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Transcript
Petra Amoudruz
Maternal immune characteristics
and innate immune responses in the
child in relation to allergic disease
Petra Amoudruz
Maternal immune characteristics and innate immune
responses in the child in relation to allergic disease
ISBN 978-91-7155-655-4
The Wenner-Gren Institute
Doctoral Thesis in Immunology at Stockholm University, Sweden 2008
Doctoral thesis from the Department of Immunology, the Wenner-Gren Institute,
Stockholm University, Stockholm, Sweden
Maternal immune characteristics and innate
immune responses in the child in relation
to allergic disease
Petra Amoudruz
Stockholm 2008
All previously published papers were reproduced with permission from the publishers
© Petra Amoudruz, Stockholm 2008
ISBN 978-91-7155-655-4
Cover illustration: Sculpture made by my father Bertil Arlert
Printed in Sweden by Universitetsservice AB, Stockholm 2008
Distributor: Stockholm University Library
The most exciting phrase to hear in science, the one that heralds the most discoveries, is not
"Eureka!" (I found it!) but "That's funny... "
Isaac Asimov (1920-1992)
To Ebba and Ines
SUMMARY
The mechanistic factors responsible for the increase in allergic diseases are still not fully
understood, but a reduced microbial stimulation seems to be one of the key issues. Research is now
aiming at investigating the relationship between the innate immune system, involving the toll-like
receptors, and allergy development. Further, the maternal influence on the child, possibly through in
utero effects, but also through the breast milk, has shown to be of great importance. This thesis aimed
at understanding how the maternal immune system is influenced by early exposures and allergic
disease, but also to investigate the consequences of the maternal phenotype on the innate immune
system of the developing child.
The Th1/Th2 cytokine pattern in allergic diseases has been extensively studied. Here we were
interested in comparing the innate cytokines in allergic and non-allergic women, and to see if the
allergic status was influencing the effect of pregnancy differently. We demonstrate that IL-1β, IL-6,
IL-10 and IL-12 production in cells from adult women are not influenced by allergic status, neither
during pregnancy nor 2 years after. However, pregnancy had an apparent effect on cytokine levels,
regardless of allergic status. Also, total IgE levels in allergic women were significantly lower 2 years
after pregnancy in comparison with the levels during pregnancy, pointing to the fact that pregnancy
indeed has an immunomodulatory role.
We further wanted to investigate the immune system of mothers who had migrated to Sweden in
comparison with indigenous mothers. The reason for our interest here was that children born from
immigrated mothers have shown to have an increased risk of developing diseases such as allergy and
Crohn’s disease. The results showed that immigrants from a developing country had significantly
higher levels of breast milk IL-6, IL-8 and TGF-β1. Further, regardless of maternal country of birth, a
larger number of previous pregnancies was associated with down-regulation of several substances,
statistically significant for soluble CD14 and IL-8. The results suggest that maternal country of birth
may indeed influence adult immune characteristics, potentially relevant to disease risk in offspring.
The influence of allergic status of the mother on the expression of CD14, TLR2 and TLR4 was
further investigated in monocytes from mothers and their newborn babies upon microbial stimulation.
We could not find any differences in monocytic TLR levels between the groups. No significant
differences regarding cytokine levels between allergic and non-allergic mothers in response to stimuli
were found either. However, the cytokine and chemokine release triggered by TLR2 stimulation in CB
revealed that CBMC from children with maternal allergic disease released significantly less IL-6, and a
trend towards less IL-8.
As we could not find differences in TLR levels attributed to maternal allergy, but an impaired
IL-6 response, we turned our focus on an intracellular event taking place after TLR ligation. The
results confirmed our results of decreased IL-6 levels in CB from children to allergic mothers. At 2
years of age, the children of allergic mothers still displayed a diminished IL-6 response. Additionally,
they also had a decreased activity of p38 MAPK. p38 has an important role in driving Th1 responses,
suggesting that the p38 pathway could be one of the responsible mechanisms behind the impaired
responses correlated to allergic heredity found in CB as well as at 2 years of age.
Infancy is a crucial time period for the developing immune system. Further, the relative
composition of the two major monocytic subsets CD14++CD16- and CD14+CD16+ is altered in some
allergic diseases. TLR levels are different in the two subsets, proposing a possible link to the reduced
responding capacity of monocytes from children with allergic heredity. We followed up our earlier
studies of children at birth and at 2 years of age by looking at 5 year old children. There were no
differences regarding monocytic subsets, nor in TLR levels in unstimulated cells. However, when
stimulating the cells with PGN, both monocytic subsets in allergic subjects were less capable of
upregulating TLR2 compared to the age-matched controls.
Taken together, the work in this thesis suggests that the maternal immune system is affected by
the process of pregnancy and childhood exposures. It further suggests that maternal allergy affects the
young child, in terms of impaired responses to microbial stimuli, which later in infancy correlates with
allergic disease in the child. These impaired innate responses could lead to a diminished Th1 response,
or alternatively to a deficiency in regulatory mechanisms, and thereby cause allergic disease.
ARTICLES
This thesis is based on the following original articles, which will be referred to by their
Roman numerals:
І
Amoudruz P, Minang T J, Sundström Y, Nilsson C, Lilja G, Troye-Blomberg
M, Sverremark-Ekström E. Pregnancy, but not the allergic status, influences
spontaneous and induced IL-1β, IL-6, IL-10 and IL-12 responses. Immunology
2006 Sep;119(1):18-26.
ІІ
Amoudruz P, Holmlund U, Schollin J, Sverremark-Ekström E, Montgomery S
M. Maternal country of birth and previous pregnancies are associated with breast
milk characteristics. Pediatr Allergy Immunol 2008. In press.
III
Amoudruz P*, Holmlund U*, Malmström V, Trollmo C, Bremme K, Scheynius
A, Sverremark-Ekström E. Neonatal immune responses to microbial stimuli: is
there an influence of maternal allergy? J Allergy Clin Immunol 2005
Jun;115(6):1304-10. *These authors contributed equally to the work.
IV
Saghafian-Hedengren S, Holmlund U, Amoudruz P, Nilsson C, SverremarkEkström E. Maternal allergy influences p38-mitogen-activated protein kinase
activity upon microbial challenge in CD14(+) monocytes from 2-year-old
children. Clin Exp Allergy 2008 Mar;38(3):449-57.
V
Amoudruz P, Holmlund U, Saghafian-Hedengren S, Nilsson C, SverremarkEkström E. Impaired TLR2 signaling in monocytes from 5 year old allergic
children. Submitted 2008.
TABLE OF CONTENTS
INTRODUCTION............................................................................................................. 9
INNATE AND ADAPTIVE IMMUNITY ..................................................................... 9
PATTERN RECOGNITION ........................................................................................ 11
Toll-like receptors................................................................................................. 12
Intracellular signaling .......................................................................................... 13
Lipopolysaccharide recognition............................................................................ 14
Peptidoglycan recognition .................................................................................... 16
IMMUNE CELLS AND MEDIATORS....................................................................... 17
Monocytes/macrophages....................................................................................... 17
Dendritic cells ....................................................................................................... 18
NK cells ................................................................................................................ 19
Mast cells .............................................................................................................. 20
Granulocytes ......................................................................................................... 20
T cells.................................................................................................................... 21
B cells.................................................................................................................... 22
Cytokines and chemokines ................................................................................... 23
Type 1/type 2 responses ........................................................................................ 25
TNF ....................................................................................................................... 26
IL-1β...................................................................................................................... 27
IL-6........................................................................................................................ 27
IL-10...................................................................................................................... 28
IL-12...................................................................................................................... 29
TGF-β.................................................................................................................... 30
IL-8........................................................................................................................ 31
ALLERGY.................................................................................................................... 32
The allergic reaction ............................................................................................. 32
The hygiene hypothesis and the role of innate immunity ..................................... 34
Monocytes/macrophages and disease ................................................................... 36
Pattern-recognition receptors and disease............................................................. 37
Gut......................................................................................................................... 38
Gene-environment interactions............................................................................. 39
Epigenetics............................................................................................................ 40
Microbial recognition for therapeutic interventions ............................................. 40
IMMUNOLOGY OF PREGNANCY........................................................................... 42
Maternal influences on the child........................................................................... 43
In utero.................................................................................................................. 43
Breastmilk ............................................................................................................. 45
The sibling effect ................................................................................................... 47
THE PRESENT STUDY ................................................................................................ 48
AIMS............................................................................................................................. 48
METHODOLOGY ....................................................................................................... 50
RESULTS AND DISCUSSION ................................................................................... 51
Impact of pregnancy and allergic status on cytokine responses (І) ...................... 51
Maternal exposures and breast milk characteristics (II) ....................................... 53
Neonatal immune responses to microbial stimuli (ІII) ......................................... 56
Maternal allergy and monocyte signaling in 2-year-old children (IV) ................. 59
TLR2 signaling in 5 year old allergic children (V)............................................... 61
CONCLUDING REMARKS AND FUTURE PERSPECTIVES ............................... 65
ACKNOWLEDGEMENTS ........................................................................................... 67
REFERENCES................................................................................................................ 70
List of Abbreviations
APC
Antigen-presenting cell
MHC
Major histocompatibility complex
ASM
Airway smooth muscle cells
MyD88
Myeloid differentiation factor 88
BCR
B-cell receptor
NBS
Nucleotide-binding site
CB
Cord blood
NF-κB
Nuclear factor kappa –B
CBA
Cytometric bead array
NK
Natural killer
CBMC
Cord blood mononuclear cells
NKT
Natural killer T cell
CMV
Cytomegalovirus
NOD
Nucleotide-binding oligomerization
CTL
Cytotoxic T cells
DC
Dendritic cell
OVA
Ovalbumin
EBV
Epstein-Barr virus
PAMP
Pathogen-associated
EDN
Eosinophil-derived neurotoxin
ERK
Extracellular signal regulated kinase
PBMC
Peripheral blood mononuclear cells
gp
Glycoprotein
pDC
Plasmacytoid dendritic cell
GM-CSF
Granulocyte-macrophage colony-stimulating
PGN
Peptidoglycan
factor
PIN
Peptidyl-prolyl isomerase
GVHD
Graft-vs-host disease
PRR
Pattern-recognition receptor
HAV
Hepatitis A virus
sCD14
Soluble CD14
HMGB1
High-mobility group box 1
sIgA
Secretory IgA
IEC
Intestinal epithelial cells
sIL-6R
Soluble IL-6 receptor
IBD
Inflammatory bowel disease
SNP
Single-nucleotide polymorphisms
IFN
Interferon
SOCS
Suppressor of cytokine signaling
Ig
Immunoglobulin
SPT
Skin prick test
IL
Interleukin
STAT
Signal transducer and activator of
IL-1R
Interleukin-1 receptor
IL-12R
IL-12 receptor
TCR
T-cell receptor
JAK
Janus kinase
TGF
Transforming growth factor
JNK
c-Jun N-terminal kinases
Th
T helper
LBS
LPS-binding protein
TIM
T cell, Ig- and mucin domain
LPS
Lipopolysaccharide
TIR
Toll/IL-1R
LRR
Leucine-rich-repeat
TLR
Toll-like receptor
LTA
Lipoteichoic acid
TNF
Tumor necrosis factor
MAL
MyD88 adaptor-like
TRAM
Toll-receptor associated molecule
MAMP
Microorganism-associated molecular pattern
Treg
Regulatory T cell
MAPK
Mitogen-activated protein kinase
Trif
Toll-receptor associated activator of
mDC
Myeloid dendritic cell
domain
molecular
pattern
transcription
interferon
Innate and adaptive immunity
INTRODUCTION
INNATE AND ADAPTIVE IMMUNITY
The immune system of vertebrates consists of two interrelated components, the
innate and adaptive responses, which are jointly required for the resolution of most
infections. The innate immune response is the first line of host defense, and is
responsible for an immediate recognition and control of microbial invasion. It is
comprised mainly of phagocytic cells, such as macrophages and neutrophils, which
can ingest and kill the invading pathogens. The effector phase of innate immunity is
the process of inflammation, where the immediate response to a pathogen gives rise
to a reaction characterized by the migration of cell types with defensive functions.
Further consequences are alterations in vascular permeability and the secretion of
soluble mediators such as cytokines and chemokines, leading ultimately to the
initiation of the adaptive arm.
The specificity and memory of the adaptive immune response are mediated by
T- and B cells. The specificity is generated through genetic rearrangements and
selection of receptors that best recognize the specific antigens. Through the history of
immunology, scientists have had difficulties integrating the innate and the adaptive
systems, but the discovery of toll-like receptors (TLRs) [1] was a huge step towards
bridging the two systems together. The fact that innate immune responses not only
provided a first line of defense, but also were critical for setting the adaptive immune
system into action, changed the former view of the innate immune system as being
primitive and unspecific. Multiple studies have now revealed that it is indeed specific
in the sense that different stimuli give different signals to the adaptive immune
system, thereby “deciding” how the most effective response should be designed to
combat a specific intruder [2]. One of the best illustrations of the now established
concept that innate and adaptive immunity are not completely independent entities
comes from studies showing that optimal T-cell responses require help from mast
cells, cells belonging to the innate immune system [3].
A recent finding re-challenged the view of immunological memory of the
adaptive immune system. It showed that the memory upon a secondary infection not
only depends on memory cells of the adaptive immune system, but also on parts of
the innate immune system [4].
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Petra Amoudruzvvvv
Moreover, it was recently shown that not only does adaptive immunity have a
role in combating infections, but it is also important for dampening the strong
inflammatory reactions that the innate immune system gives rise to upon infection.
Thus, mice unable to mount an adaptive immune response died rapidly after infection.
Unexpectedly, the mice were shown not to die of unchecked microbial infection, but
from damage caused by uncontrolled inflammatory cytokines released by the innate
immune system [5].
10
Pattern recognition
PATTERN RECOGNITION
The innate immune response relies on evolutionarily ancient germline-encoded
receptors, the pattern-recognition receptors (PRRs), which recognize highly
conserved microbial structures, traditionally known as pathogen-associated molecular
patterns (PAMPs). These microbial patterns are not uniquely found in pathogenic
organisms, but also in nonpathogenic commensal microorganisms. This type of
recognition enables the host to quickly identify and respond to a broad range of
pathogens. The most studied PRRs are the TLRs. However, PAMP-PRR interactions
are not restricted to TLRs. There are several other PPRs functioning in a similar
manner in that they also recognize microbial components, although they are located
in the cytosol rather than at the cell surface or in vesicles. Nucleotide-binding site and
leucine-rich repeat (NBS-LRR) proteins are examples of such receptors where the
nucleotide-binding oligomerization domain (NOD) is one of the most studied [6].
Examples of microbial ligands recognized by pattern-recognition molecules include
lipopolysaccharide (LPS), lipoteichoic acid, flagellin, mannans, nonmethylated CpG
sequences and peptidoglycan (PGN). There is further increasing evidence that TLRs
also recognize host-derived ligands from the damage or death of host cells [7]. The
recognized compounds are known as damage-associated molecular-pattern molecules,
where the high-mobility group box 1 (HMGB1) [8] and heat shock proteins [9]
belong to those that are the best studied.
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Toll-like receptors
Toll-like receptors is the best characterized class of PRRs, and today 13
mammalian TLRs are known (Table 1), of which TLR2 and TLR4 are two of the
most studied [10].
What the Toll-Like Receptors See
TLR
Natural Ligand
TLR1 (partnered with Bacterial triacyl lipopeptides and certain proteins in
TLR2)
parasites
TLR2 (partnered with Bacterial diacyl lipopeptides, lipoteichoic acid from
TLF6)
Gram-positive bacteria, and zymosan from the cell
wall of yeast
TLR3
Double-stranded RNA from viruses (e.g., West Nile
virus)
TLR4
Endotoxin (lipopolysaccaride) from Gram-negative
bacteria
TLR5
Flagellin from mobile bacteria
TLR7
Single-stranded RNA from viruses (e.g., HIV)
TLR8 (inactive in mice)
Same as TLR7
TLR9
CpG DNA from bacteria or viruses
TLR10
(found
in Unknown
humans but not mice)
TLR11 (found in mice;
human form is truncated
and thought to be
inactive)
Profilin, a protein from the protozoan pathogen
Toxoplasmosis gondii that can cause miscarriage; may
also respond to components of bacteria that cause
bladder and kidney infections
TLR12 and TLR13 Unknown
(found in mice but not
humans)
Table 1. The known exogenous binding partners for each TLR (Science 2006; 312:184187). Reprinted with permission from AAAS.
TLRs are membrane glycoproteins characterized by the extracellular domains,
containing varying numbers of LRR motifs, and a cytoplasmic signaling domain
homologous to that of the interleukin 1 receptor (IL-1R), termed the Toll/IL-1R (TIR)
domain. TLRs may be expressed extra- or intracellularly. While some TLRs (TLRs 1,
12
Pattern recognition
2, 4, 5, and 6) are expressed on the cell surface, others (TLRs 3, 7, 8, and 9) are found
almost exclusively in intracellular compartments such as endosomes. TLRs are
important for dendritic cell (DC) maturation and function. Additionally, it has been
shown that generation of T-cell dependent antigen-specific antibody responses also
requires activation of TLRs in B cells [11]. However, later studies suggest that TLR
signaling in B cells amplifies, but are not required for antibody production or
maintenance of memory [12]. Microorganisms, via their PAMPs, may also contribute
directly to the perpetuation and activation of long term T-cell memory as T cells also
express TLRs [13]. In addition to ligand specificity, the functions of individual TLRs
differ in their expression patterns and the signal transduction pathways they activate.
Taken together, an increasing body of evidence emphasize the important crosstalk between different TLRs for enabling an optimal and secure response to stimuli
[6;14;15]. TLR activation is essential for the ability of the immune system to combat
invading pathogens, however a strict regulation is of major importance, as lack of
inhibition can lead to detrimental and inappropriate inflammatory responses. TLR
signaling has been shown to affect several disorders, including immunodeficiencies,
autoimmune- and allergic diseases [16;17].
Intracellular signaling
As different responses are needed for the elimination of different microbes,
TLRs operate in concert with several adaptor molecules to acquire maximum
sensitivity and specificity. The most studied adaptor proteins, shown to transduce
signals via the intracellular TIR domains of the different TLRs, are Myeloid
differentiation factor 88 (MyD88), MyD88 adaptor-like (MAL), Toll receptorassociated activator of interferon (Trif) and Toll receptor-associated molecule
(TRAM). Most TLRs activate the MyD88-dependent pathway, leading to activation
of mitogen-activated protein kinases (MAPKs) and nuclear factor -kappa B (NF-κB),
a transcription factor induced within minutes after microbial challenge. NF-κB plays
a critical role in the coordination of both innate and adaptive immune responses by
regulating the gene expression of many cellular mediators [18].
MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun Nterminal kinases (JNK) and p38 regulate the activities of several transcription factors.
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By altering the levels and activities of transcription factors, MAPKs influence
transcription of genes that are important for cytokine production and involved in the
cell cycle. MAPKs play different roles in IL-10 and IL-12 production [19]. A
regulatory DC recently described, diffDC, have an impaired IL-12p70 production and
enhanced IL-10 production compared to immature DCs. The low levels of IL-12p70
were dependent upon a suppressed p38 pathway, while the high levels of IL-10 were
caused by an increased ERK activation [20]. The results from that study highlight the
important role of MAPKs in directing different immune responses towards a more
Th1, Th2 or regulatory response. MAPKs, especially p38, can enhance the expression
of pro-inflammatory cytokines at the transcriptional level, but are also able to act on
the post-transcriptional level. p38 acts on cytokine levels after transcription through
mechanisms that enhance the stability and the translation of the cytokine mRNA [19].
Airway smooth muscle cells (ASM) play an important role in both
hyperreactivity and remodeling in asthmatic patients. A study by Shan et al [21]
showed that the different MAPKs can affect each other. If the ASM cells were
pretreated with inhibitors of ERK1/2 signaling, the induced NF-κB activity and
changes in ASM responsiveness in response to LPS were dampened, whereas
inhibition of p38-MAPK augmented the proasthmatic responses to LPS. Their data
demonstrate that p38 can have an important regulatory function, by acting on the
ERK1/2 pathway after TLR4 stimulation [21]. The role of MAPK in inflammation
makes them attractive targets for new therapies, when efforts are being made to
identify newer, more selective, inhibitors for inflammatory diseases [22].
Lipopolysaccharide recognition
Host mechanisms that recognize gram-negative bacterial LPS are among the
most sensitive and best studied. All gram-negative bacteria express the glycolipid
component called LPS or endotoxin at their surface. There is evidence to suggest that
there are structural and functional differences between LPS molecules originating
from different bacterial species [23]. Myeloid cell activation by LPS involves the
signaling receptor complex MD-2/TLR4 receptor, which receives LPS from CD14; a
membrane bound receptor anchored by a glycerophosphatidylinositol tail. The plasma
LPS-binding protein (LBP) catalytically transfers single LPS molecules from LPS
14
Pattern recognition
aggregates onto CD14 (Figure 1). Upon binding of the ligand, the TLR4 signaling
consists of two different pathways; the MyD88-dependent and the MyD88–
independent [24].
MD-2 is a small secreted glycoprotein that confers LPS responsiveness to
TLR4. MD-2 associates with TLR4 in the endoplasmic reticulum, and is necessary
for translocation of TLR4 from Golgi to the surface [25]. After LPS has been
recognized by the receptor-mediated mechanism, it is endocytosed and transported
into Golgi-like structures together with TLR4 [26]. After signaling has taken place
the TLR4 is trafficked to lysosomes where it is degraded. This endosomal trafficking
of the LPS receptor complex is essential for antigen presentation to Th cells as well as
for termination of the signaling, thus controlling both the innate and the adaptive
immune system [27].
Figure 1. Cell signaling in response to LPS (Nature 2002; 420:885-891). Reprinted by
permission from Nature copyright Macmillan Publishers Ltd.
Another form of CD14, without the lipid tail, circulates as a soluble plasma
protein; soluble CD14 (sCD14). sCD14 participates in cell activation by transferring
LPS to CD14 on the cell membrane, or by transferring the LPS directly to the MD2/TLR4 receptor complex on cells that do not express CD14 on the cell membrane,
such as endothelial and epithelial cells [28]. LBP and CD14, which play key roles in
enabling sensitive responses to LPS can also have inhibitory activities that help to
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control LPS responses by limiting LPS interactions with MD-2/TLR4 [29]. sCD14 is
highly expressed in breast milk and has an important role for the neonatal intestine, as
it enables gut epithelial cells to respond to microbial stimuli in terms of LPS [30], a
stimulation believed to be important for postnatal gut tolerance and homeostasis [31].
Excessive stimulation of monocytes and macrophages by LPS leads to
endotoxin shock, a systemic disorder with a high mortality rate in humans. Studies
have shown that pre-exposure to LPS reduces sensitivity to a second exposure to LPS,
a phenomenon known as LPS tolerance or LPS hyporesponsiveness. This tolerance is
not fully understood, but it has been suggested that the decreased TLR4 expression
after LPS stimulation could be one of the underlying mechanisms [32].
Peptidoglycan recognition
As a major constituent of the cell wall of virtually all bacteria, and absent from
eukaryotes, PGN represents an excellent target for innate immune recognition. PGN
is highly abundant in gram-positive bacteria. In gram-negative bacteria the thin PGN
layer is found underneath the LPS containing outer membrane [33]. Extracellular
recognition of PGN is mediated by membrane-bound CD14 and TLR2, while the
intracellular recognition is mediated by NOD1 and 2. There are also soluble PGN
recognition molecules involved, such as sCD14 and C-type lectins. The CD14
receptor is not required for PGN signaling, even though the responses are usually
enhanced by CD14 binding [33].
Peptidoglycan was traditionally believed to signal through surface TLR2, but
this signaling was questioned after Travassos et al found that PGN sensing through
TLR2 was lost after removal of lipoteichoic acids (LTAs) from commercial S. aureus
PGN [34]. However, a more recent reevaluation has confirmed that it is indeed acting
upon TLR2 [35]. The current belief is that there seems to be important interactions
between TLR2- and NOD2 signaling pathways [6;36]. Of the 11 characterized TLRs
in humans, TLR2 is unique by virtue of its ability to heterodimerize with either TLR1
or TLR6, resulting in a relatively broad ligand specificity. The signaling pathway
after TLR2 ligation is similar to the TLR4 MyD88-dependent pathway, which leads
to the production of pro-inflammatory cytokines.
16
Immune cells and mediators
IMMUNE CELLS AND MEDIATORS
Monocytes/macrophages
Peripheral blood monocytes are circulating myeloid precursors of antigenpresenting cells originating from the bone marrow. In humans, they constitute ~ 510% of the blood leukocyte pool. They vary in size and have different degrees of
granularity. After being released into the periphery, where they circulate for several
days, they enter tissues and replenish the tissue macrophage population. Immune
stimuli, such as pro-inflammatory mediators, elicit increased recruitment of
monocytes to the tissues, where differentiation into macrophages and DCs takes
place.
Monocytes were initially identified by their expression of large amounts of
CD14, which is highly expressed by monocytes and macrophages. Monocytes are
nowadays regarded as a heterogeneous cell population that after differentiation not
only contributes to host defense, but also is important for tissue remodeling and repair
[37]. The monocytic subdivisions have sometimes been indistinct, but it has now been
convincingly shown that the most prominent populations are the classical
CD14++CD16- subpopulation and the pro-inflammatory CD14+CD16+ subpopulation,
the latter representing about 10% of all monocytes in healthy individuals [38]. CD16
is the low affinity receptor for immunoglobulin (Ig) G, therefore also named FcγRIII.
Monocytes are the cells within the innate immune system that exhibit the highest
density of TLRs on their surface [39]. The two monocytic subsets have been shown to
have different basal expressions of TLR2/TLR4 and to behave differently in response
to microbial stimuli [40].
Macrophages are being divided into classically activated (M1), and
alternatively activated (M2) macrophages, where M2 is a generic name for various
forms of activated macrophages (M2a, M2b and M2c). In general, M1 cells are the
consequence of stimulation by substances such as LPS and interferon (IFN) -γ, while
M2 have been exposed to IL-4, IL-10, IL-13 or transforming growth factor (TGF) -β.
M1 promote strong IL-12 mediated Th1 responses, while M2 support the Th2 –
associated effector functions. M2 also have a role in resolution of inflammation [41].
Interestingly, the division of macrophages into M1 and M2 is not a permanent
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phenomenon as it has been shown that both forms can be re-polarized by Th2 or Th1
cytokines, respectively [42].
The role of monocytes in disease, and more particularly in connection to
allergic disease, is central in this thesis and will be described in detail in a separate
section.
Dendritic cells
Dendritic cells are crucial mediators of immune defense, but have also been
implicated in tolerance induction [43]. They are the most potent antigen-presenting
cells (APC) due to their constitutive expression of high levels of Major
Histocompatibility Complex (MHC) class II molecules and other co-stimulatory
receptors. There are different subsets of DCs with distinct pattern-recognition
receptors and functions. Most mature DCs however, have the same major function;
presentation of antigen to Th cells. The myeloid dendritic cells (mDC) are derived
from monocytes arising from the myeloid pathway, while plasmacytoid dendritic
cells (pDC) arise from the lymphoid pathway. pDCs produce high amounts of IFN-α
and mDCs mainly produce IL-12.
DCs are present in small amounts in tissues that are in contact with the external
environment, mainly the skin and the inner lining of the nose, lungs, stomach and
intestines. They can also be found in an immature state in the blood. Once DCs are
activated, they migrate to the lymphoid tissues where they interact with T- and B cells
to initiate and shape the adaptive immune responses. Located alongside epithelial
cells in the airways, DCs have an important role in determining how allergic immune
responses (described in detail later) are initiated and perpetuated [44]. Being an early
director of the immune response, it is of no surprise that DCs initiate unwanted
responses that can cause disease. However, different DC subsets also have the
potential of being used as new therapeutic tools, in order to ameliorate many of the
disease conditions [45]. Targeting DCs for effective vaccination is an important area
taking advantage of the increasing knowledge of the important role of DCs in
directing immune responses [46].
18
Immune cells and mediators
NK cells
Natural killer (NK) cells are nonspecific cytotoxic lymphocytes playing a
crucial role in the innate immune system, making up approximately 10% of the
circulating lymphocytes in humans. Their main function is to kill tumors and cells
infected by viruses. NK cells are defined as large granular lymphocytes that do not
express T-cell receptors (TCR) or the T-cell marker CD3 or surface-Ig B cell
receptors, but that usually express the surface markers CD16 (FcγRIII) and CD56 in
humans. They recognize the target cells by missing "self" markers of MHC class I,
and induce apoptosis by releasing small cytoplasmic granules of proteins called
perforin and granzyme. Human NK cells are generally subdivided into two subsets
based on the expression of CD16 and CD56, where the majority of human NK cells
express low levels of CD56 and high levels of CD16.
Another important localization of NK cells is the placenta. Human NK cells are
massively recruited at the site of embryonic implantation. These NK cells differ in
many ways from their peripheral blood NK cell counterparts in terms of gene
expression, phenotype and functionality. The function of the decidual NK cells is not
completely understood. However, they have shown to play an important role in the
control of extravillous invasion, control of uterine vascular remodeling, and local
anti-viral activity [47].
Moreover, NK cells have been shown to have a role in allergy. A study of
atopic asthmatic individuals revealed a higher ratio of IL-4-producing NK cells in the
patients compared to controls [48]. Aberrant NK cell frequencies and functions have
also been observed in patients with atopic dermatitis [49]. Further, NK cells may
contribute to allergic responses by producing several cytokines and chemokines
related to an allergic reaction [50].
Natural killer T (NKT) cells are a heterogeneous group of cells, constituting
0.2% of all peripheral blood T cells. They share properties of both T cells and NK
cells, as they express the TCR, CD16 and CD56. They are considered as being
regulators of immune responses, as they rapidly produce large amounts of both Th1
and Th2 cytokines [51]. It is therefore not surprising that their dysfunction or
deficiency have been implicated in the development of autoimmune diseases [51],
cancers [52] and asthma [53].
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Mast cells
Mast cells originate from bone marrow precursors that circulate in an immature
form. Their characteristics as mature cells are determined by the specific tissue they
are recruited to [54]. They are capable of phagocytosis, and are activated through
pattern-recognition receptors to produce inflammatory mediators, a fact that makes
them an interesting cell type in several diseases. Indeed, mast cells are implicated in
autoimmune disorders where they are involved in the recruitment of inflammatory
cells to the joints and skin [55]. Another, perhaps unexpected role for mast cells, has
been elucidated in two recent murine studies showing that mast cells are important for
their ability to mediate regional immune suppression [56;57]. However, the most
studied role of mast cells is their contribution to the allergic reaction. They express
the high-affinity receptor FcεRI. This receptor is specific for the Fc region of IgE, a
class of antibody characteristic for allergic reactions (explained in a later section).
This receptor-ligand interaction is of such high affinity that binding of IgE molecules
is essentially irreversible. As a result, mast cells are coated with IgE. When activated
by direct injury, cross-linking of IgE receptors or by activated complement proteins,
they rapidly release their characteristic granules and various hormonal mediators.
Mast cells are common at sites in the body that are exposed to the external
environment, such as the skin. Their role in the allergic reaction is further explained
in the paragraph on the allergic mechanism.
Granulocytes
Granulocytes are a category of white blood cells characterized by the presence
of granules in their cytoplasm. They are also called polymorphonuclear leukocytes,
referring to the varying shapes of the nucleus, which is usually lobed into three
segments. The granulocytic cell compartment consists of neutrophils, basophils and
eosinophils. Neutrophils, normally found in the blood stream, are the most abundant
granulocytes. However, during the acute phase of inflammation, neutrophils leave the
blood and migrate toward the site of inflammation in response to different
chemotactic factors.
Eosinophils have important functions in combating infections, where their role
in immune responses to parasitic infections is the most prominent one. They are also
20
Immune cells and mediators
important cells for mechanisms associated with allergy and asthma, where they have
been shown to amplify type 2 responses by acting as APCs [58]. Basophils are the
least common granulocytes, representing about 0.01% to 0.3% of circulating
leukocytes. Functionally, they are similar to mast cells as they also store histamine,
and are implicated in allergic reactions. Indeed, a recent study confirms that basophils
contribute to the initiation of the allergic reaction, as they are directly activated by
allergens and produce cytokines that are crucial in an allergic reaction [59].
T cells
T cells are CD3 carrying cells belonging to the cellular part of the adaptive
immune system. They can be divided into CD4+ T helper cells (Th cells), CD8+
cytotoxic T cells (CTL) and regulatory T cells (Treg). T cells originate in the bone
marrow, but the development into functioning T cells takes place in the thymus. The
ability of T cells to recognize foreign antigens is mediated by the T-cell receptor. The
receptor is associated with CD3 on the membrane of the cell. CD3 does not influence
the interaction between the antigen and the TCR, but participates in the signal
transduction. The T-cell receptor undergoes genetic rearrangement during thymocyte
maturation in the thymus, resulting in each T cell bearing a unique T-cell receptor,
specific to a limited set of peptide-MHC combinations. The random nature of the
genetic rearrangement results in a requirement of central tolerance mechanisms to
remove or inactivate those T cells which bear a T-cell receptor with the ability to
recognize self-peptides.
The Th cells recognize extra cellular antigens that are taken up and presented by
APCs in association with MHC class II molecules. The recognition of this complex
activates the Th cell, which among other functions produces cytokines and supports
different events of the immune system, such as antibody production by B cells and
killing of cells by the CTLs. CTLs recognize and destroy cells that are infected with
intracellular pathogens, such as viruses. This recognition is dependent upon the
degradation and presentation of the antigen in association with MHC I molecules on
the infected cells.
Treg cells are important for peripheral tolerance, and today the Treg family
comprises many types of cells. Two major classes of Treg cells have been described;
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the naturally occurring Treg- and the adaptive Treg cells. Naturally occurring Treg cells
(also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus, whereas the
adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a
normal immune response [60]. Genetically determined or environmentally induced
abnormality in Treg development, maintenance and function has consequences for
several diseases, where allergy is one of them [61;62].
For most antigens, the primary encounter during an initial infection or
vaccination leads to an immunological memory, comprised of a memory T cell pool
with cells of different specificity. It was recently shown that during a secondary
infection, where antigen-specific memory cells are activated, the existence of memory
T cells is not enough to eliminate the pathogen itself. The pathogen removal was
dependent on innate mononuclear phagocytic cells activated by the memory T cells.
Interestingly, this re-exposure to a pathogen and activation of memory T cells,
leading to activation of the innate arm of the immune system, also had a non-specific
bystander effect on killing another simultaneous unrelated infection [4].
Another important function of T cells that was very recently proposed is the
suppression of inflammatory cytokine production that usually takes place during a
normal situation where the innate immune system has triggered an inflammatory
response. It was shown that without T cells, mice die of uncontrolled inflammatory
events. Unexpectedly, it was not only the Treg cells that could carry out this
suppression, but also the conventional T cells. The suppression was dependent on
direct contact between T cells and the MHC II complex from cells of the innate
immune system [5].
B cells
B cells are lymphocytes belonging to the humoral immune response, an
essential part of the adaptive immune system. The principal function of B cells is to
produce immunoglobulins (Ig) or antibodies against soluble antigens. Immature B
cells are produced in the bone marrow of most mammals. B-cell development occurs
through several stages, each stage representing a change in the genes coding for
immunoglobulins. Immunoglobulins exist in a membrane-bound form as the B-cell
receptor (BCR) and in a secreted form, also referred to as antibodies. After reaching
22
Immune cells and mediators
the IgM+ immature stage in the bone marrow, the B cells migrate to the spleen where
they mature into B lymphocytes. An antibody comprises two light (L) and two heavy
(H) chains. In the H chain loci there are three regions, V, D and J, which recombine
randomly in a process called VDJ recombination, to produce a unique variable
domain in the immunoglobulin of each individual B cell. Similar rearrangements
occur for the L chain locus, except there are only two regions, namely V and J. When
a B cell encounters its cognate antigen, and receives an additional signal from a Th
cell, it differentiates into either a plasma cell secreting large amount of antibodies, or
a memory B cell, which will respond quickly to a second exposure of the same
antigen. Recent studies have shown that TLR signaling in B cells is an important
event for optimal IgM production and memory [11;63].
During an allergic reaction IgE is synthesized and secreted by B cells that have
undergone heavy-chain class switching from IgM to IgE. Synthesis of IgE by B cells
occurs at a low rate compared with other immunoglobulins, even in allergic
individuals. However, in the nasal mucosa of patients with allergic rhinitis
approximately 4% of the B cells and 12-19% of the plasma cells express IgE, in
comparison with the situation in non-allergic individuals where less than 1% of the
plasma cells and 1% of the B cells express IgE [64].
Recent advances have led to the clinical use of monoclonal antibodies that
deplete or inhibit development of B cells. This relatively new strategy has shown to
ameliorate disease severity in several hematological malignancies as well as
autoimmune disorders [65].
Cytokines and chemokines
The pro-inflammatory cytokines and chemokines induced in an inflammatory
response direct the deviation of T cells towards an adaptive effector response.
Signaling by different TLRs gives rise to differential expression of cytokines.
Whereas e. g. signaling through TLR9, recognizing bacterial DNA, induces high type
I IFN [66], signaling through TLR2 can induce high levels of IL-13 [67;68].
Chemokines, named for their ability to induce directed chemotaxis in nearby
responsive cells, are responsible for the migration of leukocytes. They are released by
many different cell types and serve to guide cells of both the innate and the adaptive
23
Petra Amoudruzvvvv
immune system. Different cell types express different chemokine receptors [69],
which is a way for the immune system to direct the right cell to the right place.
Members of the chemokine family are categorized into four groups; CC-, CXC-, Cand CX3C chemokines. The work in this thesis has investigated the production of one
inflammatory chemokine; CXCL8 or IL-8. Inflammatory chemokines are produced in
response to infection to trigger the recruitment of monocytes, neutrophils and other
effector cells from the blood to sites of infection or tissue damage. Their release is
often stimulated by pro-inflammatory cytokines such as IL-1β that are highly
expressed upon TLR ligation.
24
Immune cells and mediators
Type 1/type 2 responses
The Th responses have traditionally been divided into a Th1 or a Th2 type.
Recently a third independent effector population, Th-17 cells, was discovered [70].
Other non-CD4+ cells also produce cytokines influencing the balance between the
Th1 and Th2 cells, and the concept of type 1 and type 2 responses has now been
extended to also include cells other than Th cells. These include CD8+ cells and cells
of the innate system, where many refer to polarized macrophages as M1 and M2 cells
[41] and NK cells to NK1 and NK2 [71]. Thus, the division of cells into type 1 or
type 2 cells is in many cases an oversimplification, and the distinction between the
different subsets is not always clear.
The type 1 response is mainly effective against intracellular pathogens, and IL2, IL-12 and IFN-γ are critical for this type of response. IL-4 is the main cytokine
critical for the type 2 response, which is effective against extracellular pathogens
[72]. Th-17 cells express IL-17, a cytokine that has been implicated in many
functions correlated to disease, amongst others bronchial responsiveness [73;74] and
promotion of autoimmune inflammation [75]. Our understanding of how pathogens,
such as bacteria and viruses, induce Th1-cell responses greatly exceeds our
knowledge of how Th2-cell responses are induced by allergens and parasites.
However, two very recent studies on Th2-cell development show that signals derived
from basophils and eosinophils are directly involved in the induction of Th2-cell
responses. One of the studies, dealing with the initiation of sensitization, proposes
that allergen- or helminth proteases cleave a 'sensor' that stimulates basophils to
migrate to the lymph nodes, and to produce Th2-type cytokines, leading to Th2-cell
differentiation [59]. The other study shows that a secretory protein, produced by
eosinophils [eosinophil-derived neurotoxin (EDN)], acts as an alarm signal that is a
ligand for TLR2, skewing DCs into a Th2 type upon stimulation. Splenocytes from
mice immunized with ovalbumin (OVA) and EDN show a strong Th2 skewing upon
restimulation, with strong IL-5, IL-10 and IL-13, and no interferon-γ production,
showing that the in vivo function of EDN is of importance [76].
The products of type 1 and type 2 cells act as autocrine growth factors for
further expansion of these cells, as well as reciprocal inhibitory agents for the
opposite cell type [77]. Polarized T cells also express different patterns of chemokine
receptors, where CXCR3 and CCR5 are mostly associated with Th1 cells, whereas
25
Petra Amoudruzvvvv
CCR3, CCR4 and CCR8 are mainly expressed on Th2 cells [69]. An imbalance of the
type 1/type 2 cytokines has been linked to disorders such as autoimmune [78;79] and
allergic diseases [80;81]. Even though developmental and environmental factors are
important for skewing of the adaptive responses [82-84], susceptibility to different
diseases show a clear linkage to genetic factors [85-88].
Transcription factors are of importance for the cytokine-induced development
of naïve CD4+ T cells into Th1 or Th2 type. Signal transducer and activator of
transcription (STAT) 6 and GATA3 are important for the induction of Th2 cells and
IgE responses [89], while STAT4 and T-bet are important for Th1-cell development
[90]. GATA-3 and T-bet can act directly on each other to regulate the balance of
Th1/Th2 cells [90;91].
As the work in this thesis deals with expression of particular cytokines and
chemokines there is a more thorough background on each of them in the following
section.
TNF
Tumor necrosis factor (TNF) is a pleiotropic pro-inflammatory cytokine
synthesized primarily by macrophages and monocytes, but also by activated T- ,
mast- and NK cells. It exists as either a transmembrane or a soluble protein, where the
soluble form is the most potent one [92]. TNF is produced early in an immune
reaction, and induces the production of other pro-inflammatory mediators. It is an
important factor for the induction of labor, but is also produced by intrauterine tissues
in response to microbial products. TNF is of major importance for the onset of
premature labor in the context of infection [93].
In connection to allergic disease, polymorphisms in the promoter region of
TNF, leading to higher TNF levels, have been shown to be overrepresented in allergic
patients who are sensitized to multiple allergens, compared to those being
monosensitized and to controls [94]. Further, TNF is involved in chronic immunemediated inflammatory diseases, such as asthma, rheumatoid arthritis, Crohn's
disease and psoriasis. As a result of these observations, neutralizing monoclonal
antibodies specific to human TNF or the TNF receptor have been developed. They are
showing promising results [95], even though the treatment of asthma and rheumatoid
26
Immune cells and mediators
arthritis seems to benefit patients with more chronic stages of disease, and be less
efficient in early disease. This is a very recent area of therapeutic interventions where
the modes of action in vivo still should be carefully evaluated.
IL-1β
Originally described as the endogenous fever molecule, IL-1β is a proinflammatory cytokine, with the ability to induce several genes usually not expressed
in healthy individuals. IL-1β increases the expression of many cytokines, in particular
TNF and IL-6, as well as chemokines and adhesion molecules [96]. It is produced by
many cells and exerts its biological effects on almost all cell types [97]. IL-1β is a key
mediator of many pathophysiological events characterized by inflammation and hostenvironment interactions, such as graft-vs-host disease [98], gut cancer [97] and
rheumatoid arthritis [99].
IL-6
IL-6 is a cytokine with a broad range of functions on immune and nonimmune
cells [100]. It is produced by several cell types including APCs such as macrophages,
DCs and B cells, at sites of tissue inflammation where it can have a pro-inflammatory
as well as an anti-inflammatory effect.
Classic signaling of IL-6 involves the binding of the cytokine to its membrane
bound receptor IL-6R on target cells. The receptor complex for IL-6 also consists of
at least one subunit of the signal transducing glycoprotein (gp)130, that also exists in
a soluble form (sgp130) in human serum [101]. However, many of the biological
activities assigned to IL-6 are performed in a process known as trans-signaling,
where IL-6 binds to a soluble form of the receptor; sIL-6R, and thereby activate cells
via membrane-bound gp130 [102]. Thus, the complex is an agonist for cell types that,
although expressing gp130, are normally non-responsive to IL-6 itself [103]. The
naturally occurring combination of sIL-6R and sgp130 can act as a regulatory system
that modulates systemic responses to circulating IL-6 [104]. IL-6 activation leads to
the activation of the Janus kinase (JAK)/STAT and MAPK cascades [101].
27
Petra Amoudruzvvvv
IL-6 has a regulatory role in the process of bridging innate and adaptive
immunity through its influence on leukocyte recruitment, activation and apoptosis
[105]. Impaired regulation of the transition from innate to adaptive immunity where
IL-6 plays a pivotal role may affect disease outcome [2]. Indeed, dysregulation of IL6 signaling has been shown to play an important role in the onset and maintenance of
several autoimmune diseases, as well as cancer and osteoporosis [102;106;107]. IL-6
has further been implicated in determining the balance between the suppression and
activation of allergic responses [108;109]. A role for IL-6 trans-signaling in
supporting inflammation has been demonstrated in mice where mucosal T cells
become less prone to go into apoptosis upon IL-6 signaling [110]. A possible
mechanistic explanation for this IL-6 regulation was demonstrated in 2003 by Pasare
& Medzhitov. They showed that, in the murine system, microbial signaling via TLRs
blocked the suppressive effect of CD4+CD25+ Treg cells by inducing IL-6 release from
DCs and macrophages [111]. Thus, they concluded that IL-6 seems to play a critical
role in activation of T cells by overcoming the suppressive effect of Treg cells. In line
with this, the de novo induction of adaptive Treg cells was recently found to be
abrogated in mice due to IL-6 trans-signaling in T cells [112]. Th2 differentiation is
promoted by IL-6 production, probably due to the ability of IL-6 to induce IL-4 and
IL-5 production [113]. Simultaneously, IL-6 inhibits Th1 polarization by upregulating
suppressor of cytokine signaling (SOCS)-1 expression to interfere with IFN-γ
signaling [114]. Anti-IL-6R antibody therapy has shown promising results for
treatment of inflammatory disorders [115].
IL-10
IL-10 is an anti-inflammatory cytokine, due to its ability of suppressing the
release of pro-inflammatory cytokines by macrophages [116], but also through its
ability to induce the synthesis of IL-1β receptor antagonist and soluble TNF receptors
[117]. The suppression of IL-12 production by IL-10 is well established [118]. IL-10
is a product of multiple cell types, including Th1 and Th2 lymphocytes, cytotoxic-T
cells, B cells, mast cells, monocytes and DCs. However in humans, monocytes and B
cells are the major sources of IL-10 [119]. Treg cells exert their suppressive activity
primarily via the release of IL-10 [120]. Isotype switching to IgG4 is enhanced by IL-
28
Immune cells and mediators
10 while IL-4–induced IgE is inhibited. IL-10 has been considered for therapeutic use
in relation to various diseases, mainly for its ability to suppress inflammatory
conditions and type 1 as well as type 2 related pathways [121].
IL-10 can have different effects depending on environment and timing. Several
studies have shown that IL-10 could play a role in the perpetuation of allergic
inflammation, as high levels of IL-10 mRNA have been detected in the airways of
asthmatic patients, as well as in the skin of patients with atopic dermatitis [122;123].
An IL-10 producing monocyte population, differentiating into alternatively activated
macrophages (M2), has also been seen to be over represented in atopic individuals
[124].
IL-12
IL-12 is a pro-inflammatory cytokine produced by DCs and phagocytes early in
response to infection. It forms a link between innate and adaptive immunity through
its powerful effects on NK-cell function and T-cell development, where it promotes
the production of IFN-γ and favors the differentiation of Th1 cells. IL-12 is a
heterodimeric cytokine of 70kDa comprising of two subunits, p40 and p35. The genes
encoding the two subunits are unrelated and located on different chromosomes. The
p40 chain is often secreted in large excess over the p70 heterodimer. IL-12p35 is
retained inside the cells and only secreted when associated with the p40 chain [72].
Many of the functions earlier attributed to IL-12 have now been shown to be carried
out by a recently discovered cytokine, IL-23. IL-23 consists of two subunits, p40 that
is shared with IL-12 and p19 that is specific for IL-23 [125].
The IL-12 receptor (IL-12R) is expressed mainly by activated T- and NK cells.
However, DCs [126] and B-cell lines [127] have also been shown to express the
receptor, demonstrating the importance of IL-12 in the autocrine and paracrine effects
on APCs. T-cell stimulation also drives IL-12 production in APCs through CD40CD40L interactions [128;129]. IL-12 expression is controlled by upregulation of the
IL-12R in response to a variety of cytokines including IFN-γ, IL-18, granulocytemacrophage colony-stimulating factor (GM-CSF), IL-4, as well as IL-12 it self. A
combination of microbial, CD40, and cytokine stimuli [130] gives an optimal
induction of IL-12 production.
29
Petra Amoudruzvvvv
A reduced ability to produce IL-12 has been shown to correlate with an allergic
phenotype [131-134]. However, studies have also shown that the connection between
IL-12 and allergy is not that clear, and can even be of opposite results, very much
dependent upon the age of the subjects [134-136].
TGF-β
Transforming growth factor beta (TGF) -β1 and TGF-β2 are members of the
TGF-β superfamily characterized by the presence of common sequence and structural
features. TGF-β was first described as having an inhibitory action on cellular
processes, but it was soon discovered that it can have both stimulatory and inhibitory
effects depending on cell type and other signals present [137]. TGF-β is involved in
the recruitment of blood monocytes and neutrophils to the lamina propria where they
become intestinal macrophages, playing an important role in gut inflammation [138].
Eosinphil-derived TGF-β1 is critical in pulmonary immunity and fibrosis, and
therefore plays an important role in asthma development, where it among other
functions acts as a potent chemoattractant for monocytes [139]. A recent study,
examining the role of TGF-β1 produced by eosinophils in connection to asthma,
found that the production was regulated by a protein named peptidyl-prolyl isomerase
(PIN) 1. Interestingly, from a therapeutic view, the authors found that inhibition of
PIN1 in mouse and rat models of chronic pulmonary inflammation reduced allergic
lung fibrosis [140].
TGF-β signaling is further a potent stimulus for driving the development of
naïve CD25- T cells in the periphery into adaptive CD4+CD25+ Treg cells, which have
shown to, among other functions, be able to suppress Th1-mediated experimental
colitis [141]. Recent data further show that TGF-β produced by Treg cells interferes
with polarization of the secretory machinery in Th cells toward APC, thus
suppressing a crucial step of Th-mediated amplification of the immune response
[142].
TGF-β1 is relevant to immunological development in offspring, as it is known
to stimulate IgA synthesis [143;144]. In the context of an inflammatory cytokine
milieu, TGF-β1 supports de novo differentiation of IL-17 producing T cells [145],
cells that have been shown to be relevant in autoimmunity [75] and allergy [74]. Due
30
Immune cells and mediators
to its ability of acting stimulatory as well as inhibitory, the role of TGF-β in relation
to disease is somewhat complex. TGF-β1 levels in breast milk have been
positively/negatively associated with wheezing [146] and allergic disease [147] in
offspring. TGF-β2 levels in breast milk have also been shown to be associated with
both more and less sensitization in breast-fed infants [147;148]. A very prominent
role for breast-milk TGF-β in the induction of tolerance to allergens was recently
described in a mouse model of allergic asthma [149].
IL-8
IL-8 is a chemokine produced by various types of cells upon stimulation with
inflammatory stimuli. It exerts a variety of functions on leukocytes, particularly in
acute inflammation where it recruits and activates neutrophils [150].
IL-8 is present in high concentrations in breast milk, and is believed to have a
function in the human neonatal gut as it remains measurable throughout simulated
neonatal gastric and proximal intestinal digestion. When human fetal intestinal cells
are stimulated with rhIL-8 in vitro, there is indeed an increase in cell migration,
proliferation, and differentiation [151]. Despite concerns about potential adverse
effects of IL-8 on the intestinal mucosa, there is evidence that normal levels of IL-8
have a physiological role in the gut of the newborn [152].
31
Petra Amoudruzvvvv
ALLERGY
The allergic reaction
Allergy is defined as a hypersensitivity reaction initiated by specific
immunologic mechanisms [153]. Allergy can be antibody-mediated and/or cellmediated. In most patients with allergic symptoms from mucosal membranes in the
airways and gastrointestinal tract, the antibody belongs to the IgE isotype, and these
patients may be said to have an IgE-mediated allergy or to be IgE-sensitized. The
term atopy should be reserved to describe the genetic predisposition to become
sensitized and produce IgE antibodies in response to ordinary exposures to allergens
commonly occurring in the environment [153].
Antigen presentation under the influence of IL-4 leads to expansion of Th2 cells
and subsequent production of IL-4 and IL-13 (Figure 2). These cytokines induce Ig
class switching in B cells from IgM to IgE, the latter subclass being responsible for
allergic reactions [154].
Figure 2. Mechanisms responsible for IgE mediated allergy (Modified from Nat Rev
Immunol 2008; 8:205-17). Reprinted by permission from Nature copyright Macmillan
Publishers Ltd.
32
Allergy
The head of the IgE (Fab portion) recognizes specific allergens. The activity of
IgE is associated with a network of proteins; important among these are its two
principal receptors, Fc RI (the high-affinity Fc receptor for IgE) and CD23 (the lowaffinity Fc receptor for IgE), as well as several co-receptors for CD23, such as e. g.
CD21 (Figure 2). The IgE binds to FcεRI and sensitizes these cells to allergens.
FcεRI is expressed as a tetramer on mast cells and basophils, and as a trimer on
human APCs, monocytes, eosinophils, platelets and smooth muscle cells. CD23,
which is also found in a soluble form, is expressed on a variety of inflammatory cells,
B-cells, but also epithelial cells. CD23 is implicated in negative as well as positive
IgE regulation, where IgE-mediated feedback enhancement is one of the suggested
mechanisms [155]. During this event, IgE antibodies administered together with their
specific antigen can enhance the production of IgE recognizing this antigen by >100fold [156].
Upon a second encounter with the same antigen, IgE antibodies bound to FcεRI
are crosslinked and cause granules to rapidly empty their contents into the
surrounding tissue. The preformed granules contain a variety of inflammatory
substances such as leukotrienes, histamine, cytokines and chemokines. After the
chemical mediators of the acute response subside, late phase responses can often
occur. This is due to an inflammatory reaction causing migration of other leukocytes
such as neutrophils, lymphocytes, eosinophils and macrophages to the initial site. The
local inflammatory response can be seen as a red, itchy weal. This late reaction is
usually seen 2-24 hours after the first reaction. Cytokines from mast cells may also
play a role in the persistence of long-term effects.
33
Petra Amoudruzvvvv
The hygiene hypothesis and the role of innate immunity
The prevalence of allergic diseases has increased drastically during the past few
decades. A family history of allergic disease is clearly a strong risk factor, but in view
of the rapidity of the increase in allergy prevalence, environmental factors are likely
to play a crucial role. A notable fact is that it is not only “ type 2 diseases”, such as
allergy, that are increasing in the modern world, but there is also a strong global
correlation between childhood wheezing and diabetes, where diabetes belongs to a
more type 1 kind of disease [157]. A growing body of evidence suggests that
something may lack in our modern way of living that has the capacity to provide
protection against the development of such diseases. It is increasingly recognized that
microbial colonization of the gastrointestinal tract, linked with lifestyle and/or
geographic factors, may be important for the difference in disease prevalence
throughout the world [157]. A nowadays widely accepted theory, based on the
observations presented above and below, is the hygiene hypothesis, even though
immunological mechanisms explaining this hypothesis are still not completely
understood. Although this theory is not undisputable [158], several studies have
shown that having older siblings [159-161], attending day care centers [159;162] and
growing up on a farm [163;164] have protective effects against allergy development.
Another fact that supports the hygiene hypothesis is that the allergic prevalence seems
to increase in parallel with the affluence of a country, a recent example of this being
East Germany [165].
The initial interpretation of the hygiene hypothesis was that exposure to specific
infections during early life drives the maturation of the immune system towards the
Th1 phenotype and away from the Th2 phenotype, associated with allergic disease
[166]. Today it is believed that not only infections but also an early exposure to nonpathogenic microbes in our environment is involved in the development of the
immune system [167]. The importance of stimulation of TLR4 by intestinal
commensal flora in mice, in order to inhibit allergic responses to food allergens,
highlights the role of the innate immune system in allergy development [168]. Indeed,
it has been shown that different microbial exposures activating the innate immune
system give rise to reduced IgE-specific adaptive immune responses [169]. A
probable mechanistic explanation for this finding is that T-cell responses are usually
34
Allergy
characterized by a Th2 phenotype, unless there is a microbial stimulation of the DCs
via TLRs, polarizing the CD4+ cells to a Th1 phenotype [170].
Multiple studies have shown that exposure to LPS in early life seems to have a
protective effect against the development of allergy [171;172]. Something that has to
be stressed in this context is that LPS might only be a marker of microbial exposure
and not the causative agent. There could be other substances that are highly correlated
to LPS exposure, actually conferring the immunological protective effect of microbial
exposures. However, these substances would have to show a high correlation with
LPS, as the protective effect has been observed in many different studies.
Lately, interest has focused on the Treg- and NKT cells which suppress the
function of other cells by cell to cell contact and/or by secreting cytokines like IL-10
and/or TGF-β. An impaired activation of the Treg cells, caused by decreased exposure
to microbial agents, offers an alternative explanation for the increase in allergy
frequency [173].
Our group has found that acquisition of Epstein-Barr virus (EBV) infection
during the first 2 years of life is associated with a reduced risk of IgE sensitization,
and that this effect is further enhanced by Cytomegalovirus (CMV) coinfection [174].
Primary infection with common virus infections such as EBV and CMV occurs
within a few months to years after birth in developing countries, but only during the
second and third decade of life in industrialised countries [175-178]. Crowding,
poverty, and the widespread practice of breast feeding all encourage the early spread
of CMV [177]. As CMV and EBV in their latent forms resides inside monocytes
[179] and B cells [175] respectively, infection at a young age when the immune
system is maturing, could most probably affect important events connected to allergic
susceptibility.
Increasing epidemiological evidence show that obesity increases the risk of
allergic and autoimmune diseases [180]. The reason for this link between obesity and
allergy could also be caused by some other underlying factors, such as dietary factors
or physical inactivity. However, it is known that the increasing body weight has an
influence on the immune system. Obesity increases the levels of circulating IL-6,
leptin, and TNF, secreted by white adipose tissue. Adiponectin, which decreases with
increasing obesity, down-regulates the secretion of IL-10 from macrophages and
35
Petra Amoudruzvvvv
adipocytes. These changes in IL-6, leptin, and IL-10 are known to decrease the
regulatory effect of Treg cells resulting in decreased immunological tolerance to
antigens, which would make individuals more prone to be sensitized to allergens. It is
further speculated that in pregnant women, these obesity-induced immunological
changes might be transmitted to the fetus by epigenetic inheritance (explained below),
thereby increasing the risk of allergic disease [180].
Immigrants who move from a developing to an industrialized country maintain
their lower disease risk of allergy [165], but also some other immune-mediated
diseases such as inflammatory bowel disease (IBD) [181]. Intriguingly however, their
offspring born in an industrialized country have an even higher risk of disease than
the indigenous population of that country [182;183]. The reason for this phenomenon
is unknown, but immunological events as a result of environmental exposures in
childhood could be a clue. The difference between mother and child in childhood
exposures could explain the higher incidence of disease in children to immigrants.
One could speculate that since the immune system has been primed for multiple
generations to fit the expected burden of exposures, this “unexpected” environment
that the child grows up in could instead cause disease, as the immune system is not
adapted to the new environment.
Monocytes/macrophages and disease
Monocytes/macrophages play a pivotal role in many diseases such as cancer
[184], parasite infections [185], and rheumatoid arthritis [186]. The role of the two
different monocyte subpopulations; the classical CD14++CD16- subpopulation and the
pro-inflammatory CD14+CD16+ subpopulation, in allergic disorders is not fully
elucidated. One study showed that blood monocytes from untreated adult asthmatics
have a higher percentage of the pro-inflammatory CD14+CD16+ subset than nonallergic subjects [187]. Atopic eczema has also been linked to an increased population
of CD14+CD16+, which was diminished in connection to clinical improvement [188].
However, another study on atopic dermatitis did not find any differences in the two
monocytic subsets or in TLR levels, but an impaired IL-1β and TNF production in
response to a TLR2 ligand [189]. A recent study [124] showed that an IL-10producing monocyte subset is over-represented in allergic compared to non-allergic
36
Allergy
individuals, and that these monocytes differentiate into alternatively activated
monocytes (M2). Another study found that M2 inhibit the generation of M1, and that
this inhibition is dependent upon CCL17 and IL-10 production in M2 [185;190]. As
IL-10 has been shown to be over-expressed in other studies of allergic individuals
[191;192], and as M2 support Th2-effector functions [41], this would provide a
possible explanation for how IL-10 could act in promoting allergic disease, rather
than acting anti-inflammatory. A study in mice infected with a gastrointestinal
parasite showed that activated Th2 cells induce macrophages, required for parasite
clearance. Their study emphasizes the role of macrophages as essential effector cells
in protective Th2 responses, and provides an evolutionary role for the alternatively
activated M2 cells, believed to play a role in allergic disease [185].
Pattern-recognition receptors and disease
Multiple studies have investigated the role of TLR4, CD14 and sCD14 in
relation to allergic disease. The reason for this interest is the involvement of these
receptors in immunological responses to infections, since exposure to microbial
compounds is suggested as being a protective factor for allergy development.
Moreover, increasing evidence shows that TLRs also recognize host-derived
(endogenous) ligands, a fact that also connects TLRs to diseases that may not have an
etiology that is associated directly with infection [193].
The gene coding for TLR4 is highly polymorphic, and to date 44 TLR4 singlenucleotide polymorphisms (SNPs) have been identified [194]. Some of the
investigated polymorphisms of TLR2 and 4 have been linked to altered systemic
inflammatory reactions [195], cancer [194], viral responses [196], inflammatory
bowel disease [197;198] and allergy [199;200].
Polymorphisms within the CD14 receptor have been found to be associated
with Crohn’s disease [201] and allergy [202-204]. The results from studies of
associations of sCD14 with allergic disease are inconsistent [205-208]. This variation
in direction of association may depend upon the dual role of sCD14, depending on
concentration and environment. Thus, while it may have systemic anti-inflammatory
effects, it can also exert a pro-inflammatory influence in specific tissues to increase
resistance to bacteria [29]. Another fact, that may complicate interpretations of the
37
Petra Amoudruzvvvv
observed associations with disease, is that there are many other influencing factors
and confounders, including gender-gene-environment interactions [207]. Indeed,
multiple studies show that polymorphisms can be differently important depending on
the environmental exposure [200;209].
Gut
Immune modulation of the gut is influenced by factors derived prenatally from
the placenta, and after birth from the breast milk and oral contact with the neonatal
environment [210]. The fetal gut is structurally mature from week 19 of gestation,
and all cellular components of the gastro-intestinal immune system are already
present at birth. However, at birth, the gastrointestinal tract is functionally immature
and immunoincompetent. After birth, it undergoes rapid growth and maturational
changes, including the colonization with an immense benign biotic mass [211]. This
protects the gut from colonization of pathogens, and is also believed to be an
important stimulus for the developing immune system. During homeostatic
conditions, these commensal bacteria do not evoke inflammatory immune responses.
However, in some cases such as IBD, this flora can also cause disease [212]. The
important role of the gut for the developing immune system is underlined by the Band T cell development taking place in the fetal gut [210].
The importance of very early innate events in the context of stimulation of the
gut has been convincingly shown in several studies. One study demonstrated that
TLR4 dependent signals, provided by the intestinal commensal flora, can inhibit the
development of allergic responses to food antigens [168]. Another study [31] showed
that a rapid postnatal acquisition of epithelial tolerance to microbial ligands is
dependent upon LPS activation of intestinal epithelial cells (IEC). This homeostatic
state in the gut was dependent on LPS exposure in both studies. Factors such as
vaginal delivery, oral exposure to endotoxin, and the presence of TLR4 were coupled
to protection, while antibiotic treatment and lack of TLR4 showed opposite results.
More supporting evidence for the importance of early stimulation of the infantile gut
comes from studies using pro- and pre-biotic treatment. These studies show that probiotic treatment given to the mother during late pregnancy, and to the young infant
after birth significantly protected against atopic eczema at the age of 2 [213;214].
Indeed, differences in the gut flora between allergic and non-allergic children have
38
Allergy
been detected in several studies [215;216]. Our group recently found that children
developing allergy up to five years of age were significantly less often colonised with
lactobacilli during their first two months compared to non-allergic children [Sjögren
et al, unpublished data].
Another immunological influence of the commensal flora on the immune system
is the induction of sCD14 release from monocytes and DCs. A recent study showed
that sCD14 induced by commensal bacteria inhibits birch allergen-induced Th2
differentiation by suppressing IL-13 production [217]. IL-13 is known to diminish
LPS-mediated cell activation by downregulating TLR4 signaling [218]. Keeping in
mind that TLR4 signaling is important for gut homeostasis [31;168], this induction of
sCD14 by the commensal bacteria, leading to lower IL-13 levels, could be one of the
molecular mechanisms behind the beneficial effects of a balanced gut flora.
Gene-environment interactions
Growing up on a farm, with a constant exposure to microbial products present
in an environment with live-stock, is associated with protection against allergy
development later in life [219]. One of the main causes for this protective effect has
shown to be the consumption of farm milk [163;220]. However, Eder et al [200]
demonstrated that genetic variations in the TLR2 gene are important for the
susceptibility of farmer’s children to allergic disease. The protective effect of
endotoxin exposure in early life in relation to allergy development is further
dependent upon genetic differences regarding the LPS recognition receptor CD14
[203].
In relation to allergy development, another noteworthy finding exemplifying the
importance of gene-environment interactions was reported in 2003 [221]. McIntire et
al found that individuals with specific alleles of HAVCR1, a gene encoding the T
cell, Ig- and mucin domain (TIM)-1 glycoprotein expressed preferentially on Th2
cells, were protected against allergic disease. There was an increased protection in
individuals previously exposed to the hepatitis A virus (HAV), probably due to the
fact that TIM-1 also functions as the receptor for HAV [222].
39
Petra Amoudruzvvvv
Epigenetics
Although much effort has focused on the gene-environment interactions, there
is a growing body of evidence suggesting that environmental influences may extend
beyond the DNA sequences of our genes. Something that underscores the important
role played by the environment in shaping the genetic constitution is that
monozygotic twins are genetically indistinguishable early in life, but with age exhibit
substantial differences shown to be linked to events such as replication, transcription,
recombination and repair of DNA [223]. The emerging field dedicated to the study of
this form of biologic regulation is termed “epigenetics”. Epigenetics constitutes the
study of changes in gene expression not accompanied by alterations in DNA sequence
[224]. It has been shown that environmental factors that alter epigenetic events such
as methylation processes play a role in disease susceptibility [224]. As geneenvironment interactions play a pivotal role for allergy development, this field of
research opens up and broadens our way of looking at how gene expression is
influenced by the environment. Epigenetic events in form of cellular acetylation have
further been shown to be important for maintaining the pre-established Th1/Th2 like
responses. This fact offers an epigenetic regulatory mechanism explaining how the
environment could influence the maintenance of an excessive Th2 skewing,
characteristic for allergic diseases [225]. Another recent study in mice, confirming the
important role of epigenetics in the gene-environment context, shows that in utero or
neonatal exposure to a chemical can change the phenotype of the offspring by stably
altering the epigenome. Interestingly, this effect could be counteracted by maternal
dietary supplements [226].
Microbial recognition for therapeutic interventions
New insights of the role of TLRs in relation to disease are of interest for various
therapeutic strategies. The possibility of using TLR agonists as vaccine adjuvants and
as immunomodulators is now a very active area of research, having shown promising
results [227]. The TLR ligand/allergen vaccination approach depends on the capacity
of treated patients to respond to TLR ligands, and at the same time recognize the used
antigen. The advantage of this action is that while the allergen is being processed, the
TLR stimulation will give rise to a type 1 response counteracting a type 2 response to
the antigen used [136]. CpG, recognized by TLR9, has demonstrated preventive and
40
Allergy
therapeutic activity in murine models of atopic airway inflammation [228-231]. A
synthetic TLR4 ligand acting as adjuvant in vaccination programs has proven safe
and active in human trials [232], and TLR4 and TLR2 agonists have proven to
ameliorate allergic airway symptoms in mice [233-235]. For a successful use of TLRs
as targets in the context of allergic disease, there is still however a lot of important
knowledge lacking.
Another potential therapeutic strategy regarding allergy is to take advantage of
the interplay between some species of helminths and the immune system. During
helminth infection there is a selective immune suppression that gives rise to a
regulatory environment which also protects from other diseases such as allergies.
Helminth infections seem to modulate dendritic cells and macrophages to induce a Tcell hyporesponsiveness [236]. Studies in mice have shown that the mechanism
whereby worm infection modulates immunity is dependent on different kinds of
macrophages that can prevent inflammation [185], and that it is these macrophages
that can render infected mice refractory to induced colitis [237].
Factors that modulate the induction and function of Treg cells are other
potentially interesting areas for therapeutic interventions regarding inflammatory
diseases. IL-6 trans-signaling, that has recently been found to abrogate the induction
of Treg cells and inhibit apoptosis in T cells in Crohn’s disease [110;112], could be
one of the targeted pathways in order to restore a balance between protective and
damaging immunity. Indeed, by using neutralizing antibodies towards the IL-6R or
sIL-6R, inflammatory conditions have shown to be ameliorated [115].
41
Petra Amoudruzvvvv
IMMUNOLOGY OF PREGNANCY
Implantation is an early critical process where the fetus adheres to the wall of
the uterus. Human implantation is unique, and there is no ideal animal model for
human placentation [238]. This uniqueness is characterized on the maternal side by a
spontaneous and massive decidualization, the process by which the inner membrane
of the uterus, endometrium, transforms itself into a dense cellular matrix. On the
embryonic side there is an almost unlimited invasive process taking place [239]. The
implantation requires a synchronized cross-talk between maternal and embryonic
tissues in terms of molecular and cellular events resulting in healthy uterine growth
and differentiation, invasion and placental formation. Besides the main role of sex
steroids, the complexity of embryo implantation and placentation is exemplified by
the number of cytokines and growth factors with established roles in these processes.
Disturbances of expression and action of these factors can result in absolute or partial
failure of implantation and abnormal placental formation [240].
During pregnancy, immune responses belonging to the adaptive part are
suppressed, while the innate branch of the maternal immune system is activated
[241]. The activation of the innate system leads to an increase in numbers of
granulocytes, and both monocytes and granulocytes enhance their phagocytic
capacity [242]. The cytokine balance during pregnancy is very complex and has been
debated over the last decade. Until recently, it was postulated that successful
pregnancy induces an immune type 2 bias [82]. However, when summarizing old and
recent data, it turns out that for reproductive success, both type 1 and type 2 immunity
are important in a dynamic interplay [243]. A recent study investigated how the
maternal IFN-γ and IL-6 levels, used as markers of the type 1/type 2 immune status,
fluctuate during pregnancy. They found that the type 1 bias with higher levels of IFNγ was dominant at the beginning of pregnancy, balanced in the middle of pregnancy
and replaced by a type 2 bias with higher IL-6 levels at the end of pregnancy [244].
The higher level of IL-6 at the end of pregnancy not only promotes a type 2
immunity, but are also important for the pro-inflammatory events taking place during
parturition [245].
Inflammation has long been recognized as a key feature of both preterm, but
also term labour. An influx of inflammatory cells into the uterus and elevated levels
42
Immunology of pregnancy
of pro-inflammatory mediators such as IL-6 and IL-8 are key events during
parturition [246]. These mediators are especially important in cervical ripening, a
prerequisite for parturition [245]. The inflammatory characteristics of labor are
further demonstrated by the role for NF-κB in the physiology and pathophysiology of
labor [247].
An interesting observation, demonstrating the effect of pregnancy on the
immune status on the mother, is that the more children women give birth to, the less
likely the women are to be IgE sensitized [248-250].
The immune system of the newborn child is also believed to be Th2 skewed
[251]. However, IL-5 and IL-13, two important cytokines for the Th2 development
are showing different patterns at birth where IL-5 is being suppressed, while IL-13 is
not, illustrating the difficulty in attributing a strict Th2 type to the immune system of
the neonate [252].
Maternal influences on the child
In utero
The potential effects of environmental stimuli on the immune system are
greatest in early life including fetal life, when the immune system is going through
important developmental processes. Maternal influences during fetal life could be
particularly important for the development of immune regulation and tolerance
induction. Studies indicate that maternal allergy influences the in utero environment
[253], which could further shape neonatal immunity. The fact that maternal but not
paternal total IgE levels correlate with cord blood (CB) total IgE [254;255] also
indicates that maternal factors, placental factors, or both have an impact on perinatal
allergic sensitization. Allergens have been shown to cross the placental barrier [256].
A fact that further illustrates this event is that T cells from unborn as well as newborn
babies can exhibit a memory phenotype, specific for common allergens [257].
However, studies have failed to show a consistent dose-response relationship between
allergen exposure in fetal life and allergen specific reactivity in cord blood. A
probable cause for this lack of correlation is that most of the allergens that cross the
placenta are retained in the placenta [258]. Studies comparing exposure levels to
43
Petra Amoudruzvvvv
pollens indicate that even though there was a trend towards increased risk of
sensitization in children whose mothers were exposed to high allergen levels during
pregnancy [259], the effect was not as evident as if the children were exposed to high
levels during early infancy [260], or if the mother was suffering from allergic disease
[261]. A very recent study questioning the sensitization of the child during the in
utero period interestingly showed that the allergen-specific IgE in cord blood is NOT
produced by the fetus, but seems to be the result of transfer of maternal IgE to the
fetus [262]. Following the observations of that study, our earlier results of presence of
IgE in the placenta [263], strongly suggests that the IgE found in placental tissues is
of maternal, and not fetal origin.
However, a murine study by Hertz et al suggests that in utero effects of a
maternal allergic phenotype do exist. They could observe that offspring to OVAsensitized mothers had an increased Th2 response at birth. Further, when immunized
with a novel antigen, the young mice born to allergic mothers had an enhanced
allergic response as compared to offspring derived from non-allergic mice [264]. The
reason for this influence of maternal allergy is not fully understood, but it has been
demonstrated, among other things, that the fetus is exposed to intact amniotic fluid
IgE via the gastrointestinal tract [265].
Studies of prenatal exposure in humans have shown to result in measurable
phenotypic responses in the newborn [164;266;267]. Farming environment, levels of
endotoxin in house dust and presence of cat or dog have been suggested as factors
affecting the IFN-γ and IL-6 producing capacity during the first 3 months of life
[267], a fact that could have implications for allergy development. In a similar study
looking at exposure of stable environment, the strongest protective effect against IgE
sensitization of the child was found for maternal stable work during pregnancy [164].
They could further observe that maternal exposure to stable environment during
pregnancy, rather than the current exposure to several farm characteristics, was
associated with increased gene expression of CD14, TLR2 and TLR4 in the children.
Results, in line with this, were further established in a study where maternal allergies
were associated with lower levels of CD14, TLR2 and TLR4 mRNA in cord blood
samples [268]. The idea that LPS exposure during pregnancy can modulate the
development of allergies in the offspring has also been demonstrated in mice
[269;270]. Another indication of an in utero effect on child sensitization is that IgE in
44
Immunology of pregnancy
cord blood is reduced with increasing birth order [255]. The importance of geneenvironment interactions in the context of allergic disease also apply to the in utero
environment. A study recently suggested that different IL-13 gene polymorphisms in
the child modify the effect of in utero exposure to tobacco smoke on persistent
wheeze and asthma in childhood [271].
Understanding the maternal influences on the child is of major importance
when it comes to the important area of how to avoid sensitization in the child. The
increased knowledge of tolerance induction has shifted the former belief of allergen
avoidance into a current view of a more deliberate exposure. As an example, a very
recent study showed that maternal intake of fish oil during pregnancy decreased
several compounds, relevant to allergic susceptibility, in the CB, such as IL-4 and IL13 [272].
A murine study from 2001 proposed that breast feeding by mothers that are
immune to an antigen suppresses the development of an allergic response to the same
antigen in the pups [273]. A more recent study [274] furher investigated the influence
of preconception immunization of the mother on sensitization in the newborn.
Neonatal sensitization was induced by allergen exposure during pregnancy and
breastfeeding. Maternal immunization efficiently decreased the passage of free
antigens through breastfeeding, which inhibited the enhanced IgE antibody response
after postnatal antigen exposure in the pups. Together, these findings suggest that
early life sensitization could be avoided by preventive vaccination of the mother.
Breastmilk
Human milk is the communication medium between the maternal immune
system and the child. It has several immunological roles for the child; it promotes
maturation of the gut barrier function, protects from pathogenic insults, and can also
actively attenuate early inappropriate inflammatory reactions [275]. Colostrum is the
specific first diet of mammalian neonates, and it contains a variety of antimicrobial
substances that function to protect the lactating mammary gland, as well as the
newborn child at a time when its immune system is still immature. Human milk
contains its own immune system with a wide range of soluble and cellular factors.
The cellular compartment comprises 55-60% macrophages, 30-40% neutrophils and
45
Petra Amoudruzvvvv
5-10% lymphocytes (where >80% are T cells) [276]. In animal models, lymphocytes
from the milk are able to traverse the neonatal intestine, showing that the breast milk
indeed can have a systemic influence on the child [277].
Mammary epithelial cells are the source of chemokines such as IL-8 and
RANTES [278], and as a part of a functional immune system, lactating mammary
glands also have a local production of antibodies, mainly secretory IgA (sIgA). These
antibodies are highly directed against infectious agents in the mother’s environment,
mainly respiratory and intestinal pathogens that the child most likely will encounter
[279]. Colostrum and mature milk contain many cytokines and chemokines, including
IL- 1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, TGF-β, IFN-γ and TNF [280-283].
These cytokines and chemokines, in combination with the ingested maternal
immunoglobulins and nonspecific antibacterial components, likely play an important
role in modulating immunologic development in the newborn child.
Additionally to child survival, breastfeeding reduces the risk of developing
various immune-related conditions [276]. Its connection to allergic disease is
somewhat complex. It is speculated that in IgE-mediated allergy, manifested as
dermatitis or asthma, breastfeeding is less protective than in non-IgE-mediated
gastrointestinal disease. Breast milk has been suggested to be a transmission route of
allergic susceptibility from mother to child, even though these results are from animal
model systems [284;285]. In one of the studies both serum and cell membrane-bound
IgE were undetectable in newborn foals before colostrum uptake and peaked on days
2–5 after birth. At the peak, the IgE levels in the mothers’ milk were strongly
correlated to the serum IgE levels in offspring, demonstrating that the milk is most
probably the transference medium [284]. Breast milk contains dietary antigens [286],
and a very recent murine study showed that airborne antigens that the female is
exposed to during lactation can also be found in the breast milk [149]. In that study
they demonstrated an interesting tolerogenic effect where pups receiving breast milk
containing airborne allergens acquired an antigen-specific protection from allergic
airway disease later on.
46
Immunology of pregnancy
The sibling effect
Several studies have shown that having older siblings is protective against the
development of allergic diseases [165]. The most widely accepted explanation for this
phenomenon is that having older siblings results in higher levels of exposure to
common infectious agents at a younger age. Despite this, no specific infectious agent
or exposure has been linked convincingly and consistently with allergy risk [287]. A
study investigating the protective effect of older siblings interestingly showed that the
onset of IgE sensitization was delayed in younger siblings, but not prevented per se,
suggesting that the apparent protective effect of older siblings on allergic diseases
reported elsewhere only delays the onset of IgE sensitization [288]. Recent interest in
the mechanisms behind the sibling effect has turned the focus to prenatal events.
Thus, at birth, IgE levels in cord blood and cellular responses to allergens show a
decrease with increasing birth order, indicating that the sibling effect may instead
have its origin in utero [255].
47
Petra Amoudruzvvvv
THE PRESENT STUDY
AIMS
Increasing data suggest that immunological events taking place in a pregnant
woman have a large impact on the immune system and allergy development of the
neonate. Further, early life innate immune reactions are nowadays considered to be of
major importance in relation to allergic conditions. The work presented in this thesis
concerns several immunological aspects regarding the maternal immune system and
its influences on the immune system of the newborn. More precisely, paper I and II
consider maternal immune characteristics that could influence the child, paper III and
IV study the maternal influence on the child, while the last paper (V) considers the
children when they have a manifested allergic disease.
The precise aims of each paper are listed below
Paper I: Pregnancy is an immunological challenge for the maternal immune system.
Disease could further modulate the maternal immune system during pregnancy,
which could have implications for the fetus. In paper I, we wanted to investigate how
in vitro PBMC cytokine responses (IL-1β, IL-6, IL-10 and IL-12) are influenced by
pregnancy and allergic status in women, before and after microbial stimulation.
Paper II: Children of immigrants from developing countries have a higher risk of
certain immune mediated diseases than the indigenous population. Here, we wanted
to investigate if maternal immunological characteristics, in terms of TNF, IL-1β, IL6, IL-8, IL-10 IL-12, TGF-β1 and 2 and sCD14, are influenced by factors such as
maternal country of birth, and more recent exposures such as number of siblings.
Paper III: Maternal allergy could influence the immune system of the fetus. Here,
we wanted to examine how a maternal allergic phenotype influences the innate
immune system of the newborn. Therefore, we investigated CD14, TLR2 and TLR4
surface expression on monocytes in CB, as well as cytokine production (TNF, IL-1β,
IL-6, IL-8, IL-10 and IL-12), following in vitro culture of CBMC with LPS or PGN.
48
Aims of the study
Paper IV: In paper III we found a defective IL-6 response to PGN in newborns from
allergic mothers, in spite of unaltered TLR expression. This finding suggested that
there could be important events downstream the TLRs accounting for the decreased
IL-6 levels. Here we aimed at analyzing the important intracellular component; p38MAPK, involved in both LPS and PGN-mediated signaling. We further aimed at
investigating how the responses to innate stimuli develop with age, by examining the
same group of children at birth and at 2 years of age.
Paper V: As we in paper II and III revealed a defective response to PGN in children
of allergic mothers at birth, as well as at 2 years of age, we here wanted to investigate
children at 5 years of age when they have a more manifested disease. Further, we
wanted to study the monocyte population following microbial stimulation in more
detail, with regard to the distribution of monocyte subsets. To do this, we analyzed
monocytic CD14, CD16, TLR2 and TLR4 expression, as well as PBMC production
of TNF, IL-1β, IL-6, IL-8, IL-10 and IL-12 upon in vitro stimulation.
49
Petra Amoudruzvvvv
METHODOLOGY
The methods used for paper I-V are described in detail in the respective
“Material and methods section”. The following methods were used in this thesis:
-
Clinical evaluation (paper I, III and IV)
-
Phadiatop/Cap-FEIATM (paper I, III and IV)
-
Skin prick test (SPT) (paper I, III and IV)
-
Questionnaire (paper I-IV)
-
ELISA (paper I-III)
-
ELISpot (paper I)
-
Separation of CBMC and PBMCs (paper I, III and IV)
-
In vitro activation of CBMC and PBMCs (paper I, III and IV)
-
Cytometric Bead Array (CBA) for cytokine and chemokine analysis (paper IIIV)
-
Flow cytometric analyses (paper II-IV)
-
Statistical analyses (paper I-IV)
The studies were approved by the ethics committees of the Karolinska University
Hospital, Stockholm Söder Hospital and the Uppsala region of Sweden. All families
gave their informed consent.
50
Results and discussion
RESULTS AND DISCUSSION
Impact of pregnancy and allergic status on cytokine responses (І)
The involvement of type 1 and type 2 cytokines in allergic disorders has been
extensively studied [289;290]. In paper I we also wanted to assess the role of the
innate immune system with regards to allergy and pregnancy. This was achieved by
looking at production of IL-1β, IL-6, IL-10 and IL-12 in response to LPS in allergic
and non-allergic women. As pregnancy induces several innate and pro-inflammatory
components of the immune system [241], we also compared cytokine levels at
pregnancy with 2 years after.
We demonstrate here that IL-1β, IL-6, IL-10 and IL-12 production in PBMCs
from adult women are not influenced by the allergic status, neither for unstimulated
cells, nor for cells stimulated with a microbial substance (LPS). We also show that the
lack of differences attributed to allergic status is not affected by pregnancy.
Differences in receptors recognizing microbial products have been shown to influence
the likelihood of developing allergic diseases [200]. A subsequent diversity in
cytokine production would be a natural consequence of these differences [199;291].
However, our results indicate that either these differences are not seen in adult
women, or alternatively, differences in receptor expression does not arbitrarily mean
that the final response (here cytokines) would have to differ.
Further, we could not find any differences between allergic and non-allergic
individuals in terms of IL-10 or IL-12 production when stimulating the adaptive
immune system (PHA and allergen extracts). This lack of differences in allergeninduced responses between allergic and non-allergic individuals might be explained
by the fact that single allergen-specific T cells constitute a very small fraction of the
whole CD4+ T-cell repertoire [292]. Another possible explanation is that as the
allergen extracts used were not tested for LPS contamination, an allergen-specific
reaction might have been masked by an innate response induced by LPS. Earlier
studies in our group have shown that a diminished number of IL-12 producing cells in
CB, in response to allergen extracts, is associated with IgE sensitization at 2 years of
age [133]. This could also be the result of a failure to respond properly to LPS, and
not to the specific allergens. The finding of no differences in IL-12 production in
51
Petra Amoudruzvvvv
response to allergen extracts between allergic and non-allergic women in paper I
could be explained by the same reasoning. This would agree with results showing that
monocyte-derived DCs from highly atopic adult individuals are not impaired in their
IL-12 responses to toll-like receptor ligands [136].
Further, we compared the cytokine pattern in women during pregnancy and 2
years after pregnancy. We observed elevated spontaneous innate cytokine levels (IL1β, IL-6) during pregnancy, findings in line with previous publications illustrating the
activated innate immune components during pregnancy [241]. Also here, we found
that PHA, a T-cell stimulus, induced lower production of IL-10 and IL-12 during
pregnancy than 2 years after. This is in agreement with the theory of a suppressed
adaptive immunity during pregnancy. IL-10 and IL-12 are produced by cells of both
the innate and the adaptive immune system. Thus, the lower IL-10 and IL-12
production in response to PHA could be attributed to a diminished production of these
cytokines from T- and B cells, respectively. However, stimulation of T cells has been
observed to upregulate APC activity via stimulation through CD40-CD40L
interactions [293]. Then, the diminished IL-10 and IL-12 production during
pregnancy, in response to T-cell stimuli, could also be originating from APCs that
have received lower stimulation signals from “pregnancy-suppressed” T cells.
Whether IL-12 production is influenced by pregnancy is debated [294;295]. We
did not find any statistically significant alterations in IL-12 production due to
pregnancy, neither for unstimulated cells, nor for LPS- stimulated cells. No
differences in IL-1β and IL-10 production/producing cells in response to LPS were
detected, when comparing the pregnant state of the women versus the non-pregnant
state. The greater effect of LPS on IL-6 production 2 years after pregnancy than
during pregnancy is perhaps an unexpected result. It is possible that pregnancyassociated immune modulation results in an increased ability to respond to microbial
stimuli after pregnancy. It should also be noted that cells of the adaptive immune
system express PRRs that could respond to microbial stimuli such as LPS
[11;13;296].
Studies have pointed towards an important role of pregnancy and birth
regarding immunological changes in women [248;249]. In this study, we found
significantly lower total serum IgE levels in allergic women 2 years after than during
pregnancy. Numerous studies have shown that having older siblings is protective
52
Results and discussion
against the development of allergic diseases [165], which is usually attributed to a
“healthy” immunological shift due to early acquired infections. In the light of our
findings however, it is tempting to speculate that the protective effect of having older
siblings is at least partially explained by the modulated immune system in the mother
following multiple pregnancies.
Maternal exposures and breast milk characteristics (II)
Populations in high infectious exposure countries are at low risk of immunemediated diseases such as Crohn’s disease [181] and allergy [165]. This low risk is
maintained upon immigration to an industrialized country, but the offspring of such
immigrants have a higher immune-mediated disease risk than the indigenous
population [182;183]. In paper II we hypothesized that early life exposures in a
developing country shape the maternal immune system, which could have
implications for the offspring born in a developed country with less infectious
exposures. To measure one means of maternal influence, we measured breast milk
compounds that are abundant in colostrum, and have been shown to be of relevance to
the immune system of the newborn. It has previously been shown, in a murine study,
that breast milk is a transmission route for allergy susceptibility from mother to
offspring [285], indicating that mediators in the breastmilk indeed reflects the immune
status of the mother.
To compare women with different childhood exposures, breast milk was
collected from 64 mothers who either; 1) grew up in Sweden, considered to have had
low levels of microbial exposures in childhood or 2) spent their first ~10 years of life
in a developing country, but now living in Sweden, considered as having been
exposed to higher levels of microbial substances in childhood.
Our results show that immigrants from a developing country had statistically
significant higher levels of breast milk IL-6, IL-8 and TGF-β1 compared to the native
Swedish mothers (Figure 3). Further, regardless of maternal country of birth, a larger
number of previous pregnancies was associated with down-regulation of several
substances, statistically significant for soluble CD14 and IL-8. The results suggest that
maternal country of birth may indeed have immunological consequences still in
adulthood, which could be relevant to disease risk in offspring. Such a mechanism
53
Petra Amoudruzvvvv
may explain the higher immune-mediated disease risk among children of migrants
from a developing to developed country.
The mechanisms behind the differences in breast milk compounds due to
childhood exposures are not clear. However, one could speculate that epigenetic
events play a role. It has been shown that environmental factors alter epigenetic
events such as methylation processes [224]. LPS exposure that have been negatively
associated with allergic disease [219] plays a role in gene silencing, acting as a potent
inflammogen that stimulates another epigenetic event; acetylation of histones [297]. If
the immigrant mothers were exposed to high levels of LPS and similar substances
during childhood, a probable cause for the long time effects could be at the epigenetic
level.
With the design of our study, the possibility that contemporaneous exposures
such as dietary habits and stress could have influenced the results can not be ruled out.
However, adjustment for the person-per-room ratio (crowding) did not notably
influence the reported associations. It is known that lifestyle factors influence the
composition of the gut flora [298], which might be transferred to the offspring.
However, this fact cannot readily explain the higher incidence of inflammatory
diseases in offspring to immigrants when they are born in a developed country instead
of their native country.
To have a “control group”, we also included women who were raised and gave
birth in Mali (Africa). Unfortunately, we did not have access to complete
demographic data of these women, and the results from that group were therefore not
included in the paper. The breast milk components that were investigated in these
women living in Africa did not differ from the ones in breast milk of women who had
migrated to Sweden (Figure 3). Interestingly, and to our surprise, a comparison of the
milk from women living in Africa with the milk from native Swedish mothers,
revealed no differences regarding inflammatory mediators such as IL-6 and IL-8.
However, TGF-β1 and sCD14 levels were higher in the women living in Africa,
compared to native Swedish mothers. The observation of higher TGF-β levels in
breast milk, both from African- and immigrated mothers compared to women being
raised in Sweden, is of great interest as inflammatory diseases are more prevalent in
the western world compared to developing countries. Since breast milk TGF-β was
recently shown to be of major importance for tolerance induction and protection from
54
Results and discussion
allergic asthma in mice, these higher breast milk TGF-β levels in women raised in
developing countries might provide a partial explanation to why children born in
Africa have a lower incidence of allergy than children in the western world. The
finding of no differences between women living in Africa and women who had
migrated to Sweden, confirm our hypothesis of the important childhood exposures in
modifying the immune system. Additionally, it argues against the possibility of
current exposures as being the cause of differences found between immigrant- and
Swedish mothers.
Figure 3. Summary of results including women from 3 groups; native Swedish mothers,
immigrated mothers in Sweden and women living in Mali.
Even though we did find differences in breast milk composition due to maternal
country of birth or previous pregnancies, the major question is how these differences
affect the newborn child. To investigate this issue we stimulated cord blood
mononuclear cells (CBMCs) with the breast milk alone, or with the addition of
microbial stimuli, and analyzed the cytokine responses. Preliminary data from this
ongoing study indicate that breast milk from native Swedish mothers and African
women living in Mali show no differences in their stimulatory capacity (Figure 3).
However, breast milk from the immigrant mothers, together with LPS stimuli,
induced higher IL-6 levels than breast milk from the two other groups. As breast milk
55
Petra Amoudruzvvvv
from immigrant mothers had higher levels of IL-6 and IL-8 than the Swedish native
mothers, these augmented responses could explain why children of immigrants have a
high risk of inflammatory diseases. Intriguingly though, we would have expected the
breast milk from African women to have the same stimulatory capacity as the breast
milk from mothers who had migrated from developing countries, as there were no
differences in breast milk composition. Perhaps there are other factors, not measured
in this study, which are lost or increased upon migration to a developing country,
which would explain the differences in stimulatory capacity of the breast milk.
It is possible that the well known sibling effect seen for diseases such as allergy
[159;161] and multiple sclerosis [299] influences disease risk in the child through the
action of previous pregnancies on maternal immune characteristics. Further, a
maternal influence on neonatal immunity has been shown in previous studies,
although mainly in animal studies. Both in utero [164;257;264;266;267], as well as
breast-milk related effects have been reported [149;284]. The difference in breast milk
composition in this study is of course only one part of the maternal influences that the
mother exerts on the child. However, we believe that the immune status of the mother
is reflected in the breastmilk, and together with the influences of the in utero
environment has a large impact on the immune system of the newborn. We further
suggest that the imbalance in pattern of early life infection between mother and
offspring among immigrants from a developing to an industrialized country may help
to explain the increased risk of some immune-mediated disease in such offspring.
Neonatal immune responses to microbial stimuli (ІII)
TLR-mediated signaling plays an important role in diminished IgE-specific
immune responses. This, and the fact that susceptibility to allergic diseases can be
transferred from mother to child in utero [253;257], prompted us to look at how innate
CB responses to microbial stimuli were affected by maternal allergy. More precisely,
in paper III, we investigated the influence of allergic status in the mother on the
monocytic CD14, TLR2 and TLR4 expression in their newborn babies upon microbial
stimuli (LPS and PGN). We also measured cytokine and chemokine (TNF, IL-1β, IL6, IL-8, IL-10 and IL-12) responses from stimulated PBMCs using the CBA method.
56
Results and discussion
Even though we could not detect any significant differences in CD14, TLR2 or
TLR4 receptor expression between mothers with different allergic status, the allergic
mothers showed a tendency towards a higher surface expression of TLR2 and TLR4
on their monocytes.
Further, examination of CB monocyte expression of TLR2, TLR4 and CD14
revealed no significant differences between children with allergic mothers and
children with non-allergic mothers. However, when we compared the mothers with
their children, CB monocytes from children with allergic mothers had a significantly
lower cell surface expression of TLR2 and TLR4 than monocytes from their mothers.
This was the case both before and after PGN and LPS stimulation. No corresponding
difference was observed when we compared TLR2 and TLR4 expression on CB
monocytes and maternal monocytes within the non-allergic group. In addition, there
was a positive correlation between mothers and children regarding TLR2 expression
on monocytes regardless of maternal allergy, suggesting a maternal influence. The
children’s “true” response to microbial stimuli might therefore be distinguishable only
if analyzed later in infancy and/or if investigating the effector responses.
Many studies have demonstrated a down regulation of TLR4 in response to
LPS, and this has been proposed as being a possible mechanism for LPS tolerance
[300]. In our study, the down regulation after LPS stimulation was similar in
monocytes from mothers and in monocytes from CB of their children regardless of
allergic status. The same pattern was seen for TLR2, as the upregulation upon PGN
stimulation was similar in all groups. These data suggest that the monocyte population
is functioning very well already at birth, and that the allergic status does not influence
the responding capacity of TLR2 and 4.
However, the apparent absence of significant differences in receptor expression
on monocytes from women with different allergic status, does not rule out that there
are genetic differences in TLRs or in the signaling cascades following TLR triggering.
Indeed, Poltorak et al [301] demonstrated that a mouse strain with a mutation of the
TLR4 gene and wild type mice showed a similar down regulation of TLR4 upon LPS
stimulation, suggesting that down regulation of TLR4 occurs independently of a
TLR4 dependent signaling pathway.
57
Petra Amoudruzvvvv
We did not detect any significant differences regarding cytokine levels between
PBMCs of allergic and non-allergic mothers in response to LPS or PGN. However, a
trend towards an augmented release of pro-inflammatory cytokines after PBMC
stimulation was seen for the allergic mothers. This is probably explained by the
inflammatory nature of allergic disease, i.e. that the allergic mothers had a somewhat
activated immune system even in the basal state.
However, the cytokine- and chemokine release triggered by TLR2 stimulation
in CB revealed some interesting differences. CBMC from children with maternal
allergic disease released significantly less IL-6 in response to PGN stimulation,
compared to CBMC from children without maternal allergic disease, and there was
also a trend towards less secretion of IL-8. This observation might indicate that subtle
differences at the cell surface receptor level could have implications for the signaling
cascade triggered by TLRs. It is of interest that Pasare et al [111] showed that
microbial signaling via TLRs in mice abrogated the suppression of CD4+CD25+ Treg
cells by inducing IL-6 release from DCs and macrophages.
Figure 4. Innate immune cells control Treg cell development and activation. The activation state
of APCs determines the CD4+ T cell response. Resting APCs may promote the development of
CD4+CD25+ Treg cells by inducing Foxp3 expression in CD4+ T cells. During infection by pathogens,
recognition of PAMPs by TLRs results in activation of APCs. The APCs then produce IL-6 and other
soluble factors that together override the suppressive effects of Treg cells, allowing efficient generation
of effector T-cells (TE) cells against the pathogen (Science 2003; 299:1030-1). Reprinted with
permission from AAAS.
They speculate that a reduced IL-6 production following microbial exposure
could result in an over-regulated immune response, not allowing proper Th1-type of
immune responses to develop (Figure 4). Relating this to our findings, it would
58
Results and discussion
implicate that a lowered anti-microbial response early in life could result in a
hampered/slower Th2-Th1 shift, which results in an increased risk for allergy
development. A study supporting this theory very recently showed that if monocyte
derived DCs are not exposed to microbial stimuli, the CD4+ population is Th2 skewed
[170].
For sCD14 levels in plasma, no significant differences between allergic and
non-allergic mothers or between their children could be observed, although we
observed a tendency towards higher sCD14 levels in allergic mothers and their
children, which is in agreement with previously published data from our research
group [206]. The association of sCD14 with allergic disease is hard to interpret as
studies have shown inconsistent results, possibly due to gene-environment
interactions [205].
Even though the sample size in this study was small (9/10), other studies have
provided results that are in line with ours [268;302]. In a study from 2005, maternal
allergies were shown to be associated with significantly lower levels of CD14, TLR2
and TLR4 mRNA in cord blood samples [268]. In contrast to our study, where we did
not detect any differences between the mothers in terms of cell surface expression of
CD14/TLR2/TLR4, they found that maternal allergy was associated with decreased
levels of mRNA from the same receptors. The discrepancy between their results and
ours may be due to sample size, or more likely to the fact that they investigated
mRNA expression, when we looked at receptor levels on the cell surface. Another
study found that PGN-induced IL-10 production and induction of FOXp3, a
transcription factor expressed in Treg cells, were higher in CBMC without than with
maternal allergy [302]. Interestingely, this lower Treg cell activation coupled to
allergic status of the mother could have a link to our results of lower IL-6 levels in CB
from children of allergic mothers, as IL-6 has been shown to have a regulatory role in
the process of bridging innate and adaptive immunity [105].
Maternal allergy and monocyte signaling in 2-year-old children (IV)
In paper IV we aimed at investigating if the decreased IL-6 production in
newborns of allergic mothers seen in paper III persisted during infancy, and if the
responsible mechanism could be found intracellularly. We considered p38-MAPK to
59
Petra Amoudruzvvvv
be a suitable intracellular target for this study since it is involved in both LPS [303]
and PGN signaling [304], and a key factor in inflammatory responses [22]. By
applying a novel flow cytometry-based technique [305] we analyzed p38-MAPK
phosphorylation in the same infants at birth, and at 2 years of age after microbial
(LPS, PGN) stimulation.
The results showed that there were no significant differences between the
groups of infants concerning basal levels (unstimulated cells) of IL-6, neither at birth
nor at the age of 2. However, CBMCs from newborns with allergic mothers tended to
have a lower IL-6 response following a microbial challenge compared with the group
without maternal allergy, while p38-MAPK activation levels did not differ
significantly between the groups. Although the results did not reach statistical
significance, they confirm our results in paper III. LPS stimulation did not result in
statistically significant differences between the two groups of children in either paper
III or paper IV, although trends were seen in the same direction as for PGN.
One should keep in mind that a microbial encounter in reality is not strictly
divided into LPS or PGN, but a more heterogeneous simultaneous exposure from
several compounds. By mixing several microbial substances in these types of
experiments one would receive further information about an individual’s capacity to
respond to this type of challenge.
Further, stimulation of PBMCs from the same children at the age of 2 revealed a
significantly lower IL-6 production upon PGN stimulation in children with allergic
mothers compared with age-matched infants with non-allergic mothers. Increasing the
sample size of 2 year olds made the significance stronger, which reinforces the
observed results. Confirming the inability of children with maternal allergic disease to
respond, a tendency towards a similar difference was also seen for LPS stimulation.
The extended group of 2 year olds further strengthened this tendancy.
To investigate whether the decreased release of IL-6 after LPS and PGN
stimulation could be a consequence of reduced activity of p38-MAPK in monocytes,
we analyzed the p38-MAPK phosphorylation ability in the stimulated CD14+
monocytes. Two-year old infants with allergic mothers displayed markedly reduced
CD14+ monocyte p38-MAPK phosphorylation after LPS and PGN challenge
compared to children with non-allergic mothers (Figure 5).
60
Results and discussion
Figure 5. Representative histogram plots displaying phosphorylated p38-MAPK upon
peptidoglycan challenge (unshaded lines) in 2-year-olds with allergic mothers (a) and with non-allergic
mothers (b). Unstimulated controls are shaded.
This altered anti-microbial response was attributed to maternal allergy rather
than to being IgE-sensitized at 2 years of age. The activity of p38-MAPK is important
for driving Th1 responses [306]. Thus, the lower p38 activity found in connection to
allergic heredity in this study would suggest that there is a reduced capacity to induce
Th1 responses in these children. As Th1 and Th2 responses are known to counteract
each other [77], a consequence of the reduced p38 activity would be an increased Th2
bias, which is a hallmark of allergic diseases [80;307]. The strong correlation between
p38-MAPK phosphorylation and IL-6, following both LPS and PGN stimulation,
suggests that an altered monocyte function may play an important role in the reduced
anti-microbial IL-6 response in PBMCs from children with allergic mothers.
TLR2 signaling in 5 year old allergic children (V)
In paper III and IV we could show that children of allergic mothers have a
reduced capacity to respond to microbial stimulation in terms of decreased IL-6
responses at birth as well as at 2 years of age, possibly caused by the observed
reduction of p38-MAPK phosphorylation in CD14+ monocytes. In line with these
results, other studies have also demonstrated a selective impairment of TLR2
responses in terms of IL-10 production in children with allergic heredity [302], and in
adults with allergic disease [189].
Age has shown to be of importance for innate immune responses in mice
[310;311]. Also, the immune system of the growing child is going through multiple
61
Petra Amoudruzvvvv
important maturation steps, making interpretations from different studies in humans
difficult due to study populations with different ages. As an example, age is known to
effect responses to LPS in terms of IL-12p70 production [312].
The fact that at 2 years, the IgE sensitization of the children was not correlated
to defective signaling (paper IV) prompted us to look at children with a more
manifested allergic disease. To do this, we looked at the innate immune responses in
relation to allergy development of the child by stimulating PBMCs from 5 year old
allergic/non-allergic children with LPS and PGN. We thereafter analyzed the CD14,
CD16, TLR2 and TLR4 expression as well as the p38-MAPK activity in monocytes.
The inflammatory cytokine responses after 24h of in vitro stimulation were also
measured.
The results showed that for unstimulated cells there were no differences in
frequency of the monocytic subsets or their toll-like receptor levels between allergic
and non-allergic children. Another study on atopic dermatitis (AD), confirming our
results, also failed to detect any differences in the two monocytic subsets or in TLR
levels that could be correlated to disease [189]. Interestingly, they found an impaired
IL-1β and TNF production in response to a TLR2 ligand in the subjects suffering from
AD. In line with this, we could show that children in the allergic group did not
upregulate the TLR2 receptor upon stimuli to the same extent as the non-allergic
children (Figure 6).
Figure 6. Stimulation index (ratio of TLR expression in stimulated/unstimulated monocytes) for
TLR2 after PGN stimulation in the different monocyte populations in allergic (n=10) and non-allergic
(n=10) 5 year old children. °=outlier. The line represents stimulation index=1, which means no
up/downregulation.
62
CD14+CD16+
CD14++CD16-
All CD14 positive cells.
Results and discussion
A very recent study found the same defective upregulation of both TLR2 and
TLR4 together with a reduced upregulation of HLA-DR in monocytes from patients
with AD in response to Staphylococcus aureus enterotoxin B [312], a compound that
act upon both TLR4 and TLR2 in human monocytes. Even though the allergic
children in our study had a reduced TLR2 upregulation in response to PGN we did not
observe any differences in p38-MAPK activity. The reason for this remains unclear,
however it should be emphasized that the signaling cascade in response to PGN
involves many other pathways that could have compensatory roles [6].
We could not observe any differences in TLR4 signaling attributed to allergic
disease in this study, showing that the allergic phenotype here seems to have a larger
impact on the TLR2- rather than the TLR4 signaling.
Another interesting result concerning the TLR4 expression in response to LPS is
that in the CD14+CD16+ subset TLR4 was upregulated, while in the CD14++CD16subset TLR4 was downregulated, regardless of allergic status. There has been
conflicting results regarding the regulation of TLR4 in response to LPS [300]. A
reason for the diverging results could be that a lot of evidence for TLR regulation is
based on mRNA levels, and the cell surface expression of TLR4 do not parallel data
obtained at the mRNA level [313]. Our results of different regulation of TLR4 in the
two monocytic subsets also show that gating and phenotyping of monocytes when
performing this type of experiments are crucial events for the interpretation of the
results obtained.
As we had earlier found a defective IL-6 response to PGN in cells from children
with allergic heredity, at birth (paper III) and at 2 years of age (paper IV), we also
analyzed the production of cytokines in response to PGN at the age of 5. For
unstimulated cells there was a higher production of IL-6 for allergic children. IL-8
and IL-1β levels were also slightly elevated for the allergic children, however not
statistically significant. The pattern of higher spontaneous pro-inflammatory cytokines
in allergic children suggests that once the disease is declared, there is a more
inflammatory state of the immune system in allergic children compared to nonallergic. Further, there were no differences between the two groups regarding p38MAPK activity or cytokine and chemokine production upon stimulation. Our results
are supported by a study in 2006 showing that TNF secretion following in vitro
stimulation of PBMCs was similar in women with and without allergy [302].
63
Petra Amoudruzvvvv
However another study using a different ligand (Pam3Cys) for TLR2 activation could
observe a diminished TNF response in allergic adults compared to non-allergic
individuals [189]. This discrepancy could be the affect of the different microbial
stimuli used in the studies.
Taken together, the results of higher spontaneous cytokine production, and a
decreased TLR2 responsive capacity in monocytes from the allergic subjects in this
study suggests that there is an activated monocytic state in allergic individuals, that
seems to lead to a hypo-responsiveness to further stimulation. This type of hyporesponsiveness in monocytes has previously been demonstrated in patients with atopic
dermatitis [312].
64
Concluding remarks and future perspectives
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Even though reduced stimulation of TLRs might effect the development of
different diseases, the immunological mechanisms responsible for this protective
effect are still largely unknown. The fact that non-immune cells and cells of the innate
immune system [10] as well as the adaptive immune system [11;13] all express PRRs,
further highlight the important interaction and regulation of the traditionally separated
innate- and adaptive immune systems.
The importance of age for the outcome of adaptive immune responses is readily
acknowledged. The results in this thesis further show that age is crucial also for innate
immune responses, as TLR regulation and cytokine production differed between
children at different ages.
A main finding from paper I and III is that the maternal immune system is
affected by the process of pregnancy. This is demonstrated by the association of lower
breast milk cytokine levels in mothers with previous pregnancies (paper II), plus the
lower IgE levels of mothers two years after giving birth (paper I). These results
indicate that the immune system of the mother is modulated as a consequence of
giving birth, but that this modulation also seems to increase with increasing number of
births. Thus, it is possible that the apparently protective effect of older siblings in
allergy [165] and multiple sclerosis [299] may be mediated though maternal immune
characteristics and their influence on the developing infant. Today, women in the
Western world tend to have fewer children, and also to give birth later in life, which
might be important for disease development, both in the women themselves and in
their children.
Summarizing the results from study III, IV and V, one should note a decreased
capacity to respond to microbial stimuli in individuals with allergic heredity, or later
in infancy with an allergic phenotype. This is interesting in relation to the discussion
regarding microbial exposure and allergic disease. Stimulation through PRRs seems to
inhibit allergic disorders, but the findings from this thesis also show that maternal
allergy confers an altered capacity to respond to microbial stimuli; implying that not
only exposures are important. This might also have important implications for
immunomodulatory therapy, as compounds stimulating toll-like receptors are being
aimed at ameliorating inflammatory diseases in the future. The observed influence of
65
Petra Amoudruzvvvv
maternal allergy on the immune system of the neonate is fascinating, but future
studies are needed to further dissect the responsible immunological mechanisms, and
if the influence of the mother disappear with time.
In conclusion, our results from the children show that monocytes from
newborns with allergic mothers are less responsive to bacterial challenge than
monocytes from children with non-allergic mothers, and that this impairment persists
during the first 2 years of infancy. However, at 5 years of age, the manifested allergic
disease seems to be more important for the immune responses than the allergic
heredity. It seems as if the events responsible for allergy development in infancy are
dependent upon heredity, but once the disease is declared, the additional inflammatory
state that the individual suffers from is of more importance.
In a future perspective one would have to take into consideration additional
factors when investigating the maternal influences on the child. Results of the
influence of breast milk in our study, as well as in most other studies, comes from in
vitro set-ups, where only the substances included in the liqous fraction are
investigated. It would be of great interest to also investigate the cellular fractions, as
these most surely play a role for the maturation of the neonatal immune system, but
perhaps also are implicated in susceptibility to disease. Moreover, understanding how
the epigenome responds to environmental exposures will be of great importance for
understanding the gene-environment interactions that seem to be of great importance
for allergy development. Epigenetics also provides an important link between early
developmental environment and later disease [314], and elucidation of these events
will be an important contribution to the knowledge of maternal influences on the
child. A plausible mechanism behind the effects of pregnancy on the immune system
of the mother might indeed be found in epigenetic events, as these effects seem to be
kept in time. Another question mark that needs further investigations is whether the
observed impairment in response to PGN, found in connection to allergic disease in
this thesis, also extends to other microbial ligands and receptors. A hint of that it
might be the case comes from a very recent study showing that DCs from allergic
adults have a reduced capacity to produce IFN-α after stimulation with TLR9 ligands
[315].
66
Acknowledgements
ACKNOWLEDGEMENTS
I would like to express my warm gratitude to everybody that has contributed in any
way in making this thesis a reality, and most especially I would like to thank;
Eva Sverremark-Ekström, for simply being the best supervisor you could ever
imagine. Working with her is easy and inspiring. And thanks also to Mattias for
letting her work so much late at night ☺. Revisions were usually done to the next
day…
All colleagues at the department; especially my room mates Nora Bachmayer,
Yvonne Sundström and Ylva Sjögren for making our room into a nice place to come
to in the morning. I really appreciated our mutual support during the tougher days
Yvonne. John Arko-Mensah, Nnaemeka Iriemenam, Salah Eldin Farouk, Amre
Nasre, Jubayer Rahman, Stefania Varani, Nancy Awah, Maria Johansson, Ebba
Sohlberg, Judith Anchang, Charles Arama, Hedvig Perlmann, Khosro Masjedi,
Margareta “Maggan” Hagstedt, Ann Sjölund, Gelana Yadeta and Anna-Leena
Jaarva. Shanie Saghafian-Hedengren for her class and enthusiasm whether it is
science, wine or life. Ulrika Holmlund for our co-operation and social stuff which
have always been very valuable. Lisa Israelsson, my toast madame, thanks for
everything!! The Tip Tops, the joy, your true interest and help with my dissertation
and thesis, but also for the sharing of our common passion; horses! You know how to
be a good scientist without forgetting other things in life. Halima Balogun for being
so cool and nice to work with. Manijeh Vafa Homann for our discussions about
science, dissertations, love and cultural differences. You also provided us with the
name of Zelzele, we are SO greatful. Pablo Giusti, you were the coolest ☺
newcomer. The “boys” at the physiology department, Olle, Damir and Thomas, for
suddenly giving the Wenner-Gren “meetings” a new meaning. Former students at the
department; Anna Tjärnlund, Camilla Rydström, Ariane Rodríguez, Nina-Maria
Vasconcelos, Qazi Khaleda Rahman, Mounira Djerbi and Anna-Karin Sigfrinius
(Larsson).
67
Petra Amoudruzvvvv
The “seniors” at the department; Klavs Berzins, Marita Troye-Blomberg, Carmen
Fernández and Eva Severinsson for making me a better scientist.
My co-authors Ulrika Holmlund, Shanie Saghafian-Hedengren, Jacob T Minang,
Marita Troye-Blomberg, Gunnar Lilja, Caroline Nilsson, Christina (Tina)
Trollmo, Vivianne (Vivi) Malmström, Katarina Bremme, Annika Scheynius and
Jens Schollin. Scott M Montgomery for his professionalism, our fruitful discussions
and nice co-operation.
My outstanding conference partners (best privileges for a PhD student): Gunnar,
Jacob, Anna-Karin, Lisa, Manijeh, Halima, Shanie, Ylva, Yvonne, Per
Thunqvist, Monica Nordlund, Nicolas Brodszki and Philippe Cabelduc.
Gunnar Lilja, for always being there for me, whether it is work issues or only a
concerned mother who needs to discuss childhood diseases. I’m forever grateful that
you encouraged me to choose the course “Immunology” back in 1999, that’s where
this journey all started.
Thanks to Vivi and Tina at CMM for offering me an exiting future in a stimulating
and nice environment.
The “Uppsalagirls”; Ewa Wredle, Anna Larsson, Mikaela Patel, Monica Roman
and Linnea Nygren-Babol for our regular get togethers that are filled with laughter,
food, chocolate and friendship. We are still waiting for Ewa to get that job close to a
beach so we can have even nicer meetings in the future…
I would be a less happier person if it wasn’t for things and people that light up the
sometimes harder days, special thanks to Peter, Lotta, Tuva, Täby galopp with the
whole Bendik Bö stable, the GAFF board, the SÅEF people and Stall Zygot.
Special thanks to Leif Wretman, and let us hope for Zelzele to be a star.
Thanks to Pär for your professionalism and support. Let this thesis inspire you to
finish yours, it will probably be so good that even your supervisor won’t
understand….
68
Acknowledgements
All my other friends that are a part of why I am what I am; Pia, Katta, Caroline,
Karin, Marie, Christian, Daniel, Jeanette, Krille, Kidd, Anders, Johan F, John,
Yann, Johan L, Jocke and Patrik.
Thanks to my family; Pappa, for the sculpture and your growing interest in my world
of horses, Irene, Jonas, Ullis, Fanny, Iris, Valter, Hugo, the Boëthius family and
most of all to my mother Karin for her warm heart and for always being there for me.
Best of them all, filling my life with so much love, Ebba and Ines. The future is
yours.
Modified, and used with permission from © ScienceCartoonsPlus.com
This work was supported financially by grants from the Swedish Research Council (grants: 57X15160-05-2, 74X-15160-03-2, 74XD-15160-01A, K2004-74X-15042-01A), the Swedish Medical
Society, The Swedish Asthma- and Allergy Research Foundation, Örebro University Hospital Research
Committee; KI Fonden; Visceral; the Cancer- and Allergy-, The Golden Jubilee Memorial-, HRH
Crown-princess Lovisa & Axel Tielman-, Hesselman-, Vardal-, Golje-, Jeansson-, Magnus Bergvall-,
Swärd/Eklund-, Apotekare Hedberg-, Salén- and the Åhlén foundations.
69
Petra Amoudruzvvvv
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