie nu echt ging afronden… Dank aan
de posse Aswin, Bas, Daan, Edin, Guido, Tom, Tim de V, dank Arjen en Tim L, dank TP’ers voor
het elke keer opnieuw vragen of ik dan toch mee ga stappen in de bergen… Yasser, Andreas,
Bas, en ook dank Jessie, natuurlijk Fonnet voor onze onderzoeksweddenschap, Jenneke, Niels
voor het delen van onderzoeksperikelen en Marieke en Arash voor de fietstocht naar Rome.
Andere stappen heb ik gemaakt in Haarlem bij de kindergeneeskunde met de gezellige ver-
pleging, leuke kinderartsen en aniossen, met de aiossen, docenten en opleiders bij de huis-
artsenopleiding, in Oosterblokker en Osdorp, en nu verder in onder andere Amsterdam en
Hoorn. Speciale dank aan Nynke, Annemarie en Monique voor de aanhoudende steun en kritische noot.
A special thanks to the Kagens and to Tom Platts-Mills. The Kagens, Steffi, Tommy, Mike, Missy,
Gayle and Steve have made me feel at home many steps away from Amsterdam. The round
table discussions with Marv, Steve, Charlie and Mike have contributed to my steps to become
a doctor and a researcher. I have fond memories of the lab with Mutthiah. Also special thanks
to Tom and his lab and the doctors in Charlottesville who have shown how serious research
can be combined with fun in the lab. I have fond memories of the mountain walks to clear the
mind. With the latest walk during the WIRM in over three feet high snow. Big steps!
Lieve familie en schoonfamilie, Pauline, Rob, Suzanne, Jeroen, Pauline en Floor, Tanneke, Lodewijk, Fenna, Arthur, Steven en Taeke, Laurien, Marius en Willem, Noor, Paul, Lucas en Philine. Al-
tijd vroegen jullie lief naar mijn promotie, hoe het ging, zonder irritant te zijn. Altijd geinteres-
seerd, elke keer weer luisteren en doorvragen. Dankzij jullie bleef elke stap toch weer bijzonder.
Beste Tonny dank voor je kritische blik en je inspanningen om het proefschrift te begrijpen.
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Lieve Rob, dank je wel voor jouw meerdere rollen in dit onderzoek. Je liet me mijn eigen stap-
pen maken, je liet me zwemmen, maar zorgde er ongemerkt voor dat ik niet verdronk. Je was
er op de achtergrond, altijd aanwezig voor de discussie, altijd met heel veel geduld. Jij bent
vaak al vele stappen verder, maar kan ook weer zien waar ik nog sta. Ik ben er ontzettend trots
op dat wij samen een gedeelte van dit onderzoek hebben mogen doen met prachtige artikelen
als resultaat. Lieve Pauline, dank je wel voor de ruimte die jij schiep voor de tafeldiscussies
met Rob over onze pinda-stukken. Veel dank voor je lieve mailtjes met vrolijke noot en je
steun. Heel fijn.
Lieve Stijn en Roos, jullie stapjes zijn de mooiste. Het is een groot geluk. Lieve Maaike, dank
voor alle liefde en steun, het volhouden en relativeren. Met jou is dit boekje gelukt.
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Joost Aalberse werd geboren op 22 januari 1977 te Amsterdam. In 1986 heeft hij in Charlottesville,
Virginia gewoond. Hij is getrouwd met Maaike van Dijk. Zij hebben samen twee kinderen, Stijn en Roos.
In 1996 behaalde hij het VWO diploma aan het Fons Vitae lyceum te Amsterdam. Datzelfde jaar
begon hij met de studie Geneeskunde aan de Universiteit van Amsterdam. Tijdens de opleiding
heeft hij een extracurriculair vak gevolgd bij de faculteit Geesteswetenschappen: “Introductie
tot de filosofie”. Daarnaast heeft hij verschillende cursussen gevolgd waaronder Muziekgeschiedenis bij CREA, Expirementele Oncologie aan het NKI en Medische Ethiek aan de VU.
Als student assistent was hij betrokken bij een onderzoek over obstipatie bij kinderen onder supervisie van Dr. R. van Ginkel en Dr. M. Benninga. Daarna was hij een aantal jaar betrokken bij
onderzoek van Trinette Steenhuis en Dr. M. Hoekstra, waarbij onderzoek werd gedaan naar een
mogelijk vaccin tegen allergie.
Hij heeft tijdens zijn studie onderzoek gedaan in Charlottesville, Virginia, terug op de plek waar
hij eerder naar de basisschool ging. Dit onderzoek richtte zich op IgG anti-lichamen tegen tetanus toxoid, zowel in moederlijk- als in navelstrengbloed. Het onderzoek vond plaats bij Prof. Dr.
T. Platts-Mills , waarbij in het AMC ook Prof. Dr. J. van der Zee betrokken was. Vervolgens werd
dat onderzoek voortgezet in het CLB (nu Sanquin), te Amsterdam bij Dr. S. Stapel en Prof. Dr.
R. Aalberse. Op dat lab heeft hij ook onderzoek gedaan naar kruisreactiviteit tussen hond en
koe-allergenen, en naar de rol van IgG4 bij kinderen met een allergie voor katten. Bij de presen-
tatie van enkele van deze resultaten op een summerschool in Cagliari, Italie, werd hij uitgeno-
digd door Prof. Dr. S. Bonini om een studenten-congres voor allergie te initiëren in Europa voor
Europese studenten. Dit heeft geresulteerd in het Chiron Project, welke hij vier jaar lang organi-
seerde samen met Claudio d’Ambrosio, met congressen in Napels, Berlijn, Parijs en Amsterdam.
Na het behalen van het doctoraal examen in 2002 heeft hij onderzoek gedaan in Australie bij Prof. Dr. D.
Lehmann en Dr. C. Jeffries. Dit onderzoek richtte zich op de oorzaken van middenoorontsteking bij Aboriginals. Deze tijd heeft hem ook veel kennis gegeven over de geschiedenis en cultuur van Aboriginals.
Hierna is hij gestart met zijn coschappen, met onderzoek naar Henoch Schonlein purpura bij
Prof. Dr. J. Davin van de afdeling kinder-nierziekten. In 2005 heeft hij zijn coschappen afgerond.
Direct aansluitend is hij gestart met het promotieonderzoek. Het onderzoek is aanvankelijk gestart onder supervisie van Prof. Dr. B. Prakken en Dr. M. Hoekstra. Vlot daarna is ook Dr. F. van
Wijk copromotor geworden bij de studie. Vanwege meerdere interesses van Dr. M. Hoekstra en
werkzaamheden elders, is Dr. E. Knol halverwege het traject copromotor geworden in plaats van
Dr. M. Hoekstra. Het promotieonderzoek heeft geleid tot dit proefschrift.
Tijdens de promotie heeft hij lesgegeven en als vrijwilliger gewerkt op een huisartsenpost voor
onverzekerden. Sinds 2014 werkt hij op verschillende plekken in Noord Holland als huisarts.
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List of publications
Molecular allergen-specific IgE assays as a complement to allergen extract based sensitization
assessment.
Aalberse RC, Aalberse JA. J Allergy Clin Immunol Practice 2015 accepted for publication A lesson from component-resolved testing: we need better extracts.
Aalberse JA, Aalberse RC. J Allergy Clin Immunol Pract. 2014 Sep-Oct;2(5):635-6
Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut.
Aalberse JA. et al. Clin Transl Allergy. 2013 Dec 2;3(1):40
Moving from peanut extract to peanut components: towards validation of component-resolved
IgE tests.
Aalberse JA, et al. Allergy. 2013 Jun;68(6):748-56
Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis.
Kapitein B, Aalberse JA, et al. Cell Stress Chaperones. 2013 Jan;18(1):87-95.
Association between early bacterial carriage and otitis media in Aboriginal and non-Aboriginal children in a semi-arid area of Western Australia: a cohort study.
Sun W. et al. BMC Infect Dis. 2012 Dec 21;12:366.
HSP: Bystander Antigen in Atopic Diseases?
Aalberse JA. et al. Front Immunol. 2012 May 31;3:139
Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60).
Aalberse JA. et al. PLoS One. 2011;6(9):e24119
The interaction between respiratory viruses and pathogenic bacteria in the upper respiratory tract of asymptomatic Aboriginal and non-Aboriginal children.
Moore HC, et al. Pediatr Infect Dis J. 2010 Jun;29(6):540-5.
Absent otoacoustic emissions predict otitis media in young Aboriginal children: a birth cohort study in Aboriginal and non-Aboriginal children in an arid zone of Western Australia.
Lehmann D. et al. BMC Pediatr. 2008 Aug 28;8:32
Henoch Schonlein purpura in children: an epidemiological study among Dutch paediatricians on incidence and diagnostic criteria.
Aalberse J. et al. Ann Rheum Dis. 2007 Dec;66(12):1648-50.
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