Download Cytokine responses in metal-induced allergic contact in Jacob Taku Minang

Cytokine responses in metal-induced allergic contact
dermatitis: Relationship to in vivo responses and
implication for in vitro diagnosis
Jacob Taku Minang
Stockholm, 2005
Doctoral Thesis from the Department of Immunology, The Wenner-Gren Institute,
Stockholm University, Stockholm, Sweden
Cytokine responses in metal-induced allergic contact
dermatitis: Relationship to in vivo responses and
implication for in vitro diagnosis
Jacob Taku Minang
Stockholm, 2005
ISBN 91-7155-158-1 pp 1-79
PrintCenter, Stockholm University 2005
© Jacob Taku Minang
Stockholm 2005
“Without love, benevolence becomes egotism”
-Dr. Martin Lurther King Jr. (1929-1968)
“People like you and I, though mortal of course like everyone else,
do not grow old no matter how long we live…[We] never cease to stand like
curious children before the great mystery into which we were born.”
- Albert Einstein (1879-1955): in a letter to Otto Juliusburger (1867-1952).
To my dear mum, Minang Margaret Atih
ORIGINAL PAPERS
This thesis is based on the following original papers, which are referred to in the text by their
Roman numerals.
Paper I.
Jacob T. Minang, Marita Troye-Blomberg, Lena Lundeberg and Niklas Ahlborg (2005).
Nickel elicits concomitant and correlated in vitro production of Th1-, Th2-type and regulatory
cytokines in subjects with contact allergy to nickel. Scand J Immunol. 62:289-296.
Paper II.
Jacob T. Minang, Iréne Areström, Bartek Zuber, Gun Jönsson, Marita Troye-Blomberg,
Niklas Ahlborg (2005). Regulatory effects of IL-10 on Th1- and Th2-type cytokines induced
in response to the contact allergen nickel. Submitted.
Paper III.
Jacob T. Minang, Iréne Areström, Marita Troye-Blomberg, Lena Lundeberg, Niklas Ahlborg
(2005). In vitro cytokine responses to metal ions in subjects with allergic contact dermatitis to
nickel, cobalt, palladium, chromium and gold. Manuscript.
Paper IV.
Jacob T. Minang, Niklas Ahlborg and Marita Troye-Blomberg (2003). A Simplified ELISpot
assay protocol used for detection of human interleukin-4, interleukin-13 and interferon-γ
production in response to the contact allergen nickel. Exogenous Dermatol. 2:306-313.
Reprints were made with permission from the publishers.
TABLE OF CONTENTS
BRIEF OVERVIEW OF THE IMMUNE SYSTEM
The immune system
Innate immunity
Acquired immunity
T-lymphocyte subsets
The Th1/Th2 concept
T-regulatory cells (Tr1 and Tr2/Th3)
B lymphocytes and IgE class switching
HYPERSENSITIVITY/ALLERGIC REACTIONS
Classification of hypersentivity/allergic reactions
CONTACT DERMATITIS
Allergic contact dermatitis
Atopic contact dermatitis
Irritant contact dermatitis
Hapten immune system interaction in allergic contact dermatitis
RELATED BACKGROUND
Biochemistry of transition (heavy) metals
Transition metals and allergic contact dermatitis
Molecular and cellular mechanisms underlying metal-induced
allergic contact dermatitis
Cross reactivity versus co-sensitisation in metal-induced allergic
contact dermatitis
Cytokines investigated in this study
In vivo diagnosis of allergic contact dermatitis
In vitro diagnosis of allergic contact dermatitis
Cytokine detection assays
PRESENT STUDY
Objectives
Methodology
Study subjects
Measurement of cytokine levels in cell supernatants by ELISA
(papers I, II and III)
Evaluation of the detection sensitivity of one-step and two-step
reagents in capture ELISA (paper IV)
Enumeration of cytokine-producing cells by ELISpot
Phenotypic characterisation of Ni2+-specific cytokine producing
cells using depletion experiments (paper II)
Statistical methods
Results and Discussion
Paper I
Paper II
Paper III
Paper IV
CONCLUSIONS AND PERSPECTIVES
FIGURE LEGENDS
ACKNOWLEDGEMENTS
REFERENCES
APPENDICES: PAPERS I-IV
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ABBREVIATIONS
ACD
ALP
APC
Au1+/3+
BcR
CD
CLA
Co2+
Cr3+/6+
CTL
DC
DTH
ELISA
ELISpot
FACS
FBS
FCS
FSC
GM-CSF
IFN
IL
KC
LC
LPS
LTT
mAbs
MHC
Ni2+
NK
PAMP
PBMC
PBS
Pd2+
PHA
pNPP
PPD
PRR
PVDF
SA
SSC
Tc
TcR
TGF
Th
TLR
TNF
Tr
TT
Allergic contact dermatitis
Alkaline phosphatase
Antigen-presenting cell
Gold ions
B-cell receptor
Cluster of differentiation
Cutaneous lymphocyte antigen
Cobalt ion
Chromium ions
Cytotoxic T-lymphocyte
Dendritic cell
Delayed-type hypersensitivity
Enzyme-linked immunosorbent assay
Enzyme-linked immunospot assay
Flourescent activated cell sorted
Fetal bovine serum
Fetal calf serum
Forward scatter
Granulocyte monocyte colony stimulating factor
Interferon
Interleukin
Keratinocyte
Langerhans cells
Lypopolysaccharide
Lymphocyte-transformation test
Monoclonal antibodies
Major Histocompatibility Complex
Nickel ion
Natural killer cell
Pathogen-associated molecular patterns
Peripheral blood mononuclear cells
Phosphate buffered saline
Palladium ion
Phytohaemagglutinin
para-nitro-phenyl phosphate
Purified protein derivative
Pattern-recognition receptor
Polyvinyldiflouride
Strepavidin
Side scatter
T-cytotoxic cell
T-cell receptor
Transforming growth factor
T-helper cell
Toll-like receptor
Tumour necrosis factor
T-regulatory cell
Tetanus toxoid
Cytokine responses in metal-induced ACD
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BRIEF OVERVIEW OF THE IMMUNE SYSTEM
The immune system
The human immune system is a truly amazing constellation of responses to attacks from
outside the body. It has many facets, a number of which can change to optimize the response
to these unwanted intrusions. The immune system has a series of dual natures, the most
important of which is self/non-self recognition. The others are: natural/adaptive
(innate/acquired), cell-mediated/humoral and primary/secondary immunity. Functional
integration of the immune system is accomplished mainly by cell-to-cell communication that
relies on cell adhesion molecules that act in concert with a number of small soluble
molecules. Every immune system cell is equipped to synthesize and release a variety of these
small molecules, mostly cytokines, which travel to other cells (both immune and nonimmune) and stimulate those cells to become either more active (up-regulated) or less active
(down-regulated).
Innate immunity
Innate immunity refers to antigen non-specific defense mechanisms that a host uses
immediately or within several hours after exposure to an antigen. This is the immunity one is
born with and is the initial response by which the body eliminates microbes and prevents
infection. Innate immune responses involve; anatomical barriers (skin and mucosa),
physiological barriers (temperature, pH), molecules (complement proteins, acute phase
proteins, antimicrobial peptides and cytokines), cells that release inflammatory mediators
(basophils, mast cells, and eosinophils), phagocytic cells (neutrophils, monocytes, and
macrophages) and natural killer cells (NK cells).
Cells of the innate immune system use proteins, pattern-recognition receptors referred to as
PRR’s (e.g Toll-Like Receptors; TLRs), encoded in an organism's germ line to detect
potentially dangerous substances. These cells are thought to recognise particular highly
conserved pathogen-associated molecular patterns or PAMP’s (e.g carbohydrate structures
such as lypopolysaccharides; LPS) present on many different microorganisms (reviewed in
Franc et al., 1999; Teixeira et al., 2002). New findings show that the innate immune system,
once thought to be the unnecessary vestigial tail of ancient antimicrobial systems that have
been made redundant by the evolution of acquired immunity, is the cornerstone of the body's
ability to fight infection. In many aspects, the innate immune response is also a prerequisite
for an efficient induction of the specific (acquired) immune response. Following activation the
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innate system induces key costimulatory molecules on antigen presenting cells (APCs), which
are essential for antigen-driven clonal expansion of T and B cells (Akira et al., 2001; Pasare
and Medzhitov, 2004; Hedges et al., 2005; Xu et al., 2005). Dendritic cells (DCs) activated
by innate stimuli and loaded with foreign antigen travel to regional lymph nodes to activate
the acquired-immune system. Subsequently, the activated acquired-immune cells move into
tissues, where the innate immune system sets-off the danger signal (Matzinger, 1994). The
chemokine system is an essential regulator of this dendritic cell and lymphocyte trafficking,
which is necessary to turn an innate immune response into an adaptive response (Luster,
2002).
Acquired immunity
Acquired (adaptive) immunity includes humoral immunity (antibody-mediated) and cellmediated immunity. Acquired immunity involves; APCs such as macrophages and DCs, the
activation and proliferation of antigen-specific T and B-lymphocytes and the production of
antibody molecules, cytotoxic T-lymphocytes (CTLs), and cytokines. T and B-lymphocytes
possess specific receptors that arise from complex gene recombination reactions giving rise to
a broad spectrum of antigen specificities that is a hallmark of the adaptive immune system.
The T-cell receptor (TcR) recognizes only epitopes on antigens processed by APCs and
presented in the context of the major histocompatibility complex (MHC) molecules (reviewed
in Sebzda et al., 1999; Inaba and Inaba, 2005). Self/non-self recognition is achieved by
having every cell displaying a marker based on the MHC complex (Doherty and Zinkernagel,
1975). Any cell not displaying this marker is treated as non-self and attacked (Oberg et al.,
2004). The B-cell receptor (BcR), in contrast can recognise specific epitopes on whole
antigens (Takata et al., 1995). The adaptive immune system has the capacity to “store”
instructions obtained from the innate immune system upon first encounter with a foreign body
by changing from a naïve to an appropriate memory phenotype (reviewed in José et al.,
1999). This property, that distinguishes the adaptive immune system from the innate, is
referred to as immunological memory.
T lymphocyte subsets
Immature T lymphocytes differentiate from pluripotent stem cells in the bone marrow and
migrate to the thymus where their maturation occurs. Surface expression of the cluster of
differentiation molecule, CD3, is a unique feature of T cells. T cells are further classified by
the type of TcR they express on their surface, having either α/β or γ/δ combination (Miescher
Cytokine responses in metal-induced ACD
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et al., 1988). The α/β TcR expressing T cells only recognize antigens as peptide fragments
presented as a complex with MHC antigens on APCs (Schwartz et al., 1976; Ball and Stastny,
1984, Braciale et al., 1987). There are two distinct subpopulations of T cells within the α/β
TcR expressing lymphocytes defined based on their surface expression of either CD4 or CD8
in combination with the accessory marker CD3 (de Gast et al., 1985). The CD4+ T cells,
designated T-helper (Th) cells, recognize peptides, which are bound to MHC class II
molecules. CD8+ T cells, referred to as T-cytotoxic (Tc) cells recognize antigens bound to
MHC class I molecules, displayed as peptide-MHC class I complexes on the surface of APCs
(Van Seventer et al., 1986). Antigen recognition by γ/δ TcR expressing T cells does not
require processing and is not MHC restricted (reviewed in Chien et al., 1996).
The Th1/Th2 concept
The Th1/Th2 hypothesis emerged in the late 1980s, stemming from observations in mice of
two distinct populations of CD4+ T-helper cells differing in cytokine secretion patterns and
other functions upon activation (Mosmann et al., 1986). The concept subsequently was
applied to human immunity (Mosmann and Coffman, 1989). Both the Th1 and Th2 subsets
are produced from a non-committed population of precursor (naïve) T cells (Lafaille, 1998).
The Th1/Th2 concept rests largely on a dichotomy of cytokine profiles, with Th1 cells
described as mainly IFN-γ, IL-2 and TNF-β producers while Th2 cells are IL-4, IL-5 and IL13 producers (McKenzie et al., 1993; Wang et al., 1994; Thomas and Kemeny, 1998).
However, as with other immune cells, the array of cytokines produced by the Th1 and Th2
cells varies greatly and is influenced by a large number of experimental variables, as well as
the danger from artefacts (Romagnani, G., 2000). The differences in cytokine profiles
between Th1 and Th2 responses described in earlier studies relied heavily on mice and
cultured cells. However, more recent research involving human subjects has proved much of
this earlier work to be highly simplistic or otherwise inaccurate (Reviewed in Dent, 2002).
Currently Th1 cells are referred to as cells that produce far more IFN-γ and IL-2 than do Th2
cells, while the Th2 cells produce far more IL-4 (and perhaps IL-5) than do the Th1 cells.
CD8+ T cells (Tc cells) have also been suggested to, depending on the type of antigen and
cytokine milieu, differentiate into cells which produce IFN-γ, but not IL-4 (Tc1), and cells
that produce IL-4, but not IFN-γ (Tc2) (Mosmann and Sad, 1996; Kemeny et al., 1999). A
third population of cells, Th0 and Tc0, has been described as lymphocytes exhibiting an
unrestricted profile of cytokines; i.e., they produce both Th1 and Th2-type cytokines
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(Firestein et al., 1989; reviewed in Bendelac and Schwartz, 1991; Torii et al., 2002). There
are two key features of the Th1/Th2 hypothesis; firstly, each cell subset produces cytokines
that serve as growth factors of that subset (autocrine effects) and secondly, cytokines
produced by the two subsets cross-regulate each other's development and activity either by
blocking polarised maturation of the opposite cell type or by blocking its receptor functions
(Abbas et al., 1996). Whereas commitment to the Th1 phenotype is reversible, that to the Th2
phenotype appears to be final, since efforts to reverse such differentiated cells have not been
successful (Rogge et al., 1997; Lafaille, 1998). Recently, it has been suggested that CD4+ Thelper cells polarisation could be indicative of a more profound polarisation of the immune
system as a whole. That is, a kind of type 1/type 2 polarisation already begins with those cells
having the primary contact with antigens, including the DCs, monocytes and macrophages,
and other APCs (Moser and Murphy, 2000; Fujimura et al., 2004; Aktas et al., 2005). These
APCs likely polarise into type 1 and type 2 cells in response to the type of antigen experience,
and then subsequently bias the polarization of the T-helper population functionally
"downstream" from them.
The Th1-type cytokines
APCs secreting IL-12 upon uptake and processing of type-1 promoting (type-1 biased)
antigens promptly migrate to nearby lymph nodes. As this cytokine increases in concentration
it begins to influence naïve T cells to eventually become Th1 cells (Verhasselt et al., 1997;
Sparwasser et al., 1998; Hessle et al., 2000). NK cells also respond to the IL-12 environment
and proceed to release IFN-γ, which reinforces the APC's production of IL-12 and also helps
to drive the naïve T-cell commitment process (Hart et al., 2005; Walzer et al., 2005). As the T
cells attain maturity, Th1 cells also produce IFN-γ, which (together with the NK cells)
stimulates the APC and naïve T cells to polarise into more Th1 cells, in a self-reinforcing
"autocrine" loop. Th1-type cytokines are mainly produced in response to intracellular
pathogens, cell wall antigens or other smaller fragments of the organism, and trigger cellmediated immunity and/or production of opsonising antibodies (Table 1). Th1 cells are
hypothesised to lead the attack against viral and bacterial antigens through the classic
delayed-type hypersensitivity (DTH) reaction (Newport et al., 1996; Lienhardt et al., 2002).
Th1 cells are also important in the fight against cancer cells (Sato et al., 1998). On the
negative side, the Th1 pathway is often portrayed as being the more aggressive of the two,
and apparently, when it is over reactive, can generate organ-specific autoimmune diseases
Cytokine responses in metal-induced ACD
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(e.g., arthritis, multiple sclerosis, type 1 diabetes) (Tang et al., 1998; Pakala et al., 1997;
Schulze-Koops et al., 2001)
The Th2-type cytokines
The emergence of Th2 cells, like the Th1 cells, is also dependent on their cytokine
environment. Their maturation is likely initiated by the cytokine IL-6 (Croft and Swain, 1992;
Rincon et al., 1997) produced by APCs (monocytes and macrophages), endothelial cells,
fibroblasts and mast cells (Bradding et al., 1993; Derocq et al., 1994). IL-4 released by NK
cells, mast cells and eosinophils, also participates in driving Th2-cell maturation (Schmitt et
al., 1990; Marcinkiewicz and Chain, 1993). As the Th2 cells mature they also produce IL-4,
which together with the other participating cell types generate an autocrine loop to the naïve T
cells to make more Th2 cells (Chen et al., 2004) (Table 1). The Th2 pathway is thought to be
primarily involved in the triggering of IgE-mediated (immediate) hypersensitivity disorders,
including allergic asthma, eczema, hay fever, and urticaria (Singh et al., 1999) and also in
protection against helminthic infections (Coffman and von der Weid, 1997; reviewed in Dent,
2002).
T-regulatory cells (Tr1 and Tr2/Th3)
T-regulatory (Tr) cells were first described in the early 1970s based on their induction of
tolerance in other lymphocyte populations (Gershon and Kondo, 1970). However, failure to
define their exact phenotype led to controversy in the 1980s about their existence (Möller,
1988). Tr cells have recently become more prominent (Mason, 2001; McGuirk and Mills
2002; Chen et al., 2003) and have been shown to intervene and to block either Th1 or Th2
activities or both (reviewed in Kidd, 2003). CD4+CD25+ T lymphocytes have been suggested
to be the main cell population with immunoregulatory properties (Shevach, 2002; Nishimura
et al., 2004; Ruprecht et al., 2005). The CD4+CD25+ Tr cells comprise 5-10% of the total
peripheral T-cell pool (McGuirk and Mills, 2002) and constitutively express the CD25 marker
(IL-2Rα), already upon exit from the thymus (Sakaguchi et al., 1995). These cells are thus
referred to as “natural” Tr cells (Trn) (Table 1). Trn cells have been shown to possess a
contact-dependent cytokine-independent mechanism of immunosuppression (Fontenot et al.,
2003; Khattri et al., 2003). A second population of CD4+ Tr cells, generated in the periphery,
has been described (Baecher-Allan et al., 2001). This Tr subset has a cytokine-dependent
mechanism of action and is subdivided into Type 1 Tr (Tr1) and Type 2 Tr (Tr2) cells based
on their cytokine expression profiles. Tr1 cells secrete high levels of IL-10 and low-to-
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Jacob Taku Minang
moderate levels of TGF-β, while Tr2 cells primarily secrete TGF-β (Groux et al., 1997;
Gorelik and Flavell, 2002). Tr2 cells were previously named T helper-3 (Th3) cells (Weiner,
2001) but later renamed as Tr2 cells to highlight their suppressor function (reviewed in
Horwitz et al., 2003). CD8+ T cells with regulatory properties have also been described
(Gilliet and Liu, 2002). Unlike CD8+ effector cells, CD8+ Tr cells lack cytotoxic activity and
produce IL-10 (Tr1) and/or TGF-β (Tr2) (Rich et al., 1995; Dhodapkar and Steinman, 2002).
Functional studies indicate that Tr1 cells and other Tr populations may help to terminate Th1related inflammatory responses to pathogens, tumors, and alloantigens (Fukaura et al., 1996;
Groux et al., 1997; reviewed in Gorelik and Flavell, 2002; Ruprecht et al., 2005).
B lymphocytes and IgE class switching
B-lymphocytes mature in the bone marrow and, unlike T lymphocytes, are able to recognise
soluble antigens without the need for processing (Takata et al., 1995). Antigen recognition by
naïve immunocompetent B cells is via surface expressed immunoglobulins mainly of the IgM
and IgD classes. Activated B cells differentiate into plasma or memory B cells in highly
organised compartments of secondary lymphode organs (i.e lymph nodes and spleen) during
which time, depending on the antigen and cytokine milieu, class switching occurs in the
immunoglobulin genes to give rise to other classes; IgG, IgE or IgA (reviewed in Coffman et
al., 1993) which, display the same antigen specificity but have different biological functions.
IgE has been shown to be the main mediator of type I allergies (see section on
‘Hypersensitivity/Allergic reactions’). The switch to the IgE antibody class is induced by IL-4
and IL-13 (Del Prete et al., 1988; Punnonen et al., 1993) (see section on ‘Cytokines
investigated in this study’). Measurement of total serum IgE antibody levels is used as a
marker of the atopic status of an individual although determination of allergen specific IgE is
considered a more reliable indicator of atopic allergy (Kasaian et al., 1995; Di Lorenzo et al.,
1997; Di Gioacchino et al., 2000; Jackola et al., 2004). IgE has also been shown to play a role
in the clearance of many parasitic infections (Coffman and von der Weid, 1997; reviewed in
Dent, 2002). There are a few reports of detection of nickel (Ni2+) specific IgE antibodies in
serum samples from some Ni2+ allergic subjects (Shirakawa et al., 1992; Estlander et al.,
1993); however, the relevance of these antibodies to the skin inflammatory response seen in
allergic contact dermatitis (ACD) is not known.
Cytokine responses in metal-induced ACD
13
Table 1. General properties of CD4+ and CD8+ T regulatory and helper subsets
T cell subset
Predominant
cytokine secreted
Response to
antigena
Response to IL-2
or IL-15b
Regulatory/suppressor
CD4+ CD25+ Trn
CD4+ Tr1*
CD4+ Tr2/Th3*
CD8+ Tr1 or Tr2*
None
IL-10
TGF-β
IL-10 or TGF-β
Unresponsive
Unresponsive
Unresponsive
Hyporesponsive
Proliferate
Proliferate
Proliferate
Proliferate
Helper/Cytotoxic
CD4+ Th1
CD4+ Th2
CD8+ Tc1
CD8+ Tc2
IL-2, IFN-γ
IL-4, IL-5c, IL-13
IFN-γ
IL-4
Proliferate
Proliferate
Proliferate
Proliferate
Proliferate
Proliferate
Proliferate
Proliferate
*CD4+ or CD8+ Tr1 or Tr2 subsets can also display CD25 following activation. a, restimulation of T cells specific for
ovalbumin or peptide 110–119 of influenza HA with the same antigen. b, both cytokines use the shared common cytokine
receptor gamma chain (γc) and both function as T cell growth and survival factors. c, my addition. Culled from Horwitz et al.,
(2003).
HYPERSENSITIVITY/ALLERGIC REACTIONS
The term ‘allergy’ was first used by von Pirquet, to denote both host-protective and
potentially host-injurious immune responses (Bendiner, 1981). Today, hypersensitivity is
defined as an exaggerated reaction towards a stimulus, whereas allergy is a more specific
hypersensitivity reaction caused by an immunological sensitisation.
Classification of hypersentivity/allergic reactions
Hypersensitivity reactions are generally divided into four types depending on the effector
mechanism involved in the response (Reviewed in Rajan, 2003) (Table 2). The type I
hypersentivity response is very rapid (within minutes or hours of re-exposure to the allergen)
and is mainly IgE antibody mediated with allergen-specific IgE receptor bearing mast cells
and eosinophils playing a significant role (Siroux et al., 2004) (see also section below on
‘atopic contact dermatitis’). The clinical manifestation of type II and III hypersentivity
responses, just like the type I response, appear within a short time upon subsequent exposure
to the allergen but unlike the type I response, the IgG antibody class plays a major effector
role; either as opsonising antibodies promoting target cell lysis or in complexe with allergen
resulting in complement-mediated lysis of underlying epithelia (Skokowa et al., 2005). The
type IV response, also referred to as the DTH response, on the otherhand takes several days to
Jacob Taku Minang
14
appear upon re-exposure to the sensitising agent and is cell-mediated (Silvennoinen-Kassinen
et al., 1992; Shah et al., 1998; Grabbe and Schwarz, 1998).
The more important allergic disorders include bronchial asthma, perennial rhinoconjunctivitis
or seasonal rhinoconjunctivitis (hay fever), urticaria, extrinsic allergic alveolitis (farmer’s
lung, bird keeper’s lung), food allergies, acute generalised allergic reactions (anaphylactic
shock), allergic drug reactions, atopic dermatitis and contact dermatitis.
Table 2. The Gell-Coombs classification of hypersensitivity/allergic reactions
Gell-Coombs
scheme
Type I
Characteristics
Effector mechanism
Manifestations
IgE mediated
Immediate reaction
IgE-dependent activation of
mast cells and basophils
(Th2-cell dependent)
Atopic allergy,
anaphylaxis, hay
fever, asthma
Type II
Antibody mediated
Rapid reaction
Type III
Antibody mediated
Rapid reaction
Antibodies to microbial
antigens, C5a release
Leukocyte infiltration
Antibody-antigen complexes
activating the complement
system
T-cell mediated
(type 1 T cells implicated)
Cytotoxic reactions,
thrombocytic
purpura
Immune complex
diseases, vasculitis
Cell mediated
Delayed type
reaction
Modified from Rajan, (2003).
Type IV
Allergic contact
dermatitis
CONTACT DERMATITIS
Contact dermatitis is defined as an inflammatory skin reaction subsequent to direct contact
with noxious agents in the environment. This phenomenon was first described as early as the
first century A.D. where some individuals were reported to experience severe itching when
cutting pine trees (reviewed in Rosenberg, 1955). Depending on the offending agent and/or
the mechanism of the reaction (immediate or delayed, allergic or non-allergic), three main
types of contact dermatitis reactions have been described viz; allergic contact dermatitis
(ACD), atopic contact dermatitis and irritant contact dermatitis.
Allergic contact dermatitis
ACD results from a specific immunological reaction (type IV allergy or DTH) (Grabbe and
Schwarz, 1998) and primarily affects the hands, head/face and feet in adults (Schnuch et al.,
Cytokine responses in metal-induced ACD
15
1998; Shah et al., 1998; West et al., 1998). Common contact allergens include organic hapten
molecules found in plant products (e.g urushiol from the North American poison ivy) (Kalish
et al., 1994), drug metabolites, perfume fragrancies, rubber, plastics, additives and
preservatives (e.g methylisothiazolinones) (Gruvberger, 1997; Schnuch et al., 1997) as well as
metal ions such as nickel, chromium, cobalt, gold and palladium (Gawkrodger et al., 2000;
Thierse et al., 2005) (Table 3).
Table 3. Some important allergens included in the European standard series and mean
prevalence of positive reactions to these allergens among subjects with eczematous reactions.
Allergen name
Combined prevalencea (95% CI)
Nickel 5%
18.6 (11.1-26)
Fragrance mix 5%
10.7 (5.4-16)
Myroxylon pereirae 25%
6.7 (3.5-9.9)
Cobalt chloride 1%
5.8 (3.1-8.4)
Colophonium 20%
5.2 (3.2-6.9)
Thiuram mix 1%
3.5 (2.8-4.2)
Wool alcohols 30%
3.3 (1.3-5.3)
p-Phenylenediamine 1%
3.0 (2.1-3.9)
Neomycin 20%
2.9 (2.0-3.9)
MCI & MIb 0.01% aq
2.4 (0.8-4.0)
Formaldehyde 1% aq
2.1 (1.2-3.0)
Potassium dichromate 0.5%
2.1 (1.1-3.1)
Caine mix IIIc 10%
1.5 (1.0-2.0)
Quaternium 15 1%
1.3 (0.1-2.6)
Epoxy resin 1%
1.2 (0.1-2.3)
Mercapto mix 1%
1.1 (0-2.4)
Sesquiterpene lactone mix 0.1%
1.1 (0-2.1)
Parabens mix 16%
1.1 (0-2.3)
PTBP formaldehyde resin 1%
1.0 (0-3.0)
Mercaptobenzothiazole 2%
0.8 (0.3-1.22)
a
In petrolatum unless otherwise indicated. Combined prevalence (95% confidence interval) of positive reactions to 20 most
important contact allergens included in the European standard series based on data from seven test centres in the United
kingdom (UK).
b
methylchloroisothiazolinone and methylisothiazoline;
2.5%. Modified from Britton et al., (2003).
c
benzocaine 5%:cinchocaine 2.5%:amethyocaine
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Jacob Taku Minang
The risk for developing ACD is related to the exposure concentration (dose per unit area) and
the inherent allergic potency of the individual chemical (Boukhman and Maibach, 2001).
Results from a number of epidemiological studies suggest that ACD is common, with 20-25%
of the European population reported to be sensitised to one or more contact allergens (Brasch
and Geier, 1997; Schnuch et al., 1997; Meding et al., 2001; Meding and Jarvholm, 2002). It is
worth noting that allergic contact dermatitis can be prevented upon appropriate diagnosis and
avoidance of the offending agent as illustrated by the important examples of ACD to nickel
and chromium (Lachapelle et al., 1980; Avnstorp, 1989; Jensen et al., 2002).
Atopic contact dermatitis
Atopic contact dermatitis refers to an immediate-type (IgE-mediated) eczematous allergic
contact reaction in an atopic person (Hannuksela, 1980). The terms ‘atopic dermatitis’,
‘immediate contact reaction’, ‘contact urticaria’ or ‘contact urticaria syndrome’ have been
used interchangeably in the literature to describe different manifestations of atopic dermatitis
(De Waard-van et al., 1998; Aalto-Korte and Makinen-Kiljunen, 2003). The inflammatory
response in atopic contact dermatitis appears within minutes to an hour after contact with an
eliciting substance and usually disappears within a few hours. The reaction could be described
as immunologic or non-immunologic depending on whether prior sensitisation to the
offending agent is required. Sensitisation most commonly occurs via the respiratory or
gastroinstestinal tracts, but can also occur through the skin, as is the case of latex and some
foods (Guillet et al., 2005). Allergen-specific IgE antibodies, produced during primary contact
with the offending agent, bind to high-affinity Fcε receptors on the surface of mast cells and
basophils. Subsequent exposure to the same sensitising agent (allergen) and interaction
between the allergen and allergen-specific IgE antibodies on mast cells and basophils leads to
cross-linking of the high-affinity Fcε receptors and the release of a panel of
pharmacologically active mediators (Di Lorenzo et al., 1997; reviewed in Jackola et al.,
2004).
Atopic dermatitis (or atopic eczema) is characterised by skin infiltration of T lymphocytes
expressing a Th2-type profile (IL4, IL-5 or IL-13) of cytokines (Herrick et al., 2003). The
Th2-type cytokine, IL-4, plays a central role in the antibody class switch to IgE production
(See ‘IL-4’ under ‘cytokines investigated in this study’). Itching, tingling or burning
accompanied by erythema are the weakest types of reactions. A local wheal and flare suggest
contact urticarial reaction. Generalised urticaria is less common but has been reported in cases
Cytokine responses in metal-induced ACD
17
of contact with agents eliciting immunologic IgE-mediated contact urticaria (Reviewed in
Wuthrich, 1998). In addition to local skin symptoms, other organs are occasionally involved
giving rise to conjunctivitis, rhinitis, an asthmatic attack or anaphylactic shock (reviewed in
Harvel et al., 1994).
Irritant contact dermatitis
Irritant contact dermatitis refers to an eczematous reaction in the skin of external origin
where, in contrast to allergic contact dermatitis, no obvious eliciting allergens can be
identified (see section on ‘allergic contact dermatitis’) (Bryld et al., 2003; Duarte et al.,
2003). Depending on the nature of the irritant, the body region that is exposed or the duration
of exposure, the irritant contact dermatitis response can be referred to as; subjective, chronic
or due to chemical burns (Tupker, 2003; Smith et al., 2004; Pedersen et al., 2005). In the
past, the pathogenesis of irritant contact dermatitis was thought to be non-immunological
(Malten et al., 1979), however, it is now generally accepted that the immune system plays a
vital role in the induction and maintenance of irritant-induced skin reactions (Wakem et al.,
2000; Levin and Maibach, 2002; Lisby and Hauser, 2002). T lymphocytes expressing the
CD4+ phenotype have been shown to be the major skin infiltrating lymphocyte population in
irritant contact dermatitis (Willis et al., 1993; Kondo et al., 1996). These skin infiltrating
CD4+ T cells secrete mainly IFN-γ and IL-2 (Hoefakker et al., 1995; Kondo et al., 1996).
Noteworthy however, in contrast to allergic skin reactions, no immunological memory seems
to be involved in eliciting irritant contact dermatitis (Moed et al., 2004a) and the development
of irritant skin reactions does not require prior sensitisation (see preceding section on ‘allergic
contact dermatitis’).
Hapten immune system interaction in allergic contact dermatitis
Haptens are small organic molecules or metal ions that are non-immunogenic by themselves
and must be able to react to or bind to proteins (larger carrier molecules) to become contact
allergens (Landsteiner and Jacobs, 1935). Thousands of potential contact sensitizers exist.
However, parameters such as whether the hapten is released from compound mixtures,
whether people are exposed, if it permeates the skin or if it has a strong sensitising capacity,
narrow down the number of interesting haptens to a few dozens of high relevance (Wahlberg,
2001). The interaction between a hapten-carrier complex and the immune system and the
ensuing inflammatory response can be divided into two phases. There is a ‘sensitization
(induction) phase’ which describes the reactions (generally asymptomatic) following primary
18
Jacob Taku Minang
application of the hapten to the skin and then an ‘elicitation (effector) phase’ during which the
clinical symptoms of ACD are manifested upon re-exposure to the hapten.
The Sensitisation phase
Following primary application to the skin, DCs, mainly epidermal LCs that reside in normal
skin in a ‘resting’ functional state, take up the hapten and process it (Fig. 1). Hapten
application also results in activation of KCs, which together with LCs and other skin-cell
types secrete inflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-12 and GM-CSF (Enk
and Katz, 1992b; Kimber and Cumberbatch, 1992; Kaplan et al., 1992).
Dermis
Tsens
Figure 1.
Sensitisation phase of contact hypersensitivity. Topical application of contact allergen induces cytokine
secretion by keratinocytes (KCs), langerhans cells (LCs) and other skin cell types. The secreted cytokines activate resting
LCs and promote the migration of antigen (Ag)-carrying LCs towards regional lymph nodes (step 1). In the lymph node, LCs
establish contact with naïve T cells (Tnaive) (step 2) and activate them via expression of Ag-MHC complexes along with
costimulatory and adhesion molecules (step 3). Primed T cells (Tsens) alter their migration pathways and begin to recirculate
through peripheral tissues (step 4). Modified from Grabbe and Schwarz, (1998) with permission from Elsevier.
These cytokines activate more LCs and upregulate their expression of cell-surface molecules
(e.g MHC class I and II, B7, CCR7 etc) and secretion of cytokines and chemokines (e.g. IL-8)
(Enk et al., 1993). Activated antigen-carrying LCs migrate via afferent lymphatic vessels to
the paracortical zone of regional draining lymph nodes where they establish contact with and
activate T cells (Sozzani et al., 1995). Antigen-specific activation alters the migration
pathways of primed T cells through increased expression of skin homing markers such as
cutaneous lymphocyte antigen (CLA) leading to their recirculation through peripheral tissues
Cytokine responses in metal-induced ACD
19
(Barker et al., 1991). Other APCs, such as dermal DCs have also been implicated in priming
of naïve T cells after epicutaneous contact with hapten (Morikawa et al., 1992).
The elicitation phase
Re-exposure to a hapten leads to the same initial direct effects on the skin as primary hapten
contact during sensitization (i.e. LC activation, proinflammatory effects). Activated LCs
release cytokines that activate endothelial cells and upregulate their expression of adhesion
molecules with a resultant attraction of leukocytes to the site of hapten application (Fig. 2).
Antigen binding and
internalization
Epidermis
7
Dermis
Figure 2.
Elicitation phase of contact hypersensitivity (CHS). Secondary hapten application on the skin leads to the
activation of Langerhans cells (LCs) and subsequent secretion of proinflammatory cytokines (step 1). The released cytokines
upregulate the expression of adhesion molecules on LCs and activate endothelial cells (step 2) with a resultant attraction of
leukocytes to the site of hapten application (step 3). Among these are primed T cells (Tsens), which are activated upon antigen
(Ag) presentation either by resident cells or by infiltrating APCs (step 4). Ag-specific T-cell activation (step 5) induces
mediator release by hapten-specific T cells (step 6), which amplifies the inflammatory process leading to further
accumulation of infiltrating cells (step 7), resulting in clinically manifest allergic contact dermatitis (ACD). The time between
application of allergen and elicitation of ACD is approximately 24-72 h. Modified from Grabbe and Schwarz, (1998) with
permission from Elsevier.
Among these are primed T cells, which are activated upon antigen presentation either by
resident cells or by infiltrating APCs and/or LCs (Ishii et al., 1994; Ishii et al., 1995).
Activated hapten-specific T cells induce mediator release (Ptak et al., 1991a). This amplifies
the response by generating an inflammatory process that leads to further accumulation of
infiltrating cells including neutrophils, mononuclear cells and antigen-nonspecific but MHCrestricted late acting CD3+CD4+CD8- T cells (Ptak et al., 1991b; Moed et al., 2004b). These
cells are thought to be responsible for the eczematous reaction resulting in clinically manifest
ACD. γδ TcR expressing cells have also been shown to play a role in the elicitation of contact
20
Jacob Taku Minang
hypersensitivity (Askenase et al., 1995). The time between application of allergen and
elicitation of ACD is approximately 24-72 h.
RELATED BACKGROUND
Biochemistry of transition (heavy) metals
Transition metals represent a block of elements (~50 metals) in the periodic table that show
great chemical similarities within a given period as well as within a given vertical group. This
contrasts to the representative elements whose chemistry changes markedly across a given
period as the number of valence electrons changes, with chemical similarities occurring
mainly within vertical groups (Zumdahl, 1997). This difference occurs because the last
electrons added for transition metals are inner (d or f) electrons. This inner d and f electrons
cannot participate easily in bonding, as can the valence s and p electrons of the representative
elements. In forming ionic compounds with nonmetals, the transition metals exhibit two
typical characteristics; 1) more than one oxidation state is often found (e.g Cr3+ and Cr6+), 2)
the cations are often complex ions i.e. species where the transition metal ion is surrounded by
two or more ligands. For example, the compound [Co(NH3)6]Cl3 contains the [Co(NH3)6]3+
cation and Cl- anions where NH3 serves as the ligand. When dissolved in water, the solid
behaves like any ionic solid; the cations and anions are assumed to separate and move about
independently.
Transition metals turn to produce geometrically highly defined coordination complexes with
four or six electron-donators such as nitrogen or oxygen in amino acid side chains of
appropriate proteins or peptides (Fausto da Silva and Williams, 2001; Zhang and Wilcox
2002). For example, a complex of Co2+ ions has a typical tetrahedral arrangement, Ni2+ and
Pd2+ a square planar tetra-coordinated arrangement (Fig. 3) and Cr3+ a six-ligand octahedral
arrangement.
Transition metals and allergic contact dermatitis
Although several transition metal ions are known to play a vital role in living organisms as
well as in industry, a number of transition metals and their compounds also present important
occupational and health hazards (Waalkes, 1995; Garner, 2004). Some of these metals are
well known contact allergens capable of inducing a delayed type hypersensitivity response in
susceptible individuals upon prolonged direct exposure.
Cytokine responses in metal-induced ACD
++
L-----------------L
Ni
L------------------L
21
L
L-------------------L
Ni
L--------------------L
++
++
L-----------------L
Pd
L------------------L
L
Nickel (II)
Nickel (II)
Palladium (II)
Square planar
Tetrahedral
Square planar
Figure 3. Examples of coordination bonds to transition metal salts. L refers to a ligand i.e an electron rich hetero-atom
such as nitrogen, oxygen, sulphur or phosphorous capable of donating a pair of electrons to form a coordination bond with a
metal ion.
The number of ligands and the geometry of the coordination complexes, formed between the
metal ions and the electron rich atoms in amino acid side chains of some proteins or peptides,
seem to be the major factors determining the allergenicity of these metals as well as their
cross reactivities (Lepoittevin, 2001). Transition metal ions in this context serve as haptens,
with high immunogenic potential, only when in complex with cellular or matrix proteins of
the skin (Walton, 1983; Gawkrodger et al., 1986; de Fine Olivarius and Menne, 1992).
Unlike the irreversible convalent bonds formed by classical haptens and their protein carriers,
the coordination complexes formed by transition metal ions (‘non-classical’ haptens), are
reversible and allow for the exchange of the allergenic metal ions between different acceptor
sites (Thierse et al., 2005). One metal may have allergenic properties at two or more oxidation
states e.g. while Cr6+ has been assumed to be the major form of chromium involved in ACD
and is used for patch testing, Cr3+ has also recently been shown to elicit ACD (Hansen et al.,
2003). The distribution in nature, uses in industry and oxidation state relevant to sensitisation
for each of the metals investigated in the studies reported in this thesis are reviewed below.
Ni2+ is a component of many different types of alloys including white gold, German silver,
nickel plating, monel solder, gold plating and stainless steel. Its presence in such a wide
variety of products makes it an especially common contactant difficult to avoid (Liden, 1992).
Ni2+ is the single most common cause of ACD (see Table 3) (Hansen et al., 2003; reviewed in
Garner, 2004). Jewelry is the main source of Au1+ exposure (often seen as dermatitis at the
site of jewelry contact e.g. earlobes, fingers) but amalgams used in gold dental restoration are
a major cause of sensitisation (Vamnes et al., 2000). Co2+ is found in abundance in our
22
Jacob Taku Minang
environment and used with e.g. Ni2+ in various alloys but also found in pigments and paints
(Liden and Wahlberg, 1994). Cr6+ is present in cement but also found in metal alloys with e.g.
Co2+ and Ni2+ (Liden and Wahlberg, 1994). Pd2+ on the other hand, is a precious metal used in
the telecommunications industry, dental alloys, high temperature solders and jewelry. White
gold may contain up to 20% Pd2+, and dental alloys may contain up to 10% Pd2+ (Vincenzi,
1995).
Molecular and cellular mechanisms underlying metal-induced allergic contact
dermatitis
A number of models have been proposed to explain metal-protein interactions underlying the
transport and delivery of metal ions (non-peptide hapten) to APC and their interactions with
HLA and TcRs. In the first model referred to as processing-independent presentation, a metal
ion (Ni2+ in this case) either directly interacts with endogenous or exogenous peptides and the
resulting Ni2+-peptide complex then binds to MHC molecules (Romagnoli et al., 1991) or on
the other hand, Ni2+ may complex directly to an MHC-bound peptide or to the MHC molecule
itself thus circumventing processing (Fig. 4, left panel) (van den Broeke et al., 1999). The
Ni2+-MHC/peptide complexes result in conformational changes of these proteins that may
create new epitopes (neoantigens), which may be recognized by T cells (Romagnoli et al.,
1991).
In the second model referred to as processing-dependent presentation, Ni2+ is thought to form
coordination bonds with membrane-bound or soluble proteins, principally human serum
albumin (HSA), on the skin via cysteine or histidine residues (Sadler et al., 1994). The Ni2+carrier protein complex is then taken up by epidermal APCs (mainly LCs), processed and
presented to T cells as Ni2+-peptide complexes on MHC molecules (Fig. 4, right panel)
(Moulon et al., 1995).
A third model has recently been proposed (Thierse et al., 2004; reviewed in Thierse et al.,
2005). Here the metal, Ni2+, is suggested to bind to the protein HSA (a known shuttling
molecule for Ni2+), and the presence of the HSA- Ni2+ complex in the vicinity of transient
contacts between TcR and APC-exposed HLA molecules is thought to facilitate a specific
transfer of Ni2+ from the protein (HSA) to high-affinity coordination sites created at the
TcR/HLA-interface.
Cytokine responses in metal-induced ACD
23
Figure 4.
Mechanism of metal ion (e.g Ni2+) presentation. Two pathways of metal ion presentation are outlined: A) To
become a complete antigen, the metal ion binds directly to a MHC-bound peptide (processing-independent presentation). B)
The metal ion forms coordinative bonds to cysteine or histidine residues of soluble or membrane-bound proteins. The
modified proteins are taken up by antigen-presenting cells (APC), and are processed and presented to T cells as metal-peptide
complexes on MHC molecules. Modified from Büdinger and Hertl, (2000) with permission from Blackwell Publishing.
Despite evidence showing HLA restriction of Ni2+ recognition by Ni2+-specific T cells, Ni2+induced ACD has not been associated with the distribution of any HLA alleles (Emtestam et
al., 1993; Moulon et al., 1998). However, a preference for certain TcR-Vβ elements by both
skin- and blood derived Ni2+-reactive T cells has been demonstrated (Werfel et al., 1997;
Cederbrant et al., 2003).
Cross reactivity versus co-sensitisation in metal-induced allergic contact dermatitis
Santucci et al., reported more frequent associated patch test positive reactions among
transition metals than between these metals and other substances normally included in the
standard patch test series (see ‘in vivo diagnosis of allergic contact dermatitis’) (Santucci et
al., 1996). This may be due to concurrent exposure to different metals (co-sensitisation) or
concomitant responses of T cell clones (cross reactivity) resulting from similar chemical
properties of the metals and the consequent interactions inside the skin. Furthermore,
sensitisation to one metal ion has been suggested to increase the chances of being sensitised to
additional metals (Brasch et al., 2001).
Coordination complexes formed by Ni2+ and Pd2+ ions display marked spatial
(geometric)
conformational similarity as oppose to Co2+ or Cr3+ (see ‘Biochemistry of transition (heavy)
metals’). The antigen determinants created by either metal ions with certain matrix or cellular
proteins in the skin could be very similar and hence potentially interact with the same set of
24
Jacob Taku Minang
specific TcR/MHC complex. Indeed, Moulon et al., demonstrated cross-reactivity between
some Ni2+-specific T cell clones and Pd2+ but not Co2+ or Cr3+ (Moulon et al., 1995). Later
studies also showed patch test reactivity to Pd2+ to be almost exclusively found in subjects
with reactivity to Ni2+ (Gawkrodger et al., 2000). A strong Ni2+ response may thus be
indicative of potential Pd2+ reactivity. Subjects sensitised to one metal that induces a cellmediated response cross-reactive to other metals, would thus be generally assumed to stand
the risk of a relapse of contact dermatitis when exposed to the cross reacting metals.
Associated patch test reactivity to other transition metals has also been suggested to be due to
co-sensitisation (concurrent exposure). Wahlberg and co-worker, using the repeated open
application tests (ROATs) in guinea pigs, showed that animals induced by Co2+ reacted in the
patch test and ROATs with Co2+ but not Ni2+. Those induced with Ni2+ also reacted in patch
testing to Ni2+ but not to Co2+ and in the ROATs to Ni2+ and less to Co2+ (Wahlberg and
Liden, 2000). A significant number of subjects with Cr3+ and almost all with Co2+ reactivity
have been shown to also react with Ni2+ in the in vivo patch test (Hegewald et al., 2005;
Gawkrodger et al., 2000). It is worth noting that, isolated Co2+ reactivity is rare and reportedly
linked, in some instances, to possible Ni2+ contamination of Co2+ patch test material (Eedy et
al., 1991; Lisi et al., 2003).
Immune cells in metal-induced allergic contact dermatitis
Antigen presenting cells
The skin is the site of immediate contact with agents capable of inducing an ACD reaction.
The epidermal LC, originally described in 1868 by Paul Langerhans, is the only cell type in
normal epidermis that exhibits all accessory cell functions. LCs form a contiguous network
within the epidermis and constitute 2%-5% of the total epidermal cell population (reviewed in
Teunissen, 1992). LCs originate from CD34+ bone marrow progenitors that enter the
epidermis via the blood stream (Dieu et al., 1998). Their numbers in the epidermis is
maintained by local proliferation (Czernielewski and Demarchez, 1987). LCs express high
levels of molecules mediating antigen presentation (e.g MHC Class I and II, CD1), as well as
cellular adhesion and costimulatory molecules (e.g CD54, CD80 and CD86) (reviewed in
Romani and Schuler, 1992). Thus, LCs represent the ‘professional’ APCs in the skin (Nestle
and Burg, 1999) and play a pivotal role in the induction of cutaneous immune responses to
infectious agents as well as contact sensitizers (Kimber et al., 1998).
Cytokine responses in metal-induced ACD
25
Epidermal KCs, fibroblasts, and infiltrating mononuclear cells (e.g macrohages that mature
from blood monocytes) can upregulate their expression of MHC class I or class II molecules
in the presence of LC derived IL-1β or TNF-α (Kondo and Sauder, 1995) and can hence serve
as ‘non’professional’ APC (Enk and Katz 1992b). Haptenised KCs produce IL-1β, TNF-α and
lymphocyte-attracting chemokines, like CXCL10 (IP-10) (Flier et al., 1999) thus amplifying
the ACD reactivity in the epidermis (Sterry et al., 1991).
CD4+ T cells
T cells expressing CD4 molecules recognize hapten in complex with MHC class II molecules
(Van Seventer et al., 1986). CD4+ T cells have been shown to have a clear preponderance in
cutaneous infiltrates during skin inflammatory responses induced by allergen contact. Hence
this T cell lymphocyte subset is mostly associated with the effector phase of the ACD reaction
(Moed et al., 2004b). Recently, a lot of attention has been focused on CD4+ CD25+ T cells,
defined as Tr cells, suggested to be critical for the outcome of the ACD response in humans
(Cavani et al., 1998; Cavani et al., 2000; Sebastiani et al., 2001; Cavani et al., 2003; Moed et
al., 2005).
CD8+ T cells
CD8+ T cells recognize hapten in complex with MHC class I molecules (Van Seventer et al.,
1986). Lipophilic haptens are generally associated with MHC class I molecules due to their
ability to directly penetrate into LC, conjugate with cytoplasmic proteins and be processed
along the ‘endogenous’ processing route (Kalish et al., 1994). However, metal (e.g Ni2+)specific CD8+ T cell clones have been successfully generated both from skin and peripheral
blood of contact allergic subjects (Cavani et al., 1998, Moulon et al., 1998; Traidl et al.,
2000). Thus, this suggests a possible mechanism of MHC class I presentation even for
hydrophilic metal ions. CD8+ T cells have been demonstrated in epidermal mononuclear cell
infiltrates during an ongoing skin inflammatory response to contact sensitisers (Zanni et al.,
1998). CD8+ T cells are thought to mediate skin inflammation through killing of haptenbearing target cells (Moulon et al., 1998; Traidl et al., 2000).
γδ T cells
In humans, less than 5% of T cells bear the γδ heterodimer, and the percentage of γδ T cells in
the lymphoid organs of mice has been reported to range from 1% to 3% (Miescher et al.,
1988). Surprisingly, γδ TcR expressing cells appear to represent a major T-cell population in
26
Jacob Taku Minang
the skin, intestinal epithelium, and pulmonary epithelium (Pawankar et al., 1996). The
localization of γδ T cells at epithelial sites, suggests that they are especially suited to combat
epidermal or intestinal antigens and thus form a surveillance system that monitors the external
milieu of the epithelial cells (Reviewed in Kabelitz et al., 2005). γδ TcR expressing cells have
been shown to play a role in the elicitation of contact hypersensitivity (Askenase et al., 1995)
probably in a non-antigen-specific and non-MHC-restricted manner (Ptak et al., 1991b; Dieli
et al., 1998).
Cytokines investigated in this study
In the present study we investigated metal-induced cytokine-expression profiles representing
different polarisations of the immune response; Th1- (IL-2 and IFN-γ), Th2-type (IL-4, IL-5
and IL-13) and T-regulatory (IL-10) (Table 1).
IFN-γ
IFN-γ, the key Th1-type cytokine, is involved in the induction or upregulation of cell adhesion
molecules (Dustin et al., 1988) and exerts inflammatory effects mainly through effects on
macrophages (Arenzana-Seisdedos et al., 1985). IFN-γ has been shown to be important in
protection against mycobacterial infections (Newport et al., 1996) through the induction of
tumour necrosis factor (TNF), NO- and H2O2. The latter involved in the killing of the
intracellularly living bacteria. Several reports point to a critical role for IFN-γ in the induction
and elicitation of metal-induced ACD (Kapsenberg et al., 1992; Traidl et al., 2000).
IL-2
IL-2, produced by activated CD4+ T helper cells (Carter and Swain, 1997) has been shown to
be a growth and survival factor for antigen primed CD4+ and CD8+ T cells as well as NK cells
(Horwitz et al., 2003). IL-2 has been described as both a Th0- and a Th1-type cytokine
(Mosmann and Sad, 1996; O’Garra, 1998) due to its pleiotropic growth promoting properties
on activated Th1-, Th2-, Tc1- and Tc2-cells. Increased production of IL-2 has been reported
in ex vivo stimulated PBMC cultures from subjects allergic to Ni2+ but not control non-
Cytokine responses in metal-induced ACD
27
allergics (Falsafi-Amin et al., 2000; Jakobson et al., 2002; Lindemann et al., 2003) suggesting
a role, also for this cytokine in the ACD response to metals.
IL-4
IL-4 is the signature cytokine for Th2-type immune responses. It has been implicated in a
broad spectrum of biological responses which include; regulation of the differentiation of
naïve CD4+ T cells into a Th2 phenotype (O’Garra, 1998; Chen et al., 2004) and control of
humoral immune responses by regulating switching in B cells from IgM/G to IgE and IgG4, in
humans (Del Prete et al., 1988; Punnonen et al., 1993). IL-4 is a key cytokine in the
development of IgE-mediated allergic inflammation (Steinke and Borish, 2001). IL-4 has
been shown to cause erythema and induration when released in the skin (Asherson et al.,
1996; Rowe and Bunker, 1998) suggesting a role for IL-4 in the inflammatory response to
haptens by sensitised individuals upon subsequent exposure to the offending metal hapten.
IL-5
IL-5, considered to be a Th2-type cytokine, has been shown to play a role in the
differentiation and maturation of cells involved in IgE-mediated allergic reactions, such as
mast cells and eosinophils. IL-5 also inhibits certain macrophage functions (Sanderson, 1992)
and acts as a growth factor for IgA-producing B cells (Sonoda et al., 1989). Rustemeyer et al.
recently demonstrated elevated levels of IL-5 in PBMC cultures from Ni2+ allergic but not
control subjects after stimulation with Ni2+ (Jakobson et al., 2002; Rustemeyer et al., 2004).
However, the relationship between this and the inflammatory response in vivo is not yet
known.
IL-10
IL-10, formerly described as a Th2-type cytokine that functions by down regulating Th1-cell
activity, has been shown to be produced in comparable amounts by other cell types such as
monocytes, macrophages, DCs, mast cells and keratinocytes (Enk and Katz, 1992a; Moser
and Murphy, 2000). IL-10 displays immunomodulatory effects on both Th1 and Th2 cells
(Bettelli et al., 1998) by inhibiting the production of IL-1α, IL-1β, IL-6, IL-8, IL-12 and
TNF-α, as well as its own production, and by down regulating the expression of costimulatory molecules required for appropriate antigen presentation (de Waal et al., 1991;
28
Jacob Taku Minang
Akdis and Blaser, 2001). IL-10 has been shown to be produced by activated keratinocytes
during the induction (sensitisation) phase of ACD and induce clonal anergy in hapten-specific
T cells via effects on langerhans cells (LC) (Enk et al., 1993) and keratinocytes (KC) (CurielLewandrowski et al., 2003). IL-10, produced by Ni2+-specific CD4+CD25+ Tr cell clones, has
also been shown to inhibit the ability of DCs to stimulate Ni2+-specific Th1 and Tc1 responses
(Cavani et al., 2000).
IL-13
IL-13, like IL-4, has been described as a Th2-type cytokine, which promotes switching in B
cells from IgM/G to IgE (the effector molecule in IgE-mediated allergy) and IgG4 in humans
(Zurawski and de Vries, 1994; Punnonen et al., 1997). IL-13 has been shown to exert antiinflammatory functions on monocoytes and macrophages (McKenzie et al., 1993).
Significantly higher IL-13 production has been demonstrated in cultures of PBMC from
subjects allergic to metal (Ni2+) or organic (methylisothiazolinones) haptens but not control
subjects (Jakobson et al., 2002; Masjedi et al., 2003). However, similar to IL-5, the
relationship between the magnitude of the IL-13 response in vitro and the ACD reaction in
vivo, in terms of the patch test reactivity remains to be elucidated.
In vivo diagnosis of allergic contact dermatitis
The present diagnosis of ACD relies on the patch test first described by Jadassohn in 1895
(reviewed in Lachapelle, 2001). The patch test is a provocation test where the skin is exposed
to a panel of haptens and the ensuing reaction is graded. The patch test is performed on
normal skin on the back and involves the administration of a standard series of the most
common contact allergens, which commonly include approximately 25-30 organic
compounds as well as metal salts (Brasch and Geier, 1997) (see Table 3). There are specially
designed patch test panels ‘tailor-made’ for individuals in different occupations/professions or
with different types of dermatological problems. For example, for assessment of ACD in
subjects with lichenoid reactions in the mouth, a dental series of haptens is often used and this
panel includes, in addition to Ni2+, Co2+ and Cr6+, various acrylates and metals such as Au1+,
Pd2+ and mercury (Hg2+). Small volumes of a defined concentration of the allergen in a
suitable vehicle (usually 5% petrolatum) are applied epicutaneously and then covered with
specific strips. The strips are removed 48 h after application and the results read, scored and
recorded on days 2 and 4 or days 3 and 6-7. Patch test results are read based on a scoring
system recommended by the International Contact Dermatitis Research Group (ICDRG)
Cytokine responses in metal-induced ACD
29
(Table 4) (Wahlberg, 2001). Positive reactions are defined as the appearance of erythema and
oedema and with stronger reactions also papules and vesicles. Conclusive clinical diagnosis of
ACD is based not only on patch testing but also the history of the patient, assessment of
exposure as well as clinical examination.
Table 4. The scoring system for patch test reactions recommended by the International
Contact Dermatitis Research Group (ICDRG)
Score
Definition
Clinical manifestations
+
Weak positive reaction
Erythema, infiltration, possible papules
++
Strong positive reaction:
Erythema, infiltration, papules, possible
vesicles
+++
Extreme positive reaction:
Intense erythema and infiltration and
coalescing vesicles
-
Negative reaction†
IR
Irritant reactions of different types.
Discrete patchy erythema or
homogenous erythema without
infiltration, patchy follicular erythema*
?
Doubtful reaction
Faint macular or homogenous erythema,
no infiltration
†Patients who show negative reaction to the allergens may be sensitive to substances other than those tested. Testing with
complementary substances may be indicated.
*Petechiae and follicular pustules are usually irritant and do not normally indicate allergy. It is sometimes very difficult to
distinguish between an irritant reaction and a very weak positive reaction. Retesting maybe necessary to certify contact
allergy. Modified from Wahlberg, 2001.
Apart from the potential risk for the patient to be sensitised due to direct skin exposure to the
contact allergens, a number of problems have been associated with the in vivo patch test. The
use of certain drugs, especially corticosteriods, or exposure to UV radiation has been reported
to result in negative patch test reactions in subjects with a well-documented history of ACD
(Bruze, 1986; reviewed in Wahlberg, 2001). Patch test results have also been shown to
fluctuate in women with different results obtained at different times during their menstrual
cycle (Hindsen et al., 1999). Most intriguing, complete discordant reactions have been
reported when subjects are patch tested at the same time point with the same contact allergen
preparation on opposite sides of the upper back (Bourke et al., 1999). Furthermore,
interpretation of patch test results, especially with regards to discriminating between a
negative, weak (+) or doubful (?) reaction, is very difficult and tends to be subjective and
depends on the observer’s experience (reviewed in Wahlberg, 2001). Frequent reports of
30
Jacob Taku Minang
false-positive and false-negative reactions are a direct consequence of some or all of these
drawbacks. There is therefore a need for reliable in vitro diagnostic test methods.
In vitro diagnosis of allergic contact dermatitis
A number of studies has been carried out aimed at developing in vitro assays that can replace
or complement the patch test. The lymphocyte transformation test (LTT) exploits the
proliferative responses of T cells upon in vitro activation with allergens (Nordlind, 1984;
Masjedi et al., 2003). However, the LTT has been shown to have a low specificity, i.e.
proliferative responses also reported in individuals with negative patch test reactions to
specific haptens and no history of ACD (von Blomberg-van der Flier et al., 1987; Lisby et al.,
1999). Results from recent studies aimed at characterising immune responses to metal and
organic haptens by cytokine profiling, suggest that the measurement of cytokine production in
response to contact allergens could be utilized as a diagnostic tool (Jakobson et al., 2002;
Masjedi et al., 2003; Rustemeyer et al., 2004).
Cytokine Detection Assays
The enzyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunospot
(ELISpot) assay were used to define the cytokine expression profiles in metal-induced ACD
in the studies reported in this thesis. The ELISA assay is very useful in determining the
amount (concentration) of cytokines produced as a result of an immune response. The ELISA
was first described by Engvall and Perlmann (1972) and employs high protein binding
microtitre plates commonly in a 96-well format. There is a first step of adsorption of capture
monoclonal antibodies (mAb) specific to an epitope of the cytokine of interest. The plates are
subsequently incubated with supernatants from cells cultured in the presence or absence of
defined antigens. Serial dilutions of recombinant human cytokines are assayed in parallel to
obtain a standard curve. Thereafter the plates are incubated with biotinylated mAb specific to
a second epitope on the captured cytokines followed by enzyme-labelled streptavidin (SA).
The plates are then incubated with a substrate that forms a coloured soluble product. The
colour intensity is read and standard plots drawn using the concentrations of the recombinant
human cytokines from which the concentrations of the samples are extrapolated.
The ELISpot assay was first described as a method to determine the number of antibodyproducing B cells (Czerkinsky et al., 1983; Sedgwick and Holt, 1983). The technique was
later modified (Czerkinsky et al., 1988) for measurement of the frequency of antigen-specific
Cytokine responses in metal-induced ACD
31
cytokine-producing cells and has become a useful method when elucidating immune
responses at the single cell level. The principle is similar to that of the capture ELISA. Just as
for ELISA, the assay involves a first step of adsorption of capture mAb to 96-well ELISpot
plates utilising membranes as the solid phase for attachment of the capture mAb. However,
instead of culture supernatants being used in the subsequent step, cells are incubated for
varying periods (depending on the cell population, type of stimuli or cytokine investigated) in
the plates and the mAb captures the cytokine produced. Detection of the captured cytokine is
subsequently achieved by incubation of a biotinylated mAb followed by enzyme-labelled SA.
Spots are generated by enzymatic cleavage of a substrate that yields an insoluble precipitate.
The spots, a footprint of the locally accumulated cytokine produced by one cell, can later be
counted using a dissection microscope or an image-analysis system. Since the ELISpot assay
detects single cytokine-producing cells, and thus is dependent only on a locally high
concentration of cytokine, it is possible to identify low frequencies of e.g. antigen-specific T
cells that together produce cytokine levels below the detection level of a regular capture
ELISA (Tanguay and Killion 1994; Ewen et al., 2001; Ekerfelt et al., 2002; Masjedi et al.,
2003).
32
Jacob Taku Minang
THE PRESENT STUDY
OBJECTIVES
The present study was undertaken with the overall objective of defining the relationship
between in vivo responses in terms of patch test reactivity and in vitro responses to metals
known to cause ACD in terms of cytokine production and to evaluate the potential of the
cytokine detection assays, ELISpot and ELISA, for diagnosis of metal-induced ACD. The
study had as specific objectives:
• To relate the in vitro reactivity to the in vivo reactivity to Ni2+ in order to assess if subjects
with different degrees of allergic reactions differ in their cytokine response profile and/or
magnitude [I]
• To investigate the role of IL-10 in the regulation of cytokine responses in ACD to Ni2+ [II]
• To define the profile and magnitude of cytokine responses to other common metal contact
allergens in vitro [III]
• To optimise the ELISpot assay for diagnostic purposes [IV]
Cytokine responses in metal-induced ACD
33
METHODOLOGY
More detailed descriptions of the methods used in this thesis are found in the accompanying
papers (I-IV). Below is a brief description of the subjects, the reagents and the methods used
to obtain the results discussed in this thesis.
Study subjects:
Forty subjects participated in the study described in paper I. These included 30 female
subjects with a positive patch test to NiSO4 (i.e 10 subjects each with +3, +2 or +1 patch test
reactivity) and 10 healthy volunteers (control subjects; 9 female and 1 male) with no history
of contact allergy and a negative patch test result to NiSO4. The study reported in paper III
included 31 (28 female and 3 male) subjects with a positive patch test to one or more metal
haptens and five healthy volunteers (control subjects; 4 female and 1 male) with no history of
contact allergy and a negative patch test result to metals included in the standard as well as
dental patch test series. The metal-allergic individuals and their matched controls were
recruited from the Department of Dermatology and Venereology at Karolinska University
Hospital in Stockholm. Both studies were approved by the Ethics committee at the Karolinska
Hospital, Stockholm, Sweden (Ethical permission Dnr: 00-238).
Regular blood donors (n=40) registered at the Karolinska University Hospital, Stockholm,
Sweden; either with unknown allergic status or with a clinical history of Ni2+-allergy, as
stated by the subjects, provided buffy coats used in the studies reported in papers II and IV.
Subjects included in the studies gave their informed consent to participation
Diagnosis of contact allergy by the in vivo patch test (papers I and III):
The subjects were either patch tested with a standard patch test series including the metals
nickel (NiSO4; 5% in petrolatum), cobalt (CoCl2: 1 % in petrolatum) and chromium
(K2Cr2O7: 0.5 % in petrolatum) (papers I and III) or with a dental series including the
additional metals palladium (PdCl2: 2 % in petrolatum) and gold (Na3(Au(S2O3)2); 2 % in
petrolatum) (paper III). The metal haptens in petrolatum were applied by FinnChambers
(Epitest Ltd Oy, Tuusula, Finland) epicutaneously on the backs of the subjects for 48 h. The
patches were removed and the reaction read and scored using the scoring system
recommended by the International Contact Dermatitis Research Group (ICDRG) (Brasch and
Geier, 1997) (see ‘Table 4’ p30 on the ICDRG scoring system).
34
Jacob Taku Minang
Blood sample collection and processing:
Whole blood samples were obtained from metal-allergic and healthy control subjects by
venepuncture and collected in sterile heparinised glass vials and plasma samples separated
from the whole blood [papers I and III]. Buffy coats were obtained from regular blood donors
by centrifugation to remove the erythrocyte rich fraction (papers II and IV). PBMC were
separated from the whole blood (papers I and III) or buffy coats (papers II and IV) by densitygradient centrifugation over Ficoll-Paque and kept frozen in liquid nitrogen until use.
IgE measurement (paper I):
Total plasma IgE levels (reference range 1.6-122 kU L –1) were measured with the Pharmacia
ImmunoCAP kit (Pharmacia Diagnostics, Uppsala, Sweden).
Measurement of cytokine levels in cell supernatants by ELISA (papers I, II and III):
The capture ELISA assay was used to determine the levels of cytokines in supernatants from
cell cultures with or without antigen stimulation.
Evaluation of the detection sensitivity of one-step and two-step reagents in capture ELISA
(paper IV):
Mouse anti-human IL-4, IL-13 and IFN-γ detection mAbs directly conjugated to the enzyme
alkaline phosphatase (ALP) were developed for use in a simplified ELISpot assay for
detecting allergen-induced cytokine production. The sensitivity of these directly conjugated
mAbs was compared to that using the same mAbs conjugated to biotin requiring a second
incubation step with a SA-ALP conjugate in ELISA. Briefly, ELISA plates were coated
overnight with mouse anti-human capture mAbs [82.4 (IL-4), IL13-I (IL-13) and 1-D1K
(IFN-γ)] (Mabtech, Nacka, Sweden) and subsequently with serial dilutions (1000 pg/ml to 2.0
pg/ml) of recombinant human IL-4, IL-13 (R&D Systems, Minneapolis, Minn., USA) or IFNγ (Bender MedSystems GmbH, Vienna, Austria). The detection mAbs [12.1 (IL-4), IL13-II
(IL-13) or 7-B-6-1 (IFN-γ) (Mabtech)] were either directly conjugated to ALP (one-step
reagent) or biotinylated (two-step reagent). Plates incubated with the one-step reagents were
incubated directly with the substrate [para-nitro-phenyl phosphate (pNPP)] whereas those
with the two-step reagent needed an extra incubation step with SA-ALP prior to incubation
with pNPP. The concentration of recombinant cytokine required to give an absorbance value
of 2.0 for either system was compared as an indicator for the detection sensitivity.
Cytokine responses in metal-induced ACD
35
Enumeration of cytokine-producing cells by ELISpot:
ELISpot assays were performed using either polyvinyl diflouride (PVDF) plates
(MAIPS4510; Millipore Corp, Bedford, MA, U.S.A) pre-coated with capture mAbs
(Mabtech, Stockholm, Sweden) [paper I and IV] or PVDF plates (ELIIP10SSP: Millipore
Corp, Bedford, MA, U.S.A) coated with the capture mAbs overnight (a day before the assay)
[papers II, III and IV]. Cell suspensions in culture medium containing antigens at defined
concentrations were applied in triplicates to the plates; unstimulated cells were included in
each plate to assess spontaneous production. The cytokines captured were detected using
matched secondary mAbs either directly conjugated to ALP [paper IV] (Fig 5) or biotinylated
with subsequent addition of SA-ALP [papers I, II, III and IV]. Plates were developed by
incubation with bromo-chloro-indolyl phosphate/nitro blue tetrazolium-Plus substrate (Moss,
Inc., Pasadena, MD, U.S.A) and spots analysed as described previously (Masjedi et al., 2003).
Determination of optimal concentration of metal salts for stimulation of PBMC (paper III).
The potential of different concentrations of a panel of metal salts to induce non-specific
cytokine production or to exert toxic effects on PBMC in vitro i.e. suppression of mitogeninduced cytokine production, were assessed using the standard ELISpot assay. The ELISpot
assay was set up using PBMC from non-allergic subjects with or without addition of the
different metal salts and the frequency of IL-2-, IL-4- or IL-13-producing cells determined. A
metal salt was defined as mitogenic at any given concentration if it induced a three-fold
increase in the spontaneous production of all three cytokines (i.e an increment of at least 10
spots per 2.5x105 input cells over spontaneous production) at the said concentration. The
toxicity of the metal salts was evaluated by adding PHA (PHA; 2 µg/ml) to parallel cultures
and any metal salt concentration resulting in ≥ 25 % reduction in PHA-induced cytokine
production was defined as toxic. Optimal concentrations for each metal salt (i.e
concentrations that would induce optimal cytokine production with no mitogenic or toxic
effects, as defined above) were determined using standard IL-2, IL-4 and IL-13 ELISpot
assays. However, unlike above, PBMC (3.0x105 input cells/well) from metal allergic subjects
were used.
Phenotypic characterisation of Ni2+-specific cytokine producing cells using depletion
experiments (paper II):
To determine the phenotype of Ni2+-specific cytokine-producing cells, different cell
populations were depleted or enriched in PBMC from Ni2+ reactive donors and the frequency
36
Jacob Taku Minang
of Ni2+-specific IL-4 producing cells and the levels of IFN-γ production determined. Briefly,
2.0 x 107 PBMC in sterile 2% FBS/PBS were incubated with 4.0 x 107 anti-human CD3, CD4
or CD8 (Dynal Biotech, Oslo, Norway) mAb coated beads. Cytokine production in cultures of
the different cell fractions (whole PBMC, CD4+ or CD8+ enriched or CD3+, CD4+ or CD8+
depleted) with or without 50 μM NiCl2.6H2O (Merck) or PHA (2 μg/ml; Orion Diagnostics)
was determined by ELISpot or ELISA as described above. As an additional control for cell
viability, the CD3 depleted cell fractions were stimulated separately with LPS (100 pg/ml;
Sigma Aldrich, St Louis, MO, USA) or anti-human CD3 mAb (100 ng/ml; Mabtech) and IL6, IL-10 and IFN-γ levels measured by ELISA in supernatants of 2 day cultures.
The cell depletions were confirmed by flow cytometry using phycoerythrin (PE)-conjugated
anti-human CD3, CD4 or CD8 mAb (Becton and Dickinson) staining as described previously
(Zuber et al., 2005) with saponin permeabilisation excluded from the protocol.
Intracellular cytokine measurement by flow cytometry (paper II):
PBMC, 6 x 106 cells/ml, in culture medium (Gibco BRL) with or without either NiCl2.6H2O
(50 μM; Merck, Darmstadt, Germany) or purified protein derivative (PPD; 5 μg/ml) (Orion
Diagnostics, Trosa, Sweden), were incubated in 24 well (0.5ml/well) flat-bottom tissue
culture plates (Costar Corp., Cambridge, MA, USA) at 37oc in a humidified atmosphere of
5% CO2 for 3 days. Six hours before the end of the cultures, 20 ng/ml of phorbol myristate
acetate (PMA) plus 1 μM ionomycin (Sigma Immunochemicals, St. Louis, MO, USA) were
added to the cultures. In order to promote the accumulation of de-novo synthesised cytokines
in the Golgi apparatus of the synthesising cell, Brefeldin A (BFA) (Sigma) at a 10 μg/ml final
concentration was added to the cell cultures at the same time point as PMA-ionomycin. After
stimulation, cells were processed for intracytoplasmic cytokine analysis as described
previously (Zuber et al., 2005). Briefly, to simultaneously stain the cells with directly
conjugated anti-cytokine and surface marker antibodies, 50 μl solutions of PE-labelled
antibodies to either human IL-4, IL-10 (both from Becton and Dickinson, Franklin Lakes, NJ,
USA) or IFN-γ (Mabtech) together with FITC labelled antibodies to either human CD4
(Mabtech) or CD8 (Becton and Dickinson) in saponin buffer were added to the cell
suspensions and the plates incubated at RT for 1 h. The labelled cells were washed once with
PBS containing 0.5% FCS, resuspended in 0.3 ml of the same buffer and immediately
analyzed by flow cytometry in a FACScalibur® (Becton and Dickinson) equipped with the
CellQuest® software. List mode data for 50,000 events in a “live gate” mode were acquired.
Cytokine responses in metal-induced ACD
37
Single-cell cytokine production was evaluated after forward scatter (FSC) and side scatter
(SSC) gating on lymphocytes.
Statistical methods:
Cytokine responses between groups of subjects displaying different patch test reactivity or
controls [I and III] or between blood donors whose PBMC were reactive or not with Ni2+ [II
and IV] were compared using the Mann-Whitney U-test. The Wilcoxon matched pairs test
was used to compare responses by PBMC determined by the regular and a simplified ELISpot
protocol [IV]; the same test was use to compare the change in the Ni2+-induced cytokine
responses by Ni2+-reactive PBMC in the presence or absence of human recombinant IL-10
(rhIL-10) or anti-human-IL-10 (αh-IL-10) mAb respectively [II]. To evaluate the relationship
between observed parameters, correlations were computed using the Spearman rank-order
correlation coefficient rs [I, II and IV]. A multivariate correlation analysis was performed to
test for associations between multiple parameters (patch test, cytokine and IgE levels) [I]. For
comparison of antigen-specific responses, values with background (spontaneous production)
subtracted were used. A p-value < 0.05 was considered to be statistically significant. All tests
were performed using the software STATISTICA 5.1 (StatSoft, Tulsa, OK, USA).
38
Jacob Taku Minang
RESULTS AND DISCUSSION
Ni2+ induces a mixed cytokine response profile in PBMC from Ni2+-sensitised subjects and
this correlates with the in vivo patch test score (paper I):
We investigated, in the first study, if there is an association between specific cytokine profiles
or the magnitude of the cytokines induced by Ni2+ in vitro and the severity of allergic
reactivity in vivo. The in vivo reactivity to Ni2+, defined by the subject’s patch test reactivity
to NiSO4, was graded as +1 (weak), +2 (moderate) and +3 (strong) according to
recommendations by the ICDRG (Wahlberg, 2001). The frequency of Ni2+-specific cytokine
producing cells and the levels of Ni2+-induced cytokine production in PBMC from Ni2+allergic and control non-allergic subjects was determined by ELISpot and ELISA,
respectively.
Our results showed significantly higher levels of Ni2+-induced production of all cytokines
[Th1- (IFN-γ), Th2-type (IL-4, IL-5 and IL-13) and T regulatory (IL-10)] in subjects patch
test positive to NiSO4 as compared to the controls. Most notably, there was a significant
correlation between the degree of patch test reactivity [+1 (weak), +2 (moderate) or +3
(strong)] and the magnitude of the Ni2+-induced production of all the cytokines.
Earlier studies investigating the cytokines implicated in ACD to Ni2+ suggested a major role
for Th1-type cytokines in the ensuing detrimental inflammatory response (Sinigalia et al.,
1985; Silvennoinen-Kassinen et al., 1991; Kapsenberg et al., 1992). Data from more recent
reports suggest a mixed cytokine profile elicited by Ni2+ in PBMC from Ni2+-allergic subjects
(Falsafi-Amin et al., 2000; Jakobson et al., 2002; Lindemann et al., 2003). Our observation of
concommittant induction of Th1-, Th2- and Tr-type cytokines by Ni2+ in PBMC from Ni2+sensitised subjects is thus in agreement with these recent reports. However, unique to our
study, is the fact that we further categorised the Ni2+-allergic subjects based on their different
patch test reactivities (+1, +2, +3 or controls). We observed a positive correlation between the
magnitude of the cytokine responses in vitro and the degree of reactivity in the in vivo patch
test. Thus, Ni2+ induces a similar profile of cytokine responses in subjects with different
degrees of in vivo reactivity to Ni2+ but the magnitude of the cytokine response is positively
correlated with the degree of in vivo reactivity.
Cytokine responses in metal-induced ACD
39
Noteworthy, however, despite the correlations observed between patch-test reactivity and
cytokines representing both Th1- and Th2-type responses, we observed an exclusive Th2-type
cytokine response to Ni2+ in several of the Ni2+-allergic subjects. Thus, in the context of
contemporary descriptions of Ni2+-induced ACD, our results suggest that Th2-type cytokines
may have simultaneous down-regulatory effects by counterbalancing the deleterious Th1
responses during the DTH response, characteristic of ACD. On the other hand, Th2-type
cytokines may exacerbate the proinflammatory responses in ACD already set off by Th1-type
cytokine-responses or in fact may have a direct role in the pathogenesis of ACD independent
of Th1-type cytokines as is the case in atopic allergies (Parronchi et al., 1992; McHugh et al.,
1994; Asherson et al., 1996; Rowe and Bunker, 1998; Liu et al., 2003).
To investigate if there existed any relationship between atopy and the exclusive Th2-type
Ni2+-induced cytokine response profile observed in a number of the Ni2+-allergic subjects,
total plasma IgE levels were measured. Interestingly, the IgE levels of the Ni2+–allergic
subjects were found to correlate negatively with the Ni2+-induced IFN-γ responses (p=0.026,
rs=-0.46, n=23) of these subjects. The total level of plasma IgE is a crude, but nevertheless
relevant correlate for the atopic status of an individual (Kasaian et al., 1995; Di Lorenzo et
al., 1997; Di Gioacchino et al., 2000; Jackola et al., 2004). Lower proliferative as well as IL-2
responses to Ni2+ have been reported in PBMC from subjects with ACD to Ni2+ with
concomitant atopic dermatitis but not in ACD subjects with no atopic dermatitis symptoms
(Buchvald et al., 2004). It is, thus, possible that the atopic status of a subject can influence
their cytokine response to contact allergens. However, due to the small number of Ni2+allergic subjects in this study that displayed high IgE levels, a possible relationship between
atopy and IFN-γ responses to Ni2+ can be best assessed in studies specifically designed with
this question in mind.
Diagnostic potential of in vitro cytokine responses to Ni2+ in relation to the in vivo patch
test (paper I):
Our finding of a higher magnitude of Ni2+-induced cytokine production by PBMC from Ni2+allergic compared to control subjects and the correlation to the in vivo patch test score
suggests a diagnostic value for these cytokine assays. We therefore attempted a definition of
positive and negative responses to Ni2+ by setting cut off values based on the responses
displayed by control subjects. The cut off criteria included both the cytokine increment (i.e
cytokine production in cultures with Ni2+ minus the unstimulated or spontaneous production)
40
Jacob Taku Minang
and the cytokine ratio or index (i.e ratio of cytokine production in cultures with Ni2+ and the
unstimulated or spontaneous production).
Using these cut off criteria, the number of patch positive subjects responding in one or more
of the assays was 23/30 (paper I). Eighteen out of the 20 subjects in the two groups with a
more clearly defined allergy to Ni2+ [strong (+3) or moderate (+2) patch test reactivity and a
clinical history of Ni2+ allergy] in contrast to only 5/10 of the weakly (+1) reactive subjects
were positive according to the cytokine assays. All control subjects were negative. Most
notably, the highest number of positive responses was found for the IL-4 ELISpot (n=19), IL13 ELISA (n=19) and IL-13 ELISpot (n=17) assays, respectively. This, thus, suggests a better
diagnostic value for the Th2-type (IL-4 and IL-13) cytokine asays as in vitro correlates for an
allergic reaction to Ni2+ as opposed to IFN-γ. This would be useful especially for diagnosing
ACD in Ni2+-allergic subjects with concurrent atopic symptoms especially since diminished
Th1-type cytokine responses to Ni2+ have been shown in this category of subjects (Buchvald
et al., 2004). Our findings are in line with recent results by Rustemeyer et al. reporting better
discriminaton between Ni2+-sensitised and nonsensitised subjects using IL-5, a Th2-type
cytokine, but not the Th1-type cytokine, IFN-γ (Rustemeyer et al., 2004). Worthy of mention
is the fact that, in the aforementioned study, IL-13 was not measured and IL-4 was used
instead as a supplement in combination with IL-7 in seven days PBMC cultures to boost the
levels of Ni2+-induced IL-5 production.
The cytokine ELISpot assay has been shown to have potential applications as a diagnostic
tool for infectious diseases such as Mycobacterium tuberculosis infection (Lalvani et al.,
2001a; Lalvani et al., 2001b). Our data put cytokine assays into focus as potential diagnostic
tools for contact allergy to Ni2+ that could replace or complement the currently used in vivo
patch test. While the magnitude of the IL-4 and IL-13 ELISpot responses was in accordance
with those reported by Jakobson et al. (2002) it was much higher than that reported by
Lindemann et al. (2003) wherein detectable but still low levels of Ni2+-induced IL-4 measured
by ELISpot could only be obtained after pre-incubation with Ni2+ for two days. The use of
nitrocellulose membrane plates instead of PVDF plates for the ELISpot assay (Schielen et al.,
1995) may be one of several possible explanations for this discrepancy. The Lindemann study
also identified IL-2 as an additional cytokine of interest for measurement of Ni2+-induced
responses by ELISpot.
Cytokine responses in metal-induced ACD
41
With regard to the correlation between the in vivo patch test reactivity and the magnitude of
the in vitro cytokine responses to Ni2+, it is useful to highlight the uncertainties involved when
describing subjects with weak (+1) as oppose to moderate (+2) or strong (+3) positive
responses in the patch test. The reliability/reproducibility of a patch test score of +1 was
evaluated by performing a second patch test on some of the subjects with a historic patch test
score of +1 (unpublished data). This group included seven of the ten subjects with a historic
patch test reactivity score of +1; the difference in time between patch test 1 and 2 was
between 1-3 years and in one case 5 years. Four of these subjects did not respond to Ni2+ in
vitro according to the cut off criteria described above. Strikingly, only one of these subjects
had a positive test score (+1) in the second test. The other six were scored as negative (Table
5).
Table 5. Discordance between patch tests, clinical history and in vitro cytokine test in +1
subjects
Subject Patch
Repeated
History of
IL-4
IL-13
Test
Patch test
Ni2+ allergy
ELISpot
ELISA
13
+1
-
-
+
+
19
+1
-
-
-
-
20
+1
-
+
-
-
21
+1
NDa)
-
+
+
22
+1
-
-
-
-
24
+1
-
+
-
-
30
+1
ND
-
+
-
31
+1
+1
+
+
+
40
+1
-
-
-
+
41
+1
ND
-
-
-
a) Not done.
Most reports of false positive or irritative reactions recorded as positive or vice versal at
different times, mostly involve patch test responses described as weakly positive (+1) (Bourke
et al., 1999; Hindsen et al., 1999; Wahlberg, 2001). In line with this, we observed a poor
concordance between patch test results, history of allergy to Ni2+ and in vitro reactivity to Ni2+
in the group of subjects with +1 reactivity in the patch test. Thus, the variability of the patch
42
Jacob Taku Minang
test results makes it difficult to compare patch test and in vitro test results for patch test
positive subjects with a score of +1.
The regulatory cytokine, IL-10, downregulates both Th1- and Th2-type cytokine responses
in Ni2+-induced ACD (paper II):
A key finding in the first study was our observation of elevated levels of Ni2+-induced
production of the regulatory cytokine IL-10 in the Ni2+-allergics which, just like the Th1- and
Th2-type cytokine responses, correlated with the degree of reactivity of the subjects in the in
vivo patch test. This is in line with another report (Cedebrant et al., 2003). However,
Rustemeyer et al. recently showed elevated levels of Ni2+-induced IL-10 mainly in tolerised
subjects (Rustemeyer et al., 2003). We therefore tried to define the role of IL-10 in the ACD
response to Ni2+ in relation to its effects on other cytokines.
PBMC used in this study were from blood donors with a stated history of ACD to Ni2+ and a
confirmed in vitro responsiveness to Ni2+ (ACD PBMC) and donors with unknown allergic
status but a confirmed non-responsiveness to Ni2+ in vitro (control PBMC). In vitro
responsiveness was determined using cut off definitions for IL-4 and IL-13 ELISpot as
defined in the first study. The levels of IL-13, IL-10 and IFN-γ production and the number of
IL-4-, IL-13- and IFN-γ-producing cells after Ni2+ stimulation were assessed by ELISA and
ELISpot, respectively in the presence or absence of a concentration range of rIL-10. As
expected, we observed significantly higher and correlated Th1-, Th2-type and regulatory (IL10) cytokine responses to Ni2+ in ACD PBMC as compared to control PBMC. Furthermore,
addition of rIL-10 to ACD PBMC reduced the levels of Ni2+-induced IL-13 and IFN-γ; rIL-10
had the most potent down regulatory effect on Ni2+-induced IFN-γ levels with a significant
reduction seen even at the lowest concentration of rIL-10 (0.2 ng/ml), which better reflects the
endogenous levels of IL-10 induced in response to Ni2+.
To assess if also the Ni2+-induced endogenous IL-10 exerts a similar suppressive effect on
cytokine responses to Ni2+ as seen with rIL-10, we measured Ni2+-induced cytokine
production in PBMC cultures with or without α-hIL-10 mAb (10 μg/ml). If Ni2+-induced IL10 indeed suppresses the production of other cytokines, we reasoned, then Ni2+-induced
cytokine responses would be enhanced upon its neutralisation. Whereas we observed a
significant increase in the levels of IFN-γ in response to Ni2+ in the presence of mAb to IL-10,
Cytokine responses in metal-induced ACD
43
there was no net effect on the IL-13 levels or number of IL-4-, IL-13- or IFN-γ-producing
cells upon addition of the α-hIL-10 mAb. Furthermore, a greater enhancing effect of IL-10
neutralisation on the levels of production of IFN-γ was seen in donors with high (increment
above background >30 pg/ml) IL-10 production. It is worth mentioning that, the already low
or absent production of cytokines in response to Ni2+ by control PBMC was not affected by
addition of mAb to IL-10.
Cavani and co-workers had showed previously that IL-10 produced by Ni2+-specific
regulatory T cell clones markedly diminished the capacity of monocytes to stimulate Ni2+specific type 1 T cell clones (Cavani et al., 2000). Our data confirms a key regulatory role for
IL-10 in the immune reactivity to Ni2+. Noteworthy, is the fact that we used ex vivo stimulated
PBMC, which more closely reflect the in vivo situation. A number of studies have reported a
protective role for IL-10 in IgE-mediated allergic immune responses (Akdis et al., 1998;
Bellinghusen et al., 2001; Royer et al., 2001). Akdis et al., showed an increase of both Th1(IFN-γ) and Th2-type (IL-13) allergen-specific responses in PBMC from healthy subjects
upon neutralisation of endogenous IL-10 elicited by an atopic allergen (Akdis et al., 1998).
Although we also found a pleiotropic downregulatory effect on IL-4, IL-13 and IFN-γ
responses to Ni2+ by PBMC after adding rIL-10, only the IFN-γ production levels increased
significantly upon IL-10 neutralisation. This apparent difference in the number and type of
cytokines affected by neutralisation of endogenous IL-10 in Ni2+-induced ACD compared to
atopy has a bearing on the distinct cytokine profiles elicited in atopic and non-atopic as
opposed to ACD and non-ACD subjects. Allergens causing atopic allergy would generally
elicit a mixed (Th1-, Th2- and regulatory type) cytokine response in both atopic and nonatopic subjects (Akdis et al., 2004), with a preponderance of the Th2 responses in the atopics
and the regulatory (IL-10) type responses in non-atopics. In ACD to Ni2+, elevated Ni2+induced IL-10 responses tend to coincide with increased Th1- and Th2-type responses in
allergic subjects but little or no responses in healthy controls. With respect to immune
responses to M. tuberculosis, a microbe known to induce a classical Th1-type (IFN-γ)
mediated DTH response (Lalvani et al., 2001a; Lalvani et al., 2001b), endogenous IL-10
neutralisation has been shown to induce strong CD8+ cytotoxic T cell-mediated lytic activity
together with an increased expression of costimulatory molecules on target cells (De la
Barrera et al., 2004). Endogenous IL-10 neutralisation may result in similar enhanced effector
44
Jacob Taku Minang
activity on T cells involved in ACD to Ni2+ consequent to the observed increase in the levels
of Ni2+-induced IFN-γ production.
The significant reduction both in the number of cytokine producing cells and the levels of the
secreted cytokines upon addition of rIL-10 may suggest a direct suppressive effect of IL-10
on individual cytokine producing cells. IL-10 has been shown to directly suppress T cell
activation by binding to a CD28-associated IL-10 receptor on T cells thereby inhibiting
tyrosine phosphorylation of this key costimulatory molecule (Akdis et al., 2000). IL-10
neutralisation on the other hand, seems to boost cytokine production per cell without any
major effect on spontaneous (non-specific) production; hence our observation of elevated
levels of Ni2+-induced IFN-γ production in the presence of α-hIL-10 mAb without any
corresponding increase in the number of IFN-γ-producing cells.
CD4+ T cells are the lymphocyte population in PBMC solely responsible for IL-4 and IFN-γ
production after Ni2+ stimulation (paper II):
In light of our demonstration of a downregulatory effect of IL-10 on Ni2+-induced production
of both Th1- and Th2-type cytokines coupled with reports of a direct suppressive effect of IL10 on T cells (Akdis et al., 2000), we next investigated the phenotype of the Ni2+-reactive
cells in PBMC i.e. the cells likely affected by the immunomodulatory effects of IL-10.
To achieve this aim, PBMC were enriched for CD4+ or CD8+ or depleted of CD3+, CD4+ or
CD8+ cells and the frequency of IL-4 producing cells and the levels of IFN-γ produced upon
Ni2+ stimulation were determined in these different cell fractions. Cell depletion was
confirmed by flow cytometry and 89-99% of the CD3+, CD4+ or CD8+ cells were depleted
using our protocol with the best purity obtained following CD4+ cell depletion. Depletion of
the cells expressing the pan T cell marker, CD3 (α/β and γ/δ TcR expressing T cells)
(Miescher et al., 1988), completely abrogated both IL-4 and IFN-γ production after Ni2+
stimulation. Depletion of the Th cell fraction only (i.e. CD4+ T cell) (De Gast et al., 1985) had
a similar effect on IL-4 and IFN-γ production. On the contrary, depletion of the CD8+ cell
fraction (T cytotoxic/effector cells) (Van Seventer et al., 1986) had a positive effect on the
magnitude of the Ni2+-induced production of both cytokines. Significant levels of Ni2+induced production of both cytokines were found in the CD4+ enriched cell fraction after
substraction of the spontaneous background. These were however generally lower than the
Cytokine responses in metal-induced ACD
45
levels seen in the whole PBMC (data not shown). There was no detectable Ni2+-induced
production of either cytokine above background in the CD8+ enriched cell fraction.
Previous studies involving long-term cultured T-cell lines or clones have attributed Th1- and
Th2-type cytokine responses to Ni2+ to both CD4+ and CD8+ T cells (Moulon et al., 1998;
Traidl et al., 2000). However, Moed et al., using cells directly isolated from PBMC, recently
showed that only CD4+ CLA+ CD45RO+ and not CD8+ T cells proliferate and produce both
Th1- (IFN-γ) and Th2-type (IL-5) cytokines in response to Ni2+ (Moed et al., 2004b). In this
study, we investigated the effect of depletion of the CD3+, CD4+ or CD8+ populations from
PBMC, a more direct way of confirming the role of these cells, on Ni2+-induced Th1- and
Th2-type cytokine production. Our results suggest that CD4+ T cells may be the cell
population mainly responsible for production of both IL-4 and IFN-γ in response to Ni2+
stimulation.
Analysis of Ni2+-induced IL-10 production following depletion of different lymphocyte
fractions, unlike IL-4 and IFN-γ measurements, yielded inconclusive results. We obtained
decreased levels of IL-10 after depletion of either CD4+ or CD8+ cells in some instances but
in most instances no significant effect was observed. Earlier studies by other workers, using
cell clones, suggested that CD4+ CD25+ T regulatory cells are responsible for the bulk of
antigen-induced IL-10 production (Cavani et al., 2000; Sebastiani et al., 2001). The mixed
results we obtained could be due to the generally low levels of Ni2+-induced IL-10 production
(around 30 pg/ml or less) seen in most donors in this study. On the other hand, IL-10
production has been demonstrated in a wide variety of cell types, besides T cells (Enk and
Katz, 1992a; Royer et al., 2001). This could account for the lack of any clear observable
differences in the levels of Ni2+-induced IL-10 after depleting the T (CD3+, CD4+ or CD8+)
cell fractions.
In a nutshell, our data confirms and extends previous results by us and others showing that
PBMC from subjects with ACD to Ni2+ produce IL-10 upon Ni2+ stimulation and that the
magnitude of the Ni2+-induced IL-10 correlated positively with Th1- and Th2-type responses.
Further, endogenous IL-10 levels resulting from Ni2+ stimulation have a major downregulatory effect on the IFN-γ producting capacity of Ni2+-specific CD4+ T cells.
46
Jacob Taku Minang
Transitional metals commonly used in jewelry, metal alloys in dentistry and orthopaedics,
induce significant cytokine production in PBMC from contact allergic subjects (paper III):
We as well as others have shown that Ni2+ is capable of inducing significant production of
cytokines (both Th1- and Th2-type) in ex vivo stimulated PBMC from subjects with ACD to
Ni2+ but not control non-allergics (Jakobson et al., 2002; Lindemann et al., 2003; Rustemeyer
et al., 2004). However, there is very little information regarding the cytokine profiles induced
by other transition (heavy) metals also known to cause ACD. Knowledge on the profile and
the magnitude of the cytokines induced by other metals, apart from further elucidating the
immune mechanisms involved in metal-induced ACD, could be potentially exploited in
developing in vitro methods for the diagnosis of ACD. We therefore, investigated the profile
and magnitude of cytokine responses to a panel of metals in PBMC from subjects with a
history of ACD and confirmed in vivo patch test reactivity to one or several metals. The
frequency of IL-2-, IL-4- and IL-13-producing cells and the levels of production of IL-2, IL13 and IFN-γ in PBMC stimulated with a panel of metal salts was measured by ELISpot and
ELISA, respectively.
In agreement with our previous results (paper I), Ni2+ induced significantly higher cytokine
responses in PBMC from Ni2+ patch test reactive subjects compared to the non-allergic
controls; a mixed Th1-/Th2-type response was seen in most of these subjects. Use of either
nickel salts (NiCl2 or NiSO4) yielded a similar magnitude and profile of cytokine responses.
Furthermore, both Ni2+ salts induced cytokine production in PBMC from non-allergic control
subjects (non-specific cytokine-inducing effects) at concentrations greater than or equal to
100 μM. Both chromium salts (CrCl3/Cr3+ or K2Cr2O7/Cr6+) induced significant cytokine
[Th1- (IL-2) and/or Th2-type (IL-4 and IL-13)] production in PBMC from Cr6+ patch test
positive subjects but not in the non-allergic controls. Cr3+ tended to induce responses of
higher magnitude. Furthermore, we also showed that Cr6+ but not Cr3+ is toxic at very low
concentrations. Low concentrations of Cr6+ have previously been reported to induce cell death
or inhibition of cytokine production in vitro and also cause skin irritation (Reinhardt et al.,
1985; Li and Wang, 2002). Moreover, Hansen et al., reported induction of eczema at low
concentrations of Cr6+ but not Cr3+ (Hansen et al., 2003). Thus, our data shows that Cr3+ just
like Cr6+ induces significant cytokine production in vitro in PBMC from chromium patch test
positive subjects and suggests that the salt with Cr3+ may be preferable for use in stimulation
of PBMC for in vitro cytokine analysis. Just under half of the CoCl2 (Co2+) patch test positive
subjects responded to Co2+ with significant cytokine production to Co2+ in vitro. PBMC
Cytokine responses in metal-induced ACD
47
responses to Co2+ stimulation generally involved production of either IL-2 alone or the
complete profile of cytokines (IL-4, IL-13 and IFN-γ). Co2+ has been shown to elicit irritative
and/or doubtful reactions in the patch test (Garner et al., 2004). This inherent irritative
property may account for some of the false positive results that have been reported. Thus in
vitro cytokine assays, which measure immunological memory, may be better at confirming a
true allergic reaction as well as discriminating this from any false positive reaction seen with
the patch test.
Besides our observation of positive in vitro response to Pd2+ with production of at least one
cytokine by PBMC from all subjects patch test positive to Pd2+ in the dental series, Pd2+ also
induced significant cytokine production in PBMC from some subjects tested only with the
standard series (excluding Pd2+ and Au1+). This suggests that the prevalence of ACD to Pd2+
may be considerably higher than assumed since most population/prevalence studies are based
on patch testing with the standard series (Brasch et al., 2001; Bryld et al., 2003; Hegewald et
al., 2005). Similar to Ni2+, Pd2+ induced non-specific cytokine production at concentrations
greater than 100 μM. PBMC from gold patch test positive subjects showed significantly
higher levels of IL-13 and IFN-γ responses to either salt of the metal (Na3(Au(S2O3)2/Au1+ or
HAuCl4/Au3+) in vitro compared to the non-allergic controls. Au1+ and Au3+ had toxic effects
on mitogen (PHA)-activated PBMC at concentrations above 50 μM and 70 μM, respectively.
However, despite the observed toxicity of Au1+ at lower concentrations compared to Au3+,
PBMC stimulation with Au1+, at concentrations below 50 μM, resulted in responses of higher
magnitude with a better distinction between subjects patch test positive to Au1+ and nonallergic controls.
Overall, the results show that other metals included in the standard and/or dental series of the
in vivo patch test, just like Ni2+, are capable of inducing specific cytokine production in vitro.
Furthermore, our data point to potential future applications of the in vitro cytokine assays, as
complemetary or confirmatory tools, in the diagnosis of ACD.
In vitro evidence of in vivo cross-reactivity and/or co-sensitisation patterns (paper III):
A significant number of subjects showed positive reactions in the in vivo patch test to more
than one metal in both the standard and dental series. We therefore investigated if PBMC
from these subjects responded in vitro to all the metals to which they were reactive in the
patch test.
48
Jacob Taku Minang
Approximately 45% (4/9) and 75% (9/12) of those patch test positive to Cr6+ and Co2+,
respectively, were also reactive to Ni2+ in the in vivo patch test. Interestingly, in vitro cytokine
responses were induced by Ni2+ stimulation of PBMC from a higher percentage of these
subjects i.e about 90% (8/9) and 85% (10/12) for the Cr6+ and Co2+ patch test positive
subjects, respectively. Some of the subjects that were positive for both Ni2+ and Co2+ in the in
vivo patch test showed positive in vitro cytokine responses only to Ni2+. Furthermore, all Co2+
reactive subjects with concurrent Ni2+ patch test reactivity showed a positive in vitro response
also to Pd2+ with in vitro responses to Ni2+ and Pd2+ seen even in one Co2+ patch test positive
subject that failed to respond to Co2+ in vitro; a few Cr6+ reactive subjects also responded to
Au1+/Au3+, Co2+ and Pd2+. There have been reports of concurrent Co2+/Cr6+, Co2+/Ni2+,
Cr6+/Ni2+ or Ni2+/Co2+/Cr6+ responses in the in vivo patch test (Gawkrodger et al., 2000; Uter
et al., 2004; Hegewald et al., 2005). Our results therefore confirm, in vitro, the reported in
vivo reactivities observed with these metals but also suggest that there may exist low levels of
subclinical Ni2+-cross reactivity in subjects, patch test positive to metals other than Ni2+.
Some authors have even suggested that some cases of isolated Co2+ reactivity in the in vivo
patch test may be as a result of Ni2+ contamination of Co2+ patch test material (Rystedt, 1979;
Eedy et al., 1991; Lisi et al., 2003).
Less than half of the Au6+ reactive subjects showed a positive reaction to other metals in the
in vivo patch test (1/10 to Co2+ or Cr2+ and 3/10 to Ni2+ or Pd2+). While, a significant number
of subjects that reacted to other metals, but Au6+, in the in vivo patch test tended to also react
to Au1+/Au3+ in vitro, most subjects that were exclusively patch test positive to Au2+
responded only to Au1+/Au3+ in vitro. All except one of the Pd2+ patch test positive subjects
were also positive to Ni2+. Strikingly, all the Pd2+ patch test positive subjecst showed a strong
positive reaction to Ni2+ in vitro. In a few instances, Pd2+ reactive subjects showed a positive
reaction to Au1+/Au3+ or Co2+. A number of Pd2+ patch test negative subjects (i.e those tested
with the dental series) reacted to Pd2+ in vitro; all subjects that reacted with Pd2+ in vitro also
reacted with Ni2+ in vitro. Moulon et al., showed that Ni2+-specific T cell clones cross-react
with Pd2+ but not Co2+ or Cr2+ (Moulon et al., 1995). In addition, other studies have suggested
an overlap in responses to Ni2+ and Pd2+ in the in vivo patch test with Pd2+ reactivity found
almost entirely in subjects with an already confirmed reactivity to Ni2+ (Santucci et al., 1996;
Gawkrodger et al., 2000).
Cytokine responses in metal-induced ACD
49
In conclusion therefore, apart from the potential of discriminating between metal-sensitized
and non-sensitized subjects, in vitro cytokine assays could be useful in identifying subjects
with multiple metal sensitisations. Furthermore, with respect to Pd2+ reactivity, our data
suggests that a strong Ni2+ response in vitro may be indicative of Pd2+ reactivity.
Optimisation of a simplified ELISpot assay for enumeration of allergen-specific cytokineproducing cells (paper IV):
The above results together with data from previous studies by our group (Jakobson et al.,
2002), using the ELISpot assay point to a potential use of the cytokine ELISpot as a tool to
discrimate between Ni2+-sensitised and non-sensitised subjects. In paper IV, we aimed at
evaluating a simplified ELISpot protocol for use in the detection of Ni2+-specific cytokine
producing cells.
The simplified assay consisted of precoated plates and one-step (ALP-conjugated mAb)
detection reagents as opposed to overnight adsorption of capture mAb and two-step
(biotinylated mAb followed by SA-ALP) detection reagents in the conventional assay. To
validate the simplified assay, we compared the sensitivities of the one-step and two-step
detection reagents in parallel in a capture ELISA. The one-step detection system displayed a
higher sensitivity than the two-step detection system with approximately 1.8 and 1.4 times
lower concentrations of recombinant cytokine needed to obtain absorbance values of 2.0 for
IL-4 and IL-13 respectively. On the other hand, detection with the one-step detection system
required approximately 2.3 times higher concentration of the recombinant cytokine to yield
the same absorbance value as that obtained in the two-step detection system for IFN-γ.
Although, in order to increase the detection sensitivity, the use of enzyme-labelled detection
antibodies in sandwich-based immunoassays have to a large extent been replaced by the use
of two-step detection systems (Porstmann and Kiessig, 1992; Ahlborg and Paulie, 2002), the
detection sensitivity of the one-step and two-step detection systems investigated in this study
are quite comparable.
To further investigate the usefulness of the simplified compared with the regular ELISpot
protocol, the two protocols were used in parallel to analyse the number of antigen (Ni2+, TT or
PPD) or mitogen (PHA) –induced IL-4-, IL-13- and IFN-γ-producing PBMC from blood
donors with or without a history of ACD to Ni2+. Using both protocols, significantly higher
Ni2+-induced IL-4 and IL-13 responses in PBMC from the subjects with a history of ACD to
50
Jacob Taku Minang
Ni2+ (p=0.005 and p=0.02 for the simplified and regular IL-4 ELISpot and p=0.009 and
p=0.04 for the corresponding IL-13 ELISpot, respectively) were seen. There were no
significant differences between the two protocols in the detection of cells producing IL-4, IL13 or IFN-γ after activation with any of the three antigens. A highly significant correlation
was found between the two ELISpot protocols when the number of cytokine-producing cells
in response to all three antigens was compared for all the subjects (Fig. 6).
Several studies have highlighted the value of the cytokine ELISpot as a powerful tool to
monitor and measure antigen-specific T-cell responses (Czerkinsky et al., 1988; Smith et al.,
2001) in experimental research and recently also for possible diagnostic applications (Lalvani
et al., 2001a; Lalvani et al., 2001b). PBMC from Ni2+-reactive subjects have been shown,
using the cytokine ELISpot, to produce significantly higher levels of both Th1 and Th2
cytokines in response to Ni2+ compared to non-allergic subjects (Jakobson et al., 2002;
Lindemann et al. 2003). The observation in the present study of Ni2+-induced IL-4 and IL-13
responses only in PBMC from blood donors with a history of contact allergy to Ni2+ further
supports the use of the ELISpot assay as an alternative diagnostic test.
The number of cells spontaneously producing cytokine was found to be higher using the
regular ELISpot, especially for IFN-γ. This may suggest a higher detection capacity by the
regular protocol. However, the amount of spontaneously secreted cytokines is generally low
as opposed to antigen-induced cytokines (Ekerfelt et al., 2002; Mäkitalo et al., 2002). Thus, a
decreased detection of spontaneously producing cells combined with a comparable detection
of antigen-specific spots could be an advantage when distinguishing between antigen-specific
responses in responder and non-responder individuals since a better signal-to-noise (antigenspecific/spontaneous) ratio can be obtained.
The development of a simplified ELISpot assay with an overall reduction of work and
incubation steps, apart from reducing the assay time makes it possible to use one batch of
precoated plates throughout a study, thus eliminating one source of inter-assay variation. The
simplified ELISpot assay protocol, compared to the regular assay format, would facilitate
larger clinical as well as experimental studies without any loss of sensitivity.
Cytokine responses in metal-induced ACD
51
Ni2+-sensitised and non-sensitised subjects respond to recall antigens with a similar
cytokine profile (In progress and paper IV):
We have therefore established that subjects with ACD to Ni2+ but not control non-allergic
subjects show elevated Th1-, Th2- and regulatory cytokine responses to Ni2+. However,
whether this points to a tendency for subjects with ACD to Ni2+ to manifest a generalised
proinflammatory response to recall antigens has not been elucidated. We therefore
investigated if PBMC from subjects with a history of ACD and a confirmed in vitro reactivity
to Ni2+ displayed a tendency of responding with a particular cytokine pattern also to other
stimuli such as tetanus toxoid (TT) and purified protein derivative (PPD) from M.
tuberculosis or the mitogen PHA.
Whereas TT elicited production of IFN-γ, IL-13 as well as IL-4 at similar levels, PPD elicited
a higher number of cells producing IFN-γ than IL-13 and few IL-4-producing cells. However,
unlike Ni2+, TT and PPD induced significant cytokine responses in both subjects with a
history of ACD to Ni2+ and the controls with no significant difference between the ACD
subjects, as a group, and the controls. Strikingly, when the subjects with ACD to Ni2+ were
subdivided based on their patch test reactivity score (+3, +2 and +1) the TT-induced response
was generally the highest in the +1 group and lowest in +3 group (Fig. 7). Our observation of
a positive correlation between the patch test reactivity score and the magnitude of the in vitro
response to Ni2+ implies a generally higher frequency of Ni2+-specific memory T cells in ACD
subjects with a patch test score of +3 compared to those with +2, +1 or the controls. We
therefore speculated that an increase in the number of Ni2+-specific memory T cells due to a
more recent sensitisation and/or repeated exposure to Ni2+ results in a decrease in the net
proportion of memory T cells specific to recall antigens to which subjects were immunised
decades ago. In the absence of recent boosting or exposure to the pathogen a drop in the
frequency of circulating memory T cells specific to recall antigens would compensate for the
increase in the proportion of the Ni2+-specific memory T-cell pool; a sort of requirement for
immunological homeostasis (reviewed in Seder and Ahmed, 2003). This is not unreasonable
given the mean age of the subjects that participated in our studies (mostly women above 40
years of age).
Although we observed individual variations in the response to PHA, there was no general
tendency that the PHA-induced responses differed between subjects with ACD to Ni2+ and the
controls.
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Jacob Taku Minang
These preliminary findings therefore show that subjects with ACD to Ni2+ are not prone to
elevated proinflammatory (both Th1- and Th2-type) cytokine responses to every antigen.
However, the degree of in vivo reactivity to Ni2+ may affect the balance of the memoy T cell
pool specific to recall antigens arising from old vaccination regiments. However, more studies
using a larger pool of antigens commonly used in vaccination regimens are needed to further
pursue this phenomenon.
Cytokine responses in metal-induced ACD
53
GENERAL CONCLUSIONS AND PERSPECTIVES
The following general conclusions can be drawn from the results discussed in this review and
in the individual articles that follow;
• Subjects with different degrees of patch test reactivity to Ni2+ display a similar mixed
Th1/Th2-type cytokine response to Ni2+ in vitro. However, the magnitude rather than the
profile of the cytokine response is predictive of the outcome of the allergic reactivity in vivo
[paper I].
• Ni2+ induces elevated levels of IL-10 that correlate with the Th1- and Th2-type cytokine
responses in subjects with ACD to Ni2+ and endogenous IL-10 levels resulting from Ni2+
stimulation have a downregulatory effect on Th1-type (IFN-γ) responses by CD4+ T cells
[paper II].
• Other transition metals included in the standard and/or dental series of the in vivo patch test,
similar to Ni2+, also elicit a mixed Th1/Th2-type cytokine response in vitro in PBMC from
metal allergic subjects [paper III].
• From a diagnostic point of view, our data show that in vitro cytokine assays can be used to
determine the reactivity of ACD patients not only reactive with Ni2+ but also with other
metals such as Au1+/Au3+, Co2+, Cr3+/Cr6+ and Pd2+ [papers I, III and IV]. However, to further
improve the usefulness of the ELISpot technique in the diagnosing of ACD it would be
relevant to;
1) Evaluate methods of enhancing the cytokine responses in weak responders
using different mAbs that inhibit regulatory cytokines or activate costimulatory molecules.
2) Investigate similarities in cytokine responses to organic lipohilic and
hydrophilic contact allergens to ensure that an agreed-upon in vitro test would cover all or
most of the present series of compounds included in the patch test.
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Jacob Taku Minang
Figure Legends
Figure 5
The simplified and regular ELISpot assays. There are only three major steps in the simplified
assay compared to five in the regular assay format. This saves time as well as reduces interassay variation since one batch of pre-coated plates could be used for one large study (clinical
trial or experimental study).
Figure 6
Highly significant correlation between the simplified and regular ELISpot assay formats.
Cytokine responses (IL-4, IL-13 and IFN-γ) to all three antigens tested (Ni2+, TT and PPD) by
PBMC from all 14 blood donors were pooled and analysed by the Spearman rank-order
correlation coefficient rs. All analyses were done using values with background (spontaneous
responses) subtracted (i.e. 14 blood donors x 3 cytokines x 3 antigens = 126 data points).
Figure 7
Nickel (Ni2+)-allergic and non-allergic subjects display a similar cytokine profile in reponse to
a recall antigen. Peripheral blood mononuclear cells (PBMC), 2.5 x 105 cells/well, from Ni2+allergic subjects with different patch test scores (+3, +2 and +1; n=10 per group) and nonallergic subjects (n=10) were cultured with or without Ni2+ or tetanus toxiod (TT). The
number of IL-4 (A), IL-5 (B) and IL-13 (C) producing cells were determined by ELISpot.
Results shown are the means + SD for each group. Statistical diffeences between the groups
following a following a Mann-Whitney U test are depicted with asterisks (∗p<0.05;∗∗ p<0.01;
∗∗∗p<0.001).
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Jacob Taku Minang
ACKNOWLEDGEMENTS
The success of such an endearvour is impossible without the help and support of many people
and institutions. To arrive this point, I have had to stand on the ‘shoulders of giants’. First of
all, I wish to express my sincere gratitude to my supervisor, Marita Troye-Blomberg, for
giving me a window into the fascinating world of Immunological Research and for her
constant support and guidance. My co-supervisor, Niklas Ahlborg, is greatly appreciated for
introducing me to the concept of metal (hapten)-induced immune responses and for his
unlimited patience and dedication to work. Many thanks to my second co-supervisor, Lena
Lundeberg, for all the work with organising the patients that participated in the studies
reported in this thesis as well as for the excellent academic discussions.
Thanks to my co-authors, Iréne Areström, Bartek Zuber and Gun Jönsson.
Many thanks to the seniors at the department of Immunology, Klavs Berzins, Carmen
Fernandez, Manuchehr Abedi-Valugerdi, Eva Sverremark-Ekström and Eva Severinson
for sharing their wide knowledge of immunology with me. I’m particularly grateful to Peter
Perlmann (RIP) and Hedvig Perlmann for their encouragement and their constant presence
that tells me to ‘keep going, there is no end to this’.
Former students; Salah Eldin Farouk for being my first instructor in the Laboratory, Ben
Adu Gyan thanks for opening your doors to me when I just arrived and for being such an
elder brother, Ankie Söderlund, Monika Hansson, Caroline Ekberg, Elizabeth Hugosson,
Ahmed Bolad, Mounira Djerbi and Valentina Screpanti thanks for being such an
inspiration, Alice Nyakeriga and Ariane Rodríquez Muňoz my two elder sisters at the
department, Qazi Khaleda Rahmen for all the challenging questions and the many
invitations for lunch, Gawa Bidla for being there right from the start, Shiva S. Esfahani,
Izaura Ross, Karin Lindroth and Piyatida Tangteerawatana.
To all my Colleagues (current) at the department of Immunology; Khosro Masjedi for the
very incisive questions and interesting discussions, Nina-Maria Vasconcelos for being such a
kind person, Esther Julián, Anna-Karin Larsson, Manijeh Vafa, Anna Tjärnlund,
Halima Balogun, Petra Amoudruz, John Arko-Mensah, Norra Bachmayer,Yvonne
Sundström, Shanie S. Hedengren, Camilla Rydström, Elisabeth Israelsson, Amilcar
Cytokine responses in metal-induced ACD
59
Reis, Nnaemeka Iriemenam, Magdi Ali, Nancy Awah, Jacqueline Calla and Kulachart
Jangpatarapongsa, I say thank you for the collaboration, exciting scientific discussions and
great times out of the department.
Many thanks to Margareta Hagstedt and Ann Sjölund for all the support on the bench.
Thanks to former, Gunilla Tillinger and Suzanne Åsbrink, and present, Gelana Yadeta,
secretaries of the department of Immunology for your readiness to help every time I knocked
on your office door. To the team at the MIM secretariate, Andreas Heddini, Olle Terenius
and Anna-Leena, I say, thanks for the nice discussions and for being part of every party!
The entire group at Mabtech (www.mabtech.com) is greatly appreciated for creating such a
stimulating and humane working environment.
Dr Achidi Eric Akum is gratefully acknowledged for introducing me into the world of
biomedical research.
My sincere thanks to Dyllis M. Sefeh (min kärlek, andra halva och hjärta), you have been
such a great source of support especially at moments that I felt the world was crashing on me!
Many thanks to my siblings and their families; Ni Joe, Ni John (RIP), Ni Tom, Ni Willie, Ni
Peter, sister Mary, sister Vero, Dr Paul, Maggie, their spouses and all my nieces and
nephews for their moral support. To my cousins; Victor N. Cheo, Fidelis C. Fru and Eric Y.
Tagha, I say thanks for your support and for the wonderful trips around Europe.
Special tribute to my dad, pa Alex Minang (RIP) and mum, mami Margaret Atih Minang
for pulling it off with such a full squad!
Finally, thanks to the almighty for giving me the strength to carry on.
The studies reported in this thesis were supported by grants from the Swedish Agency for
Innovation Systems (VINNOVA), the Swedish Council for Working Life and Social
Research, the Vårdal foundation for Health Care Sciences and Allergy Research (Stockholm),
The
Asthma
and
Allergy
Foundation
and
the
Swedish
Research
Council.
60
Jacob Taku Minang
REFERENCES
Aalto-Korte, K. and Makinen-Kiljunen, S. 2003. Specific immunoglobulin E in patients with
immediate persulfate hypersensitivity. Contact Dermatitis 49:22-25.
Abbas, A. K., Murphy, K. M., Sher, A. 1996. Functional diversity of helper T lymphocytes.
Nature 383:787-793.
Ahlborg, N. and Paulie, S. 2002. ELISA (Enzyme-linked immunosorbent assay) Wiley
Encyclopedia of Mol Med 5:1150-1153.
Akdis, C. A. and Blaser, K. 2001. Mechanisms of interleukin-10-mediated immune
suppression. Immunol 103:131-136.
Akdis, C. A., Blesken, T., Akdis, M., Wüthrich, B., Blaser, K. 1998 Role of Interleukin 10 in
Specific Immunotherapy. J Clin Invest 102:98-106.
Akdis, C. A., Joss, A., Akdis, M., Faith, A., Blaser, K. 2000. A molecular basis for T cell
suppression by IL-10: CD28-associated IL-10 receptor inhibits CD28 tyrosine
phosphorylation and phosphotidylinositol 3-kinase binding. FASEB J 14:1666-1668.
Akdis, C., Verhagen, J., Taylor, A., Karamloo, F., Karagiannidis, C., Crameri, R., Thunberg,
S., Deniz, G., Valenta, R., Fiebig, H., Kegel, C., Disch, R., Schmidt-Weber, C. B., Blaser,
K., Akdis, C. A. 2004. Immune responses in healthy and allergic individuals are
characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2
cells. J Exp Med 99:1567-1575.
Akira, S., Takeda, K., Kaisho, T. 2001. Toll-like receptors: critical proteins linking innate and
acquired immunity. Nat Immunol 2:675-680.
Aktas, E., Akdis, M., Bilgic, S., Disch, R., Falk, C. S., Blaser, K., Akdis, C., Deniz, G. 2005.
Different natural killer (NK) receptor expression and immunoglobulin E (IgE) regulation
by NK1 and NK2 cells. Clin Exp Immunol 140:301-309.
Arenzana-Seisdedos, F., Virelizier, J. L., Fiers, W. 1985. Interferons as macrophageactivating factors. III. Preferential effects of interferon-gamma on the interleukin 1
secretory potential of fresh or aged human monocytes. J Immunol 134:2444-2448.
Asherson, G. L., Dieli, F., Sireci, G., Salerno, A. 1996. Role of IL-4 in delayed type
hypersensitivity. Clin Exp Immunol 103:1-4.
Askenase, P. W., Szczepanik, M., Ptak, M., Paliwal, V., Ptak, W. 1995. Gamma delta T cells
in normal spleen assist immunized alpha beta T cells in the adoptive cell transfer of
contact sensitivity. Effect of Bordetella pertussis, cyclophosphamide, and antibodies to
determinants on suppressor cells. J Immunol 154:3644-3653.
Avnstorp, C. 1989. Prevalence of cement eczema in Denmark before and since addition of
ferrous sulfate to Danish cement. Acta Derm Venereol 69:151-155.
Cytokine responses in metal-induced ACD
61
Baecher-Allan, C., Brown, J. A., Freeman, G. J., Hafler, D. A. 2001. CD4+CD25high
regulatory cells in human peripheral blood. J Immunol 167:1245-1253.
Ball, E. J. and Stastny, P. 1984. Antigen-specific HLA-restricted human T-cell lines. II. A
GAT-specific T-cell line restricted by a determinant carried by an HLA-DQ molecule.
Immunogenetics. 20:547-564.
Barker, J. N., Mitra, R. S., Griffiths, C. E., Dixit, V. M., Nickoloff, B. J. 1991. Keratinocytes
as initiators of inflammation. Lancet 337:211-214.
Bellinghusen, I., Brand, U., Steinbrink, K., Enk, A. H, Knop, J., Saloga, J. 2001. Inhibition of
human allergic T-cell responses by IL-10-treated dendritic cells: differences from
hydrocortisone-treated dendritic cells. J Allergy Clin Immunol 108: 242-249.
Bendelac, A. and Schwartz, R. H. 1991. Th0 cells in the thymus: the question of T-helper
lineages. Immunol Rev123: 169-188.
Bendiner, E. 1981. Baron von Pirquet: the aristocrat who discovered and defined allergy.
Hosp Prac 16:137–144.
Bettelli, E., Das, M. P., Howard, E. D., Weiner, H. L., Sobel, R. A., Kuchroo, V. K. 1998. IL10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by
studies of IL-10- and IL-4-deficient and transgenic mice. J Immunol 161:3299-3306.
Boukhman, M. P. and Maibach, H. I. 2001. Thresholds in contact sensitization: immunologic
mechanisms and experimental evidence in humans--an overview. Food Chem Toxicol
39:1125-1134.
Bourke, J. F., Batta, K., Prais, L., Abdullah, A., Foulds, I. S. 1999. The reproducibility of
patch tests. Br J Dermatol 140:102-105.
Braciale, T. J., Morrison, L. A., Sweetser, M. T., Sambrook, J., Gething, M. J., Braciale, V. L.
1987. Antigen presentation pathways to class I and class II MHC-restricted T
lymphocytes. Immunol Rev 98:95-114.
Bradding, P., Feather, I. H., Wilson, S., Bardin, P. G., Heusser, C. H., Holgate, S. T.,
Howarth, P. H. 1993. Immunolocalization of cytokines in the nasal mucosa of normal and
perennial rhinitic subjects. The mast cell as a source of IL-4, IL-5, and IL-6 in human
allergic mucosal inflammation. J Immunol 151:3853-3865.
Brasch, J., Geier, J. 1997. Patch test results in schoolchildren. Results from the Information
Network of Departments of Dermatology (IVDK) and the German Contact Dermatitis
Research Group (DKG). Contact Dermatitis 37:286-293.
Brasch, J., Uter, W., Geier, J., Schnuch, A., and the German Contact Dermatitis Research
Group and the Information Network of Departments of Dermatology in Germany. 2001.
Associated positive patch test reactions to standard contact allergens. Am J Contact
Dermatitis 12:197-202.
62
Jacob Taku Minang
Britton, J. E. R., Wilkinson, S. M., English, J. S. C., Gawkrodger, D. J., Ormerod, A. D.,
Sansom, J. E., Shaw, S., Statham, B. 2003. The British standard series of contact
dermatitis allergens: validation in clinical practice and value for clinical governance. Br J
Dermatol 148:259-264.
Bruze, M. 1986. Seasonal influence on routine patch test results. Contact Dermatitis 14:184.
Bryld, L. E., Hindsberger, C., Kyvik, K. O., Agner, T., Menne, T. 2003. Risk factors
influencing the development of hand eczema in a population-based twin sample. Br J
Dermatol 149:1214-1220.
Buchvald, D. and Lundeberg, L. 2004. Impaired responses of peripheral blood mononuclear
cells to nickel in patients with nickel-allergic contact dermatitis and concomitant atopic
dermatitis. Br J Dermatol 150:484-492.
Büdinger, L. and Hertl, M. 2000. Immunologic mechanisms in hypersensitivity reactions to
metal ions: an overview. Allergy 55:108-115.
Carter, L. L. and Swain, S. L. 1997. Single cell analyses of cytokine production. Curr Opin
Immunol 9:177-182.
Cavani, A., Mei, D., Guerra, E., Corinti, S., Giani, M., Pirrotta, L., Puddu, P., Girolomoni, G.
1998. Patients with allergic contact dermatitis to nickel and nonallergic individuals
display different nickel-specific T cell responses. Evidence for the presence of effector
CD8+ and regulatory CD4+ T cells. J Invest Dermatol 111:621-628.
Cavani, A., Nasorri, F., Ottaviani, C., Sebastiani, S., De Pita, O., Girolomoni, G. 2003.
Human CD25+ regulatory T cells maintain immune tolerance to nickel in healthy,
nonallergic individuals. J Immunol 171:5760-5768.
Cavani, A., Nasorri, F., Prezzi, C., Sebastiani, S., Albanesi, C., Girolomoni, G. 2000. Human
CD4+ T lymphocytes with remarkable regulatory functions on dendritic cells and nickelspecific Th1 immune responses. J Invest Dermatol 114:295-302.
Cederbrant, K., Anderson, C., Andersson, T., Marcusson-Stahl, M., Hultman, P. 2003.
Cytokine production, lymphocyte proliferation and T-cell receptor Vbeta expression in
primary peripheral blood mononuclear cell cultures from nickel-allergic individuals. Int
Arch Allergy Immunol 132:373-379.
Cederbrant, K., Hultman, P., Marcusson, J. A., Tibbling, L. 1997. In vitro lymphocyte
proliferation as compared to patch test using gold, palladium and nickel. Int Arch Allergy
Immunol 112:212-217.
Chen, L., Grabowski, K.A., Xin, J. P., Coleman, J., Huang, Z., Espiritu, B., Alkan, S., Xie, H.
B., Zhu, Y., White, F. A., Clancy, J. Jr., Huang, H. 2004. IL-4 Induces Differentiation and
Expansion of Th2 Cytokine-Producing Eosinophils. J Immunol 172:2059-2066.
Chen, W., Jin, W., Hardegen, N., Lei, K. J., Li, L., Marinos, N., McGrady, G., Wahl, S. M.
2003. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T
cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 198:1875-1886.
Cytokine responses in metal-induced ACD
63
Chien, Y. H., Jores, R., Crowley, M. P. 1996. Recognition by gamma/delta T cells. Annu Rev
Immunol 14:511-532.
Coffman, R. L., Lebman, D. A., Rothman, P. 1993. Mechanism and regulation of
immunoglobulin isotype switching. Adv Immunol 54:229-270.
Coffman, R. L. and von der Weid, T. 1997. Multiple pathways for the initiation of T helper 2
(Th2) responses. J Exp Med 185:373-375.
Croft, M. and Swain, S. L. 1992. Analysis of CD4+ T cells that provide contact-dependent
bystander help to B cells. J Immunol 149:3157-3165.
Curiel-Lewandrowski, C., Venna, S. S., Eller, M. S., Cruikshank, W., Dougherty, I., Cruz, P.
D, Jr., Gilchrest, B. A. 2003. Inhibition of the elicitation phase of contact hypersensitivity
by thymidine dinucleotides is in part mediated by increased expression of interleukin-10
in human keratinocytes. Exp Dermatol 12:145-152.
Czerkinsky, C., Andersson, G., Ekre, H. P., Nilsson, L.A., Klareskog, L., Ouchterlony, Ö.
1988. Reverse ELISPOT assay for clonal analysis of cytokine production: I. Enumeration
of gamma-interferon-secreting cells. J Immunol Methods 25:29-36.
Czerkinsky, C. C., Nilsson, L. Å., Nygren, H., Ouchterlony, Ö., Tarkowski, A. 1983. A solidphase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibodysecreting cells. J Immunol Methods 65:109-121.
Czernielewski, J. M. Demarchez, M. 1987. Further evidence for the self-reproducing capacity
of Langerhans cells in human skin. J Invest Dermatol 88:17-20.
De Fine Olivarius, F. and Menne, T. 1992. Skin reactivity to metallic cobalt in patients with a
positive patch test to cobalt chloride. Contact Dermatitis 27:241-243.
De Gast, G. C., Verdonck, L. F., Middeldorp, J. M., The, T. H., Hekker, A., vd Linden, J. A.,
Kreeft, H. A., Bast, B. J. 1985. Recovery of T cell subsets after autologous bone marrow
transplantation is mainly due to proliferation of mature T cells in the graft. Blood 66:428431.
De la Barrera, S., Aleman, M., Musella, R., Schierloh, P., Pasquinelli, V., Garcia, V., Abbate,
E., Sasiain Mdel, C. 2004. IL-10 down-regulates costimulatory molecules on
Mycobacterium tuberculosis-pulsed macrophages and impairs the lytic activity of CD4
and CD8 CTL in tuberculosis patients. Clin Exp Immunol 138:128-138.
Del Prete, G., Maggi, E., Paola, P., Ghretien, I., Tiri, A., Macchia, D., Banchereau, J., De
Vreis, J.E., Romagnani, S. 1988. IL-4 is an essential factor for the IgE synthesis induced
in vitro by human T cell clones and their supernatants. J Immunol 140:4193-4198.
Dent, L. A. 2002. For better or worse: common determinants influencing health and disease in
parasitic infections, asthma and reproductive biology. J Reprod Immunol 57:255-272.
64
Jacob Taku Minang
Derocq, J. M., Segui, M., Poinot-Chazel, C., Minty, A., Caput, D., Ferrara, P., Casellas, P.
1994. Interleukin-13 stimulates interleukin-6 production by human keratinocytes.
Similarity with interleukin-4. FEBS Lett 343:32-36.
De Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C. G., de Vries, J. E. 1991. Interleukin
10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL10 produced by monocytes. J Exp Med 174:1209-1220.
De Waard-van der Spek, F. B., Elst, E. F., Mulder, P. G., Munte, K., Deviller, A. C., Oranje,
A. P. 1998. Diagnostic tests in children with atopic dermatitis and food allergy. Allergy
53:1087-1091.
Dhodapkar, M. V. and Steinman, R. M. 2002. Antigen-bearing immature dendritic cells
induce peptide-specific CD8(+) regulatory T cells in vivo in humans. Blood 100:174-177.
Dieli, F., Ptak, W., Sireci, G., Romano, G. C., Potestio, M, Salerno, A, Asherson, G. L. 1998.
Cross-talk between V beta 8+ and gamma delta+ T lymphocytes in contact sensitivity.
Immunology 93:469-477.
Dieu, M. C., Vanbervliet, B., Vicari, A., Bridon, J. M., Oldham, E., Ait-Yahia, S., Briere, F.,
Zlotnik, A., Lebecque, S., Caux, C. 1998. Selective recruitment of immature and mature
dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med
188:373-386.
Di Gioacchino, M., Cavallucci, E., Di Stefano, F., Verna, N., Ramondo, S., Ciuffreda, S.,
Riccioni, G., Boscolo, P. 2000. Influence of total IgE and seasonal increase of eosinophil
cationic protein on bronchial hyperreactivity in asthmatic grass-sensitized farmers.
Allergy 55:1030-1034.
Di Lorenzo, G., Mansueto, P., Melluso, M., Morici, G., Norrito, F., Esposito, Pellitteri, M., Di
Salvo, A., Colombo, A., Candore, G., Caruso, C. 1997. Non-specific airway
hyperresponsiveness in mono-sensitive Sicilian patients with allergic rhinitis. Its
relationship to total serum IgE levels and blood eosinophils during and out of the pollen
season. Clin Exp Allergy 27:1052-1059.
Doherty, P. C. and Zinkernagel, R. M. 1975. A biological role for the major
histocompatibility antigens. Lancet 1:1406-1409.
Duarte, I., Lazzarini, R., Kobata, C. M. 2003. Contact dermatitis in adolescents. Am J Contact
Dermat 14:200-202.
Dustin, M. L., Singer, K. H., Tuck, D. T., Springer, T. A. 1988. Adhesion of T lymphoblasts
to epidermal keratinocytes is regulated by interferon gamma and is mediated by
intercellular adhesion molecule 1 (ICAM-1). J Exp Med 167:1323-1340.
Eedy, D.J., Burrows, D., McMaster, D. 1991. The nickel content of certain commercially
available metallic patch test materials and its relevance in nickel-sensitive subjects.
Contact Dermatitis 24:11-15.
Cytokine responses in metal-induced ACD
65
Ekerfelt, C., Ermerudh, J., Jenmalm, M. C. 2002. Detection of spontaneous and antigeninduced human interleukin-4 responses in vitro: comparison of ELISPOT, a novel ELISA
and real-time RT-PCR. J Immunol Methods 260:55-67.
Emtestam, L., Zetterquist, H., Olerup, O. 1993. HLA-DR, -DQ and -DP alleles in nickel,
chromium, and/or cobalt-sensitive individuals: genomic analysis based on restriction
fragment length polymorphisms. J Invest Dermatol 100:271-274.
Engvall, E. and Perlmann, P. 1972. Enzyme-linked immunosorbent assay, ELISA. 3.
Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigencoated tubes. J Immunol 109:129-135.
Enk, A. H., Angeloni, V. L., Udey, M. C., Katz, S. I. 1993. Inhibition of Langerhans cell
antigen-presenting function by IL-10. A role for IL-10 in induction of tolerance. J
Immunol 151:2390-2398.
Enk, A. H. and Katz, S. I. 1992a. Identification and induction of keratinocyte-derived IL-10. J
Immunol 149:92-95.
Enk, A. H. and Katz, S. I. 1992b. Early molecular events in the induction phase of contact
sensitivity. Proc Natl Acad Sci U S A 89:1398-1402.
Estlander, T., Kanerva, L., Tupasela, O., Keskinen, H., Jolanki, R. 1993. Immediate and
delayed allergy to nickel with contact urticaria, rhinitis, asthma and contact dermatitis.
Clin Exp Allergy 23:306-310.
Ewen, C. and Baca-Estrada, M. E. 2001. Evaluation of interleukin-4 concentration by ELISA
is influenced by the consumption of IL-4 by cultured cells. J Interferon Cytokine Res 21:
39-43.
Falsafi-Amin, H., Lundeberg, L., Bakhiet, M., Nordlind, K. 2000. Early DNA synthesis and
cytokine expression in the nickel activation of peripheral blood mononuclear cells in
nickel-allergic subjects. Int Arch Allergy Immunol 123:170-176.
Fausto da Silva, J. J. R. and Williams, R. J. P. 2001. The Biological chemistry of the
elements. The inorganic chemistry of life, 2nd ed. Oxford University Press, Oxford UK.
Firestein, G. S., Roeder, W. D., Laxer, J. A., Townsend, K. S., Weaver, C. T., Hom, J. T.,
Linton, J., Torbett, B. E., Glasebrook, A. L. 1989. A new murine CD4+ T cell subset with
an unrestricted cytokine profile. J Immunol 143: 518-525.
Flier, J., Boorsma, D. M., Bruynzeel, D. P., Van Beek, P. J., Stoof, T. J., Scheper, R. J.,
Willemze, R, Tensen, C. P. 1999. The CXCR3 activating chemokines IP-10, Mig, and IP9 are expressed in allergic but not in irritant patch test reactions. J Invest Dermatol
113:574-578.
Fontenot, J. D., Gavin, M. A., Rudensky, A. Y. 2003. Foxp3 programs the development and
function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330-336.
66
Jacob Taku Minang
Franc, N. C., White, K., Ezekowitz, R. A. 1999. Phagocytosis and development: back to the
future. Curr Opin Immunol 11:47-52.
Fujimura, T., Chamoto, K., Tsuji, T., Sato, T., Yokouchi, H., Aiba, S., Tagami, H., Tanaka, J.,
Imamura, M., Togashi, Y., Koda, T., Nishimura, T. 2004. Generation of leukemia-specific
T-helper type 1 cells applicable to human leukemia cell-therapy. Immunol Lett 93:17-25.
Fukaura, H., Kent, S. C., Pietrusewicz, M. J., Khoury, S. J., Weiner, H. L., Hafler, D. A.
1996. Induction of circulating myelin basic protein and proteolipid protein-specific
transforming growth factor-beta1-secreting Th3 T cells by oral administration of myelin in
multiple sclerosis patients. J Clin Invest 98:70-77.
Garner, L. A. 2004. Contact dermatitis to metals. Dermatologic Ther 17:321-327.
Gawkrodger, D. J., Lewis, F. M., Shah, M. 2000. Contact sensitivity to nickel and other
metals in jewelry reactors. J. Am. Acad. Dermatol 43:31-36.
Gawkrodger, D. J., Vestey, J. P., Wong, W. K., Buxton, P. K. 1986. Contact clinic survey of
nickel-sensitive subjects. Contact Dermatitis 14:165-169.
Gershon, R. K. and Kondo, K. 1970. Cell interactions in the induction of tolerance: the role of
thymic lymphocytes. Immunology 18:723-737.
Gilliet, M. and Liu, Y. J. 2002. Generation of human CD8 T regulatory cells by CD40 ligandactivated plasmacytoid dendritic cells. J Exp Med 195:695-704.
Gorelik, L. and Flavell, R. A. 2002. Transforming growth factor-beta in T-cell biology. Nat
Rev Immunol 2:46-53.
Grabbe, S. and Schwarz, T. 1998. Immunoregulatory mechanisms involved in elicitation of
allergic contact hypersensitivity. Immunol Today 19:37-44.
Groux, H., O'Garra, A., Bigler, M, Rouleau, M., Antonenko, S., de Vries, J. E., Roncarolo, M.
G. 1997. A CD4+ T-cell subset inhibits antigen specific T-cell responses and prevents
colitis. Nature 389:737-742.
Gruvberger, B. 1997. Methylisothiazolinones. Diagnosis and prevention of allergic contact
dermatitis. Acta Derm Venereol (suppl.) (Stockh) 200:1-42.
Guillet, G., Guillet, M. H., Dagregorio, G. 2005. Allergic contact dermatitis from natural
rubber latex in atopic dermatitis and the risk of later Type I allergy. Contact Dermatitis
53:46-51.
Hannuksela, M. 1980. Atopic contact dermatitis. Contact Dermatitis. 6:30-32.
Hansen, M. B., Johansen, J. D., Menne, T. 2003. Chromium allergy: significance of both
Cr(III) and Cr(VI). Contact Dermatitis 49:206-212.
Cytokine responses in metal-induced ACD
67
Hart, O. M., Athie-Morales, V., O’connor, G. M., Gardiner, C. M. 2005. TLR7/8-Mediated
Activation of Human NK Cells Results in Accessory Cell-Dependent IFN-gamma
Production. J Immunol 175:1636-1642.
Harvel, J., Bason, M., Maibach, H. 1994. Contact urticaria and its mechanisms. Food Chem
Toxicol 32:103-112.
Hedges, J. F., Lubick, K. J., Jutila, M. A. 2005. Gamma delta T cells respond directly to
pathogen-associated molecular patterns. J Immunol 174:6045-6053.
Hegewald, J., Uter, W., Pfahlberg, A., Geier, J, Schnuch, A., for the IVDK. 2005. A
multifactorial analysis of concurrent patch-test reactions to nickel, cobalt, and chromate.
Allergy 60:372-378.
Herrick, C. A., Xu, L., McKenzie, A. N., Tigelaar, R. E., Bottomly, K. 2003. IL-13 is
necessary, not simply sufficient, for epicutaneously induced Th2 responses to soluble
protein antigen. J Immunol 170:2488-2495.
Hessle, C., Andersson, B., Wold, A. E. 2000. Gram-positive bacteria are potent inducers of
monocytic interleukin-12 (IL-12) while gram-negative bacteria preferentially stimulate
IL-10 production. Infect Immun 68: 3581-3586.
Hindsen, M., Bruze, M., Christensen, O. B. 1999. Individual variation in nickel patch test
reactivity. Am J Contact Dermat 10:62-67.
Hoefakker, S., Caubo, M., van ‘t Erve, E. H., Roggeveen, M. J., Boersma, W. J., van Joost,
T., Notten, W. R., Claassen, E. 1995. In vivo cytokine profiles in allergic and irritant
contact dermatitis. Contact Dermatitis 33:258-266.
Horwitz, D. A., Zheng, S. G., Gray, J. D. 2003. The role of the combination of IL-2 and TGFbeta or IL-10 in the generation and function of CD4+ CD25+ and CD8+ regulatory T cell
subsets. J Leukoc Biol 74:471-478.
Inaba, K. and Inaba, M. 2005. Antigen recognition and presentation by dendritic cells. Int J
Hematol 81:181-187.
Ishii, N., Sugita, Y., Nakajima, H., Tanaka, S., Askenase, P. W. 1995. Elicitation of nickel
sulfate (NiSO4)-specific delayed-type hypersensitivity requires early-occurring and earlyacting, NiSO4-specific DTH-initiating cells with an unusual mixed phenotype for an
antigen-specific cell. Cell Immunol 161:244-255.
Ishii, N., Takahashi, K., Nakajima, H., Tanaka, S., Askenase, P. W. 1994. DNFB contact
sensitivity (CS) in BALB/c and C3H/He mice: requirement for early-occurring, earlyacting, antigen-specific, CS-initiating cells with an unusual phenotype (Thy-1+, CD5+,
CD3-, CD4-, CD8-, sIg-, B220+, MHC class II-, CD23+, IL-2R-, IL-3R+, Mel-14-, Pgp1+, J11d+, MAC-1+, LFA-1+, and Fc gamma RII+). J Invest Dermatol 102:321-327.
Jackola, D. R., Blumenthal, M. N., Rosenberg, A. 2004. Evidence for two independent
distributions of serum immunoglobulin E in atopic families: cognate and non-cognate IgE.
Hum Immunol 65:20-30.
68
Jacob Taku Minang
Jakobson, E., Masjedi, K., Ahlborg, N., Lundeberg, L., Karlberg, A. T., Scheynius, A., 2002.
Cytokine production in nickel-sensitized individuals analysed with enzyme-linked
immunospot assay: possible implication for diagnosis. Br J Dermatol 147:442-449.
Jensen, C. S., Lisby, S., Baadsgaard, O., Volund, A., Menne, T. 2002. Decrease in nickel
sensitization in a Danish schoolgirl population with ears pierced after implementation of a
nickel-exposure regulation. Br J Dermatol 146:636-642.
José, A. M., Borghans., Noest, A. J., De Boer, R. J.
immunological memory be? J Immunol 163:569-575.
1999. How specific should
Kabelitz, D., Marischen, L., Oberg, H. H., Holtmeier, W., Wesch, D. 2005. Epithelial defence
by gamma delta T cells. Int Arch Allergy Immunol 137:73-81.
Kalish, R. S., Wood, J. A., LaPorte, A. 1994. Processing of urushiol (poison ivy) hapten by
both endogenous and exogenous pathways for presentation to T cells in vitro. J Clin Invest
93:2039-2047.
Kaplan, G., Walsh, G., Guido, L. S., Meyn, P., Burkhardt, R. A., Abalos, R. M., Barker, J.,
Frindt, P. A., Fajardo, T. T., Celona, R., Cohn Z. A. 1992. Novel responses of human skin
to intradermal recombinant granulocyte/macrophage-colony-stimulating factor:
Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing. J Exp
Med 175:1717-1728.
Kapsenberg, M. L., Wierenga, E. A., Stiekema F. E. M., Tiggelman A. B. C., Bos, J. D. 1992.
Th1 lymphokine production profiles of nickel-specific CD4+ T-lymphocyte clones from
nickel contact allergic and nonallergic individuals. J Invest Dermatol 98:59-63.
Kasaian, M. T., Meyer, C. H., Nault, A. K., Bond, J. F. 1995. An increased frequency of IgEproducing B cell precursors contributes to the elevated levels of plasma IgE in atopic
subjects. Clin Exp Allergy 25:749-755.
Kemeny, D. M., Vyas, B., Vukmanovic-Stejic, M., Thomas, M. J., Noble, A., Loh, L. C.,
O'Connor, B. J. 1999. CD8(+) T cell subsets and chronic obstructive pulmonary disease.
Am J Respir Crit Care Med. 160: 33-37.
Khattri, R., Cox, T., Yasayko, S. A., Ramsdell, F. 2003. An essential role for Scurfin in
CD4+CD25+ T regulatory cells. Nat Immunol 4:337-342.
Kidd, P. 2003. Th1/Th2 balance: the hypothesis, its limitations, and implications for health
and disease. Altern Med Rev 8:223-246.
Kimber, I. and Cumberbatch, M. 1992. Stimulation of Langerhans cell migration by tumor
necrosis factor alpha (TNF-alpha). J Invest Dermatol 99:48-50.
Kimber, I., Dearman, R. J., Cumberbatch, M., Huby, R. J. 1998. Langerhans cells and
chemical allergy. Curr Opin Immunol 10:614-619.
Cytokine responses in metal-induced ACD
69
Kondo, S., Kooshesh, F., Wang, B., Fujisawa, H., Sauder, D. N. 1996. Contribution of the
CD28 molecule to allergic and irritant-induced skin reactions in CD28 -/- mice. J
Immunol 157:4822-4829.
Kondo, S. and Sauder, D. N. 1995. Epidermal cytokines in allergic contact dermatitis. J Am
Acad Dermatol 33:786-800.
Lachapelle, J-M. 2001. Historical aspects of patch testing. In: Rycroft R. J. G., Menné, T.,
Frosch, P. J., Lepoittevin, J-P (eds) Textbook of contact dermatitis, 3rd edn. Springer,
Berlin Heidelberg New York pp 1-10.
Lachapelle, J. M., Lauwerys, R., Tennstedt, D., Andanson, J., Benezra, C., Chabeau, G.,
Ducombs, G., Foussereau, J., Lacroix, M., Martin, P. 1980. Eau de Javel and prevention
of chromate allergy in France. Contact Dermatitis 6:107-110.
Lafaille, J. J. 1998. The role of helper T cell subsets in autoimmune diseases. Cytokine
Growth Factor Rev 9:139-151.
Lalvani, A., Pathan, A. A., Durkan, H., Wilkinson, K. A., Whelan, A., Deeks, J. J., Reece, W.
H., Latif, M., Pasvol, G., Hill, A. V., 2001a. Enhanced contact tracing and spatial tracking
of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells.
Lancet 357:2017-2021.
Lalvani, A., Pathan, A. A., McShane, H., Wilkinson, R. J., Latif, M., Conlon, C. P., Pasvol,
G., Hill, A. V. 2001b. Rapid detection of Mycobacterium tuberculosis infection by
enumeration of antigen-specific T cells. Am. J. Respir. Crit. Care Med. 163:824-828.
Landsteiner, K., Jacobs, J. L. 1935. Studies on the sensitisation of animals with simple
chemical compounds. J Exp Med 61:643.
Lepoittevin, J. P. 2001. Molecular Aspects of allergic contact dermatitis. In: Rycroft R. J. G.,
Menné, T., Frosch, P. J., Lepoittevin, J-P (eds) Textbook of contact dermatitis, 3rd edn.
Springer, Berlin Heidelberg New York pp 59-89.
Levin, C. Y. and Maibach, H. I. 2002. Irritant contact dermatitis: is there an immunologic
component? Int Immunopharmacol 2:183-189.
Liden, C. 1992. Nickel in jewellery and associated products. Contact Dermatitis. 26:73-75.
Liden, C. and Wahlberg, J. E. 1994. Cross-reactivity to metal compounds studied in guinea
pigs induced with chromate or cobalt. Acta Derm Venereol. 74:341-343.
Lienhardt, C., Azzurri, A., Amedei, A., Fielding, K., Sillah, J., Sow, O. Y., Bah, B.,
Benagiano, M., Diallo, A., Manetti, R., Manneh, K., Gustafson, P., Bennett, S., D'Elios,
M. M., McAdam, K., Del Prete, G. 2002. Active tuberculosis in Africa is associated with
reduced Th1 and increased Th2 activity in vivo. Eur J Immunol 32:1605-1613.
Li, L. F. and Wang, J. 2002. Contact hypersensitivity in hand dermatitis. Contact Dermatitis.
47:206-209.
70
Jacob Taku Minang
Lindemann, M., Bohmer, J., Zabel, M., Grosse-Wilde, H. 2003. ELISpot: a new tool for the
detection of nickel sensitization. Clin Exp Allergy 33:992-998.
Lisby, S., Hansen, L. H., Menn, T., Baadsgaard, O. 1999. Nickel-induced proliferation of both
memory and naive T cells in patch test-negative individuals. Clin Exp Immunol 117:217222.
Lisby, S. and Hauser, C. 2002. Transcriptional regulation of tumor necrosis factor-alpha in
keratinocytes mediated by interleukin-1beta and tumor necrosis factor-alpha. Exp
Dermatol 11:592-598.
Lisi, P., Brunelli, L., Stingeni, L. 2003. Co-sensitivity between cobalt and other transition
metals. Contact Dermatitis. 48:172-173.
Liu, C. A., Wang, C. L., Chuang, H., Ou, C. Y., Hsu, T. Y., Yang, K. D. 2003. Prenatal
prediction of infant atopy by maternal but not paternal total IgE levels. J Allergy Clin
Immunol 112:899-904.
Luster, A. D. 2002. The role of chemokines in linking innate and adaptive immunity. Curr
Opin Immunol 14: 129-135.
Malten, K. E, den Arend, J. A., Wiggers, R. E. 1979. Delayed irritation: hexanediol diacrylate
and butanediol diacrylate. Contact Dermatitis 5:178-184.
Marcinkiewicz, J. and Chain, B. M. 1993. Regulation of in vitro release of TH2 type
cytokines (IL-4, IL-6) in the T cell response to the trinitrophenyl (TNP) hapten. Cell
Immunol 146:406-411.
Masjedi, K., Ahlborg, N., Gruvberger, B., Bruze, M., Karlberg, A. T. 2003.
Methylisothiazolinones elicit increased production of both T helper (Th)1- and Th2-like
cytokines by peripheral blood mononuclear cells from contact allergic individuals. Br J
Dermatol 149:1172-1182.
Mason, D. 2001. Some quantitative aspects of T-cell repertoire selection: the requirement for
regulatory T cells. Immunol Rev 182:80-88.
Matzinger, R. 1994. Tolerance, danger, and the extended family. Annu Rev Immunol 12:9911045.
McGuirk, P. and Mills, K. H. 2002. Pathogen-specific regulatory T cells provoke a shift in the
Th1/ Th2 paradigm in immunity to infectious diseases. Trends Immunol 23:450-455.
McHugh, S. M., Wilson, A. B., Deighton, J., Lachmann, P. J., Ewan, P. W. 1994. The profiles
of interleukin (IL)-2, IL-6, and interferon-gamma production by peripheral blood
mononuclear cells from house-dust-mite-allergic patients: a role for IL-6 in allergic
disease. Allergy 49:751-759.
McKenzie, A. N., Culpepper, J. A., de Waal Malefyt, R., Briere, F., Punnonen, J., Aversa, G.,
Sato, A., Dang, W., Cocks, B. G., Menon, S., De Vries, J. E. and Zurawski G. 1993.
Cytokine responses in metal-induced ACD
71
Interleukin 13, a T-cell-derived cytokine that regulates human monocyte and B-cell
function. Proc Natl Acad Sci U S A. 90: 3735-3739.
Meding, B., Jarvholm, B., 2002. Hand eczema in Swedish adults - changes in prevalence
between 1983 and 1996. J Invest Dermatol 118:719-723.
Meding, B., Liden, C., Berglind, N., 2001. Self-diagnosed dermatitis in adults. Results from a
population survey in Stockholm. Contact Dermatitis 45:341-345.
Miescher, G. C., Howe, R. C., Lees, R. K., MacDonald, H. R. 1988. CD3-associated
alpha/beta and gamma/delta heterodimeric receptors are expressed by distinct populations
of CD4- CD8- thymocytes. J Immunol 140:1779-1782.
Moed, H., Boorsma, D. M., Stoof, T. J., von Blomberg, B. M., Bruynzeel, D. P., Scheper, R.
J., Gibbs, S., Rustemeyer, T. 2004b. Nickel-responding T cells are CD4+ CLA+
CD45RO+ and express chemokine receptors CXCR3, CCR4 and CCR10. Br J Dermatol
151:32-41.
Moed, H., Boorsma, D. M., Tensen, C. P., Flier, J., Jonker, M. J., Stoof, T. J, von Blomberg,
B. M., Bruynzeel, D. P., Scheper, R. J., Rustemeyer, T., Gibbs, S. 2004a. Increased
CCL27-CCR10 expression in allergic contact dermatitis: implications for local skin
memory. J Pathol 204:39-46.
Moed, H., Boorsma, D. M., von Blomberg, B. M. E., Bruynzeel, D. P., Scheper, R. J., Gibbs,
S., Rustemeyer, T. 2005. Regulation of nickel-induced T-cell responsiveness by
CD4+CD25+ cells in contact allergic patients and healthy individuals. Contact Dermatitis
53:71-74.
Morikawa, Y., Furotani, M., Kuribayashi, K., Matsuura, N., Kakudo, K. 1992. The role of
antigen-presenting cells in the regulation of delayed-type hypersensitivity. I. Spleen
dendritic cells. Immunol 77:81-87.
Moser, M. and Murphy, K. M. 2000. Dendritic cell regulation of TH1 TH2 development. Nat
Immunol 1:199-205.
Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A., and Coffman, R. L. 1986.
Two types of murine helper T cell clone. I. Definition according, to profiles of
lymphokine activities and secreted proteins. J Immunol 136:2348-2357.
Mosmann, T. R. and Coffman, R. L. 1989. TH1 and TH2 cells: different patterns of
lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145173.
Mosmann, T. R. and Sad, S. 1996. The expanding universe of T-cell subsets: Th1, Th2 and
more. Immunol Today 17:138-146.
Moulon, C., Vollmer, J., Weltzien, H. U. 1995. Characterization of processing requirements
and metal cross-reactivities in T cell clones from patients with allergic contact dermatitis
to nickel. Eur J Immunol 25:3308-3315.
72
Jacob Taku Minang
Moulon, C., Wild, D., Dormoy, A., Weltzien, H. U. 1998. MHC-dependent and –independent
activation of human nickel-specific CD8+ cytotoxic T cells from allergic donors. J Invest
Dermatol 111:360-366.
Mäkitalo, B., Andersson, M., Areström, I., Karlen, K., Villinger, F., Ansari, A., Paulie, S.,
Thorstensson, R., Ahlborg, N. 2002. ELISpot and ELISA analysis of spontaneous,
mitogen-induced and antigen-specific cytokine production in cynomolgus and rhesus
macagues. J Immunol Methods 270:85-97.
Möller, G. 1988. Do suppressor T cells exist? Scand J Immunol 27:247-250.
Nestle, F. O. and Burg, G. 1999. Dendritic cells: role in skin diseases and therapeutic
applications. Clin Exp Dermatol 24:204-207.
Newport, M. J., Huxley, C. M., Huston, S., Hawrylowicz, C. M., Oostra, B. A., Williamson,
R., Levin, M. 1996. A mutation in the interferon-gamma-receptor gene and susceptibility
to mycobacterial infection. N Engl J Med 335:1941-1949.
Nishimura, E., Sakihama, T., Setoguchi, R., Tanaka, K., Sakaguchi, S. 2004. Induction of
antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion
of naturally arising Foxp3+CD25+CD4+ regulatory T cells. Int Immunol 16:1189-1201.
Nordlind, K. 1984. Further studies on the lymphocyte transformation test in diagnosis of
nickel allergy. Effect of fractionation of lymphocytes into different subpopulations on the
basis of density. Int Arch Allergy Appl Immunol 75:333-336.
Oberg, L., Johansson, S., Michaelsson, J., Tomasello, E., Vivier, E., Karre, K., Hoglund, P.
2004. Loss or mismatch of MHC class I is sufficient to trigger NK cell-mediated rejection
of resting lymphocytes in vivo - role of KARAP/DAP12-dependent and -independent
pathways. Eur J Immunol 34:1646-1653.
O'Garra, A. 1998. Cytokines induce the development of functionally heterogeneous T helper
cell subsets. Immunity 8:275-283.
Pakala, S. V, Kurrer, M. O., Katz, J. D. 1997. T helper 2 (Th2) T cells induce acute
pancreatitis and diabetes in immune-compromised nonobese diabetic (NOD) mice. J Exp
Med 186:299-306.
Parronchi, P., De Carli, M., Manetti, R., Simonelli, C., Piccinni, M. P., Macchia, D., Maggi,
E., Del Prete, G., Ricci, M., Romagnani, S. 1992. Aberrant interleukin (IL)-4 and IL-5
production in vitro by CD4+ helper T cells from atopic subjects. Eur J Immunol 22:16151620.
Pasare, C. and Medzhitov, R. 2004. Toll-like receptors and acquired immunity. Semin
Immunol 16:23-26.
Pawankar, R. U., Okuda, M., Suzuki, K., Okumura, K, Ra, C. 1996. Phenotypic and
molecular characteristics of nasal mucosal gamma delta T cells in allergic and infectious
rhinitis. Am J Respir Crit Care Med 153:1655-1665.
Cytokine responses in metal-induced ACD
73
Pedersen, L. K., Held, E., Johansen, J. D., Agner, T. 2005. Short-term effects of alcohol-based
disinfectant and detergent on skin irritation. Contact Dermatitis 52:82-87.
Porstmann, T. and Kiessig, S. T. 1992. Enzyme immunoassay techniques: An overview. J
Immunol Methods 150:5-21.
Probst, P., Küntzlin, D., Fleischer, B. 1995. Th2-type infiltrating T cells in nickel-induced
contact dermatitis. Cellular Immunol 165:134-140.
Ptak, W., Geba, G. P., Askenase, P. W. 1991a. Initiation of delayed-type hypersensitivity by
low doses of monoclonal IgE antibody. Mediation by serotonin and inhibition by
histamine. J Immunol 146:3929-3936.
Ptak, W., Herzog, W. R., Askenase, P. W. 1991b. Delayed-type hypersensitivity initiation by
early-acting cells that are antigen mismatched or MHC incompatible with late-acting,
delayed-type hypersensitivity effector T cells. J Immunol 146:469-475.
Punnonen, J., Aversa, G., Cocks, B. G., McKenzie, A. N., Menon, S., Zurawski, G., de Waal
Malefyt, R., de Vries, J. E. 1993. Interleukin 13 induces interleukin 4-independent IgG4
and IgE synthesis and CD23 expression by human B cells. Proc Natl Acad Sci U S A. 90:
3730-3734.
Punnonen, J., Yssel, H., de Vries, J. E. 1997. The relative contribution of IL-4 and IL-13 to
human IgE synthesis induced by activated CD4+ or CD8+ T cells. J Allergy Clin
Immunol 100:792-801.
Rajan, T. V., 2003. The Gell-Coombs classification of hypersensitivity reactions: a reinterpretation. Trends Immunol 24:376-379.
Reinhardt, C. A., Pelli, D. A., Sandvold, M. 1985. Cell detachment and growth of fibroblasts
as parameters for cytotoxicity of inorganic metal salts in vitro. Cell Biol Toxicol 1:33-43.
Rich, S., Seelig, M., Lee, H. M., Lin, J. 1995. Transforming growth factor beta 1 costimulated
growth and regulatory function of staphylococcal enterotoxin B-responsive CD8+ T cells.
J Immunol 155:609-618.
Rincon, M., Anguita, J., Nakamura, T., Fikrig, E., Flavell, R. A. 1997. Interleukin (IL)-6
directs the differentiation of IL-4-producing CD4+ T cells. J Exp Med 185:461-469.
Rogge, L., Barberis-Maino, L., Biffi, M., Passini, N., Presky, D. H., Gubler, U., Sinigaglia, F.
1997. Selective expression of an interleukin-12 receptor component by human T helper 1
cells. J Exp Med 185:825-831.
Romagnani, G. 2000. Cytokines and the Th1/Th2 paradigm. In: The Cytokine Network.
Balkwill, F. (ed), Oxford: Oxford University Press, New York pp 71-93.
Romagnoli, P., Labhardt, A. M., Sinigaglia, F. 1991. Selective interaction of Ni with an
MHC-bound peptide. EMBO J 10:1303-1306.
74
Jacob Taku Minang
Romani, N. and Schuler, G. 1992. The immunologic properties of epidermal Langerhans cells
as a part of the dendritic cell system. Springer Semin Immunopathol 13:265-279.
Rosenberg, A. 1955. An anecdotal biographical history of poison ivy. AMA Arch Derm
72:438-445.
Rowe, A. and Bunker, C. B. 1998. Interleukin-4 and the interleukin-4 receptor in allergic
contact dermatitis. Contact Dermatitis 38:36-39.
Royer, B., Varadaradjalou, S., Saas, P., Guillosson, J. J., Kantelip, J. P, Arock, M. 2001.
Inhibition of IgE-induced activation of human mast cells by IL-10. Clin Exp Allergy
31:694-704.
Ruprecht, C. R., Gattorno, M., Ferlito, F., Gregorio, A., Martini, A., Lanzavecchia, A.,
Sallusto, F. 2005. Coexpression of CD25 and CD27 identifies FoxP3+ regulatory T cells
in inflamed synovia. J Exp Med 201:1793-1803.
Rustemeyer, T., von Blomberg, B. M., van Hoogstraten, I. M, Bruynzeel, D. P., Scheper, R. J.
2004. Analysis of effector and regulatory immune reactivity to nickel. Clin Exp Allergy
34:1458-1466.
Rystedt, I. 1979. Evaluation and relevance of isolated test reactions to cobalt. Contact
Dermatitis 5:233-238.
Sadler, P. J., Tucker, A., Viles, J. H. 1994. Involvement of a lysine residue in the N-terminal
Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+.
Eur J Biochem 220:193-200.
Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M. 1995. Immunologic selftolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25).
Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.
J Immunol 155:1151-1164.
Sanderson, C.J. 1992. Interleukin-5, eosinophils, and disease. Blood 79: 3101-3109.
Santucci, B., Cannistraci, C., Cristaudo, A., Picardo, M. 1996. Multiple sensitivities to
transitional metals: the nickel palladium reactions. Contact Dermatitis 35:283-286.
Sato, M., Goto, S., Kaneko, R., Ito, M., Sato, S., Takeuchi, S. 1998. Impaired production of
Th1 cytokines and increased frequency of Th2 subsets in PBMC from advanced cancer
patients. Anticancer Res 18:3951-3955.
Schielen, P., van Rodijnen, W., Tekstra, J., Albers, R., Seinen,W. 1995. Quantification of
natural antibody producing B cells in rats by an improved ELISPOT technique using the
polyvinylidene difluoride membrane as the solid support. J Immunol Methods 188:33-41.
Schmitt, E., Van Brandwijk, R., Fischer, H. G., Rude, E. 1990. Establishment of different T
cell sublines using either interleukin 2 or interleukin 4 as growth factors. Eur J Immunol
20:1709-1715.
Cytokine responses in metal-induced ACD
75
Schnuch, A., Geier, J., Uter, W., Frosch, P. J., Lehmacher, W., Aberer, W., Agathos, M.,
Arnold, R., Fuchs, T., Laubstein, B., Lischka, G., Pietrzyk, P. M., Rakoski, J., Richter, G.,
Rueff, F. 1997. National rates and regional differences in sensitization to allergens of the
standard series. Population-adjusted frequencies of sensitization (PAFS) in 40,000 patients
from a multicenter study (IVDK). Contact Dermatitis 37:200-209.
Schnuch, A., Uter, W., Geier, J., Frosch, P.J. 1998. Contact allergies in healthcare workers.
Results from the IVDK. Acta Derm Venereol 78:358-363.
Schulze-Koops, H. and Kalden, J. R. 2001. The balance of Th1/Th2 cytokines in rheumatoid
arthritis. Best Pract Res Clin Rheumatol 15:677-691.
Schwartz, R. H., David, C. S., Sachs, D. H., Paul, W. E. 1976. T lymphocyte-enriched murine
peritoneal exudate cells. III. Inhibition of antigen-induced T lymphocyte Proliferation
with anti-Ia antisera. J Immunol 117:531-540.
Sebastiani, S., Allavena, P., Albanesi, C., Nasorri, F., Bianchi, G., Traidl, C., Sozzani, S.,
Girolomoni, G., Cavani, A. 2001. Chemokine receptor expression and function in CD4+ T
lymphocytes with regulatory activity. J Immunol 166:996-1002.
Sebzda, E., Mariathasan, S., Ohteki, T., Jones, R., Bachmann, M. F., Ohashi, P. S. 1999.
Selection of the T cell repertoire. Annu Rev Immunol 17: 829-874.
Seder, R. A. and Ahmed, R. 2003. Similarities and differences in CD4+ and CD8+ effector
and memory T cell generation. Nature Immunol 9:835-842.
Sedgwick, J. D and Holt, P. G. 1983. A solid-phase immunoenzymatic technique for the
enumeration of specific antibody-secreting cells. J Immunol Methods 57:301-309.
Shah, M., Lewis, F. M., Gawkrodger, D. J. 1998. Nickel as an occupational allergen. A survey
of 368 nickel-sensitive subjects. Arch Dermatol 134:1231-1236.
Shevach, E. M. 2001. Certified professionals: CD4(+)CD25(+) suppressor T cells. J Exp Med
193:41-46.
Shevach, E. M. 2002. CD4+ CD25+ suppressor T cells: more questions than answers. Nat
Rev Immunol 2:389-400.
Shirakawa, T., Kusaka, Y., Morimoto, K. 1992. Specific IgE antibodies to nickel in workers
with known reactivity to cobalt. Clin Exp Allergy 22:213-218.
Silvennoinen-Kassinen, S., Poikonen, K., Ikäheimo, I. 1991. Characterisation of nickelspecific T cell clones. Scan J Immunol 33:429-434.
Singh, V. K., Mehrotra, S., Agarwal, S. S. 1999. The paradigm of Th1 and Th2 cytokines: its
relevance to autoimmunity and allergy. Immunol Res 20:147-161.
Sinigalia, F., Scheidegger, D., Garotta, G., Scheper, R., Pletscher, M., Lanzavecchia, A. 1985.
Isolation and characterisation of Ni-specific T cell clones from patients with Ni-contact
dermatitis. J Immunol 135:3929-3932.
76
Jacob Taku Minang
Siroux, V., Curt, F., Oryszczyn, M.P., Maccario, J., Kauffmann, F. 2004. Role of gender and
hormone-related events on IgE, atopy, and eosinophils in the Epidemiological study on
the Genetics and Environment of Asthma, bronchial hyperresponsiveness and atopy. J
Allergy Clin Immunol 114:491-498.
Skokowa, J., Ali, S. R., Felda, O., Kumar, V., Konrad, S., Shushakova, N., Schmidt, R. E.,
Piekorz, R. P., Nurnberg, B., Spicher, K., Birnbaumer, L., Zwirner, J., Claassens, J. W.,
Verbeek, J. S., van Rooijen, N., Kohl, J., Gessner, J. E. 2005. Macrophages induce the
inflammatory response in the pulmonary Arthus reaction through G alpha i2 activation
that controls C5aR and Fc receptor cooperation. J Immunol 174:3041-3050.
Smith, J. G., Liu, X., Kaufhold, R. M., Clair, J., Caulfield, M. J. 2001. Development and
validation of a gamma interferon ELISPOT assay for quantitation of cellular immune
responses to Varicella-Zoster virus. Clin Diag Lab Immunol 8:871-879.
Smith, H. R., Rowson, M., Basketter, D. A., McFadden, J. P. 2004. Intra-individual variation
of irritant threshold and relationship to transepidermal water loss measurement of skin
irritation. Contact Dermatitis 51:26-29.
Sonoda, E., Matsumoto, R., Hitoshi, Y., Ishii, T., Sugimoto, M., Araki, S., Tominaga, A.,
Yamaguchi, N., Takatsu, K. 1989. Transforming growth factor beta induces IgA
production and acts additively with interleukin 5 for IgA production. J Exp Med 170:
1415-1420.
Sozzani, S., Sallusto, F., Luini, W., Zhou, D., Piemonti, L., Allavena, P., Van Damme, J.,
Valitutti, S., Lanzavecchia, A., Mantovani, A. 1995. Migration of dendritic cells in
response to formyl peptides, C5a, and a distinct set of chemokines. J Immunol 155:32923295.
Sparwasser, T., Koch, E. S., Vabulas, R. M., Heeg, K., Lipford, G. B., Ellwart, J. W.,
Wagner, H. 1998. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger
maturation and activation of murine dendritic cells. Eur J Immunol 28: 2045-2054.
Steinke, J.W. and Borish, L. 2001. Th2 cytokines and asthma. Interleukin-4: its role in the
pathogenesis of asthma, and targeting it for asthma treatment with interleukin-4 receptor
antagonists. Respir Res 2:66-70.
Sterry, W., Kunne, N., Weber-Matthiesen, K., Brasch, J., Mielke, V. 1991. Cell trafficking in
positive and negative patch-test reactions: demonstration of a stereotypic migration
pathway. J Invest Dermatol 96:459-462.
Takata, M., Homma, Y., Kurosaki, T. 1995. Requirement of phospholipase C-gamma 2
activation in surface immunoglobulin M-induced B cell apoptosis. J Exp Med 182: 907914.
Tang, L., Benjaponpitak, S., Dekruyff, R. M., Umetsu, D. T. 1998. Reduced prevalence of
allergic disease in patients with multiple sclerosis associated with enhanced IL-12
production. J Allergy Clin Immunol 102:428-435.
Cytokine responses in metal-induced ACD
77
Tanguay, S. and Killion, J. J. 1994. Direct comparison of ELISPOT and ELISA-based assays
for detection of individual cytokine secreting cells. Lymphokine Cytokine Res 13:259263.
Teixeira, M. M., Almeida, I. C., Gazzinelli, R. T. 2002. Introduction: innate recognition of
bacteria and protozoan parasites. Microbes Infect 4:883-886.
Teunissen, M. B. 1992. Dynamic nature and function of epidermal Langerhans cells in vivo
and in vitro: a review, with emphasis on human Langerhans cells. Histochem J 24:697716.
Thierse, H. J., Gamerdinger, K., Junkes, C., Guerreiro, N., Weltzien, H. U. 2005. T cell
receptor (TCR) interaction with haptens: metals ions as non-classical haptens. Toxicology
209:101-107.
Thierse, H. J., Moulon, C., Allespach, Y., Zimmermann, B., Doetze, A., Kuppig, S., Wild, D.,
Herberg, F., Weltzien, H. U. 2004. Metal-protein complex-mediated transport and
delivery of Ni2+ to TCR/MHC contact sites in nickel-specific human T cell activation. J
Immunol 172:1926-1934.
Thomas, M.J. and Kemeny, D. M. 1998. Novel CD4 and CD8 T-cell subsets. Allergy 53:
1122-1232.
Torii, I., Morikawa, S., Harada, T. 2002. MD41, a novel T helper 0 clone, mediates mast-cell
dependent delayed-type hypersensitivity in mice. Immunol 107: 426-434.
Traidl, C., Sebastiani, S., Albanesi, C., Merk, H. F., Puddu, P., Girolomoni, G., Cavani, A.
2000. Disparate cytotoxic activity of nickel-specific CD8+ and CD4+ T cell subsets against
keratinocytes. J Immunol 165:3058-3064.
Tupker, R. A. 2003. Prediction of irritancy in the human skin irritancy model and
occupational setting. Contact Dermatitis 49:61-69.
Uter, W., Ruhl, R., Pfahlberg, A., Geier, J., Schnuch, A., Gefeller, O. 2004. Contact allergy in
contruction workers: results of a multifactorial analysis. Ann Occup Hyg 48:21-27.
Von Blomberg-van der Flier, M., van der Burg, C. K., Pos, O., van de Plassche-Boers, E. M.,
Bruynzeel, D. P., Garotta, G., Scheper, R. J. 1987. In vitro studies in nickel allergy:
diagnostic value of a dual parameter analysis. J Invest Dermatol 88:362-368.
Vamnes, J. S., Morken, T., Helland, S., Gjerdet, N. R. 2000. Dental gold alloys and contact
hypersensitivity. Contact Dermatitis. 42(3):128-133.
Van den Broeke, L. T., Heffler, L.C., Tengvall Linder, M., Nilsson, J. L., Karlberg, A. T.,
Scheynius, A. 1999. Direct Ni2+ antigen formation on cultured human dendritic cells.
Immunol 96:578-585.
Van Seventer, G.A., Van Lier, R.A., Spits, H., Ivanyi, P., Melief, C.J., 1986. Evidence for a
regulatory role of the T8 (CD8) antigen in antigen-specific and anti-T3-(CD3)-induced
lytic activity of allospecific cytotoxic T lymphocyte clones. Eur J Immunol 16:1363-1371.
78
Jacob Taku Minang
Verhasselt, V., Buelens, C., Willems, F., De Groote, D., Haeffner-Cavaillon, N., Goldman,
M., 1997. Bacterial lipopolysaccharide stimulates the production of cytokines and the
expression of costimulatory molecules by human peripheral blood dendritic cells:
evidence for a soluble CD14-dependent pathway. J Immunol 158: 2919-2925.
Vincenzi, C., Tosti, A., Guerra, L., Kokelj, F., Nobile, C., Rivara, G., Zangrando, E. 1995.
Contact dermatitis to palladium: a study of 2300 patients. Am J Cont Dermat 6:110-112.
Waalkes, M. P. 1995. Metal carcinogenesis. In:Goyer RA, Klaassen CD, Waalkes MP eds.
Metal toxicology. Academic, New York, pp 447-464.
Wahlberg, J. E. 2001. Patch testing. In: Rycroft R. J. G., Menné, T., Frosch, P. J., Lepoittevin,
J-P (eds) Textbook of contact dermatitis, 3rd edn. Springer, Berlin Heidelberg New York
pp435-468.
Wahlberg, J. E. and Liden, C. 2000. Cross-reactivity patterns of cobalt and nickel studied with
repeated open applications (ROATS) to the skin of guinea pigs. Am J Contact Dermat
11:42-48.
Wakem, P., Burns, R. P. Jr., Ramirez, F., Zlotnick, D., Ferbel, B., Haidaris, C. G., Gaspari, A.
A. 2000. Allergens and irritants transcriptionally upregulate CD80 gene expression in
human keratinocytes. J Invest Dermatol 114:1085-1092.
Walton, S. 1983. Patch testing with copper sulphate. Contact Dermatitis 9:337.
Walzer, T., Dalod, M., Robbins, S. H., Zitvogel, L., Vivier, E. 2005. Natural killer cells and
dendritic cells: "l'union fait la force". Blood 106:2252-2258.
Wang, Z. E., Reiner, Zheng, S., Dalton, D. K., Locksley, R. M. 1994. CD4+ effector cells
default to the Th2 pathway in interferon gamma- deficient mice infected with Leishmania
major. J Exp Med 179: 1367-1371.
Weiner, H. L. 2001. Induction and mechanism of action of transforming growth factor-betasecreting Th3 regulatory cells. Immunol Rev 182:207-214.
Werfel, T., Hentschel, M, Kapp, A., Renz, H. 1997. Dichotomy of blood- and skin-derived
IL-4-producing allergen-specific T cells and restricted Vβ repertoir in nickel-mediated
contact dermatitis. J Immunol 158:2500-2505.
West, N. Y., Fitzpatrick, J. E., Jackson, E. M. 1998. Comparison testing of the irritancy of
children's liquid bubble bath using a modified human repeat insult patch test. Am J
Contact Dermat 9:212-215.
Willis, C. M., Stephens, C. J., Wilkinson, J. D. 1993. Differential patterns of epidermal
leukocyte infiltration in patch test reactions to structurally unrelated chemical irritants. J
Invest Dermatol 101:364-370.
Wuthrich, B. 1998. Food-induced cutaneous adverse reactions. Allergy 53:131-135.
Cytokine responses in metal-induced ACD
79
Xu, D., Komai-Koma, M., Liew, F. Y. 2005. Expression and function of Toll-like receptor on
T cells. Cell Immunol 233:85-89.
Zanni, M. P., von Greyerz, S., Schnyder, B., Brander, K. A., Frutig, K., Hari, Y., Valitutti, S.,
Pichler, W. J. 1998. HLA-restricted, processing- and metabolism-independent pathway of
drug recognition by human alpha beta T lymphocytes. J Clin Invest 102:1591-1598.
Zhang, Y. and Wilcox, D. E. 2002. Thermodynamic and spectroscopic study of Cu(II) and
Ni(II) binding to bovine serum albumin. J Biol Inorg Chem 7:327-337.
Zumdahl, S. S. 1997. Chemistry alternate edition. R Stratton, M Stephanie & CL Brooks eds.
Houghton Mifflin company Boston 4th Edn. pp 941-993.
Zurawski, G. and de Vries, J. E., 1994. Interleukin 13 elicits a subset of the activities of its
close relative interleukin 4. Stem Cells 12:169-174.