Doctoral thesis from the Department of Immunology,

The Wenner-Gren Institute, Stockholm University, Sweden

In vitro analyses of immune responses to metal and organic haptens in humans with contact allergy

Khosro Masjedi

Stockholm 2008

All previously published papers were reproduced with permission from the publishers. © Khosro Masjedi, Stockholm 2008 ISBN 978-91-7155-656-1 Printed in Sweden by Universitetsservice AB, Stockholm 2008 Distributer: Stockholm University Library

To my dear mother and father

SUMMARY Contact allergy is one of the most common skin diseases with great social and economical impact. The origin and nature of contact allergens (haptens) capable of inducing T-cell mediated allergic reactions are diverse, ranging from organic molecules to metal ions. Most of the current knowledge on T-cell responses to haptens in humans with contact allergy have been established by studies on the metal ion nickel (Ni), the most common cause of contact allergy, whereas reactivity to the large group of organic haptens has been less studied. Haptens are not immunogenic by themselves but must bind carrier molecules prior to their presentation on MHC class I or II molecules and subsequent recognition by T cells. Due to differences in their chemical nature, haptens interact with host molecules by different mechanisms and differences in their solubility can influence their access to different antigen-presenting pathways. The aim of the present study was to define immune responses elicited by haptens of different chemical nature including Ni (hydrophilic metal ion), methylisothiazolinones (hydrophilic organic molecule) and parthenolide (lipophilic organic molecule). The immune response displayed by subjects with allergy to these substances, and non-allergic control subjects, was assessed by measuring hapten-induced cytokine production in peripheral blood mononuclear cells (PBMC) with a focus on ELISpot analysis of T-cell type 1 (e.g. IFN-γ and IL-2) and type 2 (e.g. IL-4, IL-5 and IL-13) cytokines. For Ni and parthenolide, the phenotype of the hapten-reactive T cells was determined. The allergic status of subjects was defined by clinical history and patch testing. The latter is the established diagnostic method for contact allergy, based on applying various haptens to the subjects’ back and grading the skin reaction after 2-3 days. All three haptens elicited a concomitant T-cell type 1 and 2 response in subjects with contact allergy to the corresponding hapten, suggesting the induction of a functionally related cytokine profile, irrespective of the chemical character of the hapten. The cytokine response was related to the degree of the subjects’ patch test reactivity; PBMC from a vast majority of subjects with strong and moderate patch test reactivity displayed detectable cytokine responses to the corresponding haptens, whereas subjects with weak or no (controls) patch test reactivity did not. Despite the similar cytokine profile induced, the phenotype of the reactive T cells was found to differ between haptens with Ni eliciting CD4+ T cells and parthenolide eliciting CD8+ T cells. This difference may be explained by a better ability of a lipophilic hapten to gain access to the MHC class I-restricted antigen-presentation pathway. Moreover, the data suggest that analysis of cytokine responses to haptens may facilitate future development of in vitro-based diagnostics assay for contact allergy. Finally, the relationship between the variation over time in patch test reactivity and systemic reactivity to Ni, in terms of cytokine responses to Ni in vitro, was investigated. The degree of patch test reactivity is known to vary over time, in particular in subjects with weak reactivity. Ni-allergic subjects were patch tested three times with three month intervals and PBMC obtained at the same time points were assessed for in vitro reactivity to Ni. The overall reactivity in the patch test and the in vitro test was well correlated confirming that both methods provide a good and comparable estimate of the systemic reactivity to Ni. However, fluctuations in the patch test reactivity over time were not well correlated with variations in the cytokine response elicited in vitro suggesting that other parameters besides changes in the systemic reactivity could significantly contribute to the variation in patch test reaction over time. © Khosro Masjedi, Stockholm 2008 ISBN 978-91-7155-656-1

ORIGINAL PAPERS

This thesis is based on the following original papers, which are referred to in the text by their Roman numerals.

I. Eva Jakobson, Khosro Masjedi, Niklas Ahlborg, Lena Lundeberg, Ann-Therese

Karlberg, and Annika Scheynius (2002). "Cytokine production in nickel-sensitized

individuals analysed with enzyme-linked immunospot assay: possible implication

for diagnosis." Br J Dermatol 147(3): 442-9.

II. Khosro Masjedi, Niklas Ahlborg, Birgitta Gruvberg, Magnus Bruze, and Ann-

Therese Karlberg (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(6): 1172-82.

III. Helen Wahlkvist,* Khosro Masjedi,*, Birgitta Gruvberg, Bartek Zuber, Ann-

Therese Karlberg, Magnus Bruze, and Niklas Ahlborg (2008). "The lipophilic

hapten parthenolide induces IFN-γ and IL-13 production by peripheral blood-

derived CD8+ T cells from contact allergic subjects in vitro." Br J Dermatol

158(1): 70-7.

IV. Khosro Masjedi, Magnus Bruze, Monica Hindsén, Jacob Minang, and Niklas

Ahlborg. Is the variability of nickel patch test reactivity over time associated with

fluctuations in the systemic T-cell reactivity to nickel? Manuscript.

* These authors contributed equally to this work.

Table of contents The immune system 11 Innate and adaptive immunity 11 Dendritic cells, connection between innate and adaptive immunity 11 Antigen presentation to the immune system through MHC class I and II 12 Subsets of T lymphocytes 12 CD4+ T lymphocytes 12 CD8+ T lymphocytes 14 T-regulatory subsets 15 Memory T cells 16 Allergies 18 Classifications of allergies 18 Hypersensitivity reaction type IV and delayed type hypersensitivity 20 Mechanistic cellular events in allergic contact dermatitis 21 Irritant and allergic contact dermatitis 22 Subsets of T cells and their role in contact allergy 23 Skin biology 24 Related & specific background 26 Haptens 26 Chemical and biological properties of contact allergens 26 General properties and clinical relevance of haptens included in this study 28 Nickel 28 Methylisothiazolinone 29 Parthenolide 30 Patch testing 30 Aims of the study 32 Subjects and methods 33 Subjects [I-IV] 33 Isolation of peripheral blood mononuclear cells (PBMC) [I-IV] 33 Patch testing [I-IV] 34 The lymphocyte proliferation assay [I-II] 34 Quantification of cytokine-producing cells by ELISpot [I-IV] 34 Analysis of cytokine production by enzyme-linked immunosorbent assay (ELISA) [II] 35 Depletion of CD4+ or CD8+ cells and flow cytometry [IV] 35 Statistical methods [I-IV] 36 Results and Discussion 37 Proliferation of PBMC induced by nickel and MCI/MI [I-II] 37 Production of type 1 and type 2 cytokines induced in PBMC by nickel, MCI/MI and parthenolide [I-III]

38

Implications of the ELISpot assay for diagnosing contact allergy to nickel [I] 43 Relationships between the variability over time in nickel patch test reactivity and systemic T-cell reactivity [IV]

45

Concluding remarks 47 Acknowledgments 48 References 50

Abbreviations

ACD Allergic contact dermatitis ADCC Antibody dependent cell-mediated cytotoxicity APC Antigen-presenting cell CD Cluster of differentiation CHS Contact hypersensitivity CLA Cutaneous lymphocyte antigen CR Cytokine ratio CTL Cytotoxic T-lymphocyte DC Dendritic cell DMSO Dimethylsulfoxide DNFB Dinitrofluorobenzene DTH Delayed-type hypersensitivity ELISA Enzyme-linked immunosorbent assay ELISpot Enzyme-linked immunospot assay FCS Fetal calf serum IFN Interferon ICD Irritant contact dermatitis IL Interleukin IVDK Information network of departments of dermatology LC Langerhans cells LLNA Local lymph node assay LTT Lymphocyte-transformation test mAb Monoclonal antibodies MCI Methylchloroisothiazolinone MI Methylisothiazolinone MHC Major-Histocompatibility Complex Ni Nickel ion PBMC Peripheral blood mononuclear cells PHA Phytohaemagglutinin SI Stimulation index SL Sesquiterpene lactones Tc T-cytotoxic cells TCC T-cell clones TCR T-cell receptors TGF Transforming growth factor Th T-helper cell TNF Tumor necrosis factor TNP Trinitrophenyl TReg T-regulatory cell TT Tetanus toxoid

Immune responses to metal and organic haptens 11

The immune system Innate and adaptive immunity

The immune system consists of two parallel compartments, the ‘innate’ and ‘adaptive’

immunity. Innate immunity is a defense system without memory, with which an individual is

born, and which exists prior to exposure to an antigen [1]. Adaptive immunity is mediated by

T and B cells, which are activated after exposure to antigens, and which exhibit specificity

and memory. Different specialized organs, cells, cell products, and orchestrated mechanisms

are involved in developing the innate and adaptive immunity. Neutrophils, eosinophils,

basophils, mast cells, natural-killer cells, dendritic cells (DC) and monocytes/macrophages

are cells engaged in the innate immunity [2-4]. However, they could also play a pivotal role in

initiating the adaptive response. Inflammatory responses are usually initiated by activation of

the innate immunity followed by adaptive response. B and T lymphocytes are cells engaged in

the adaptive immunity. One key interface cell that links innate and adaptive immunity is the

DC [1]. DC, in lymphoid tissues, can initiate antigen-specific adaptive response. Many DC in

lymphoid organs are in a steady state, defined as immature, able to endocytose and process

antigens to form peptide complexes associated with major-histocompatibility complex

(MHC) class I and II products. Upon the capture of antigen, DC undergoes a maturation

process during which they acquire the capability to activate T cells.

Dendritic cells, connection between innate and adaptive immunity DC are the ‘only’ professional antigen-presenting cells (APC) that can properly prime naïve T

cells and initiate immune responses [5]. In comparison, macrophages are 100 times less

potent in stimulating T cells. Different populations of DC, including Langerhans cells (LC),

have been identified. LC comprise 2-3% of the total epidermal cells. Myeloid cells are

considered to be the origin of LC although this is still controversial [1]. Activation of LC in

the skin initiates a process of cell maturation, which is followed by production of interleukin

(IL)-1β by LC and secretion of tumor necrosis factor (TNF)-α by keratinocytes [6]. This

process plays a central role in the development of allergic contact dermatitis (ACD). Other

types of DC could be found in the dermis (dermal DC), thymus (follicular DC) and circulating

blood (CD14+ blood monocytes and plasmacytoid) [7]. In the presence of IL-4 and GM-CSF,

CD14+ monocytes can develop into monocyte-derived DC in vitro [8].

12 Khosro Masjedi

Antigen presentation to the immune system through MHC class I and II DC constitutively sample the environment and engulf extracellular antigen via phagocytosis,

pinocytosis or receptor-mediated endocytosis. Several receptors on DC facilitate antigen

binding and internalization. Such receptors include the mannose receptor, DEC205, and Fc

receptors for IgG and IgE [1]. DC also have the capacity to bind to heat shock proteins

through specific receptors. After internalization, protein antigens are proteolytically cleaved

into 12 to 25 amino acid fragments that are bound to MHC class II molecules. It is these

complexes that are presented to CD4+ T lymphocytes. MHC class I molecules are expressed

on the surface of virtually all nucleated cells, and present antigens to CD8+ T cells and NK

cells. Cytosolic foreign antigens are degraded in the cytosol. This degradation is carried out

by a multicatalytic protease complex called proteasome. With the help of TAP transporters

the degraded peptides are transported into the endoplasmic reticulum where they associate

with the MHC class I molecules.

Subsets of T lymphocytes T cells only recognize foreign antigens as a complex with MHC molecules present on APC.

Based on different subunits of the T-cell receptor (TCR), T cells can be divided into αβ or γδ

T cells. TCR in αβ T cells associates with CD3, a complex of different subunits, forming the

TCR-CD3 membrane complex. The CD3 complex in αβ TCR participates in signal

transduction after interaction of the T-cell receptor with the antigenic peptide-MHC complex.

CD4 and CD8 molecules in αβ T cells interact with non-variable parts of MHC class II and I,

respectively, on APC. In humans, the proportion of CD4:CD8 from the αβ-T cell population

is about 2:1. γδ T cells have antimicrobial as well as antitumor activity, and the ability to

produce proinflammatory cytokines [9]. γδ TCR is important for recognition of non-protein

antigens. In humans, the proportion of CD4:CD8 from the γδ-T cell population is about 1:30.

CD4+ T lymphocytes Upon encounter with antigen, naïve T lymphocytes differentiate into cells capable of exerting

effector activity. Naïve T cells release only small amounts of a limited number of cytokines,

for example IL-2. In contrast, effector cells produce a broader range of cytokines. Studies

conducted in the 1970s suggested that there are two types of response, one type associated

with inflammation, and the other related to humoral responses [10]. In the 1980s, based on

Immune responses to metal and organic haptens 13

their cytokine production profiles, it was suggested that many CD4+ T-cell clones could be

divided into two different categories of T-helper cells (Th) either producing IL-2, Interferon

(IFN)-γ and lymphotoxin but not IL-4 or IL-5 (Th1 cells) or producing mainly IL-4 or IL-5

and IL-13 but not IL-2 or IFN-γ (Th2 cells) (Table 1) [11, 12]. Over the years, more cytokines

have been discovered and categorized into the Th1 or Th2 phenotypes [13]. Th1

cells/cytokines promote inflammatory events, e.g. delayed-type hypersensitivity (DTH),

macrophage activation, and help B cells to produce certain antibodies (e.g. IgG2a in mice and

IgG3 in human). Th2 cytokines promote antibody responses (particularly IgG1 and IgE

production) of relevance for mast cell and basophil activation. Th0 cells have been described

as lymphocytes exhibiting an unrestricted profile of cytokines i.e. they produce both type 1

and type 2 cytokines [14] (Table 1).

Because Th1 and Th2 cells are crucial regulators of immune responses, proper differentiation

of naïve CD4+ Th cells into Th1 or Th2 cells is critical for T-dependent immune responses

[15]. Determination of how Th cells differentiate into Th1 or Th2 may be related to the

microenvironment surrounding the cells. It is generally agreed that the most effective inducer

of Th2 cells are the cytokines IL-4 and IL-13 in conjunction with proper costimulatory signals

[16, 17], whereas production of IL-12 and IL-18 cause naïve T cells to differentiate into the

Th1 cytokine profile [18, 19]. Moreover, IFN-γ production may block the proliferation and

differentiation of Th2 cells [20, 21].

A new Th phenotype (Th17) has been described in recent years. Th17 are IL-17-producing

cells representing a distinct subset, unrelated to Th1 or Th2 cells, capable of inducing tissue

inflammation and autoimmunity and are involved in host defense against extracellular

pathogens [22] (Table 1).

Table 1. Cytokine profile of the human CD4+ T-helper cells Th0 Th1 Th2 Th17 IL-2 IL-2 IL-3 IL-17 IL-4 IL-3 IL-4 TNF-α IL-10 TNF-β IL-5 IL-6 IFN-γ IFN-γ IL-6 GM-CSF (high) IL-9 IL-10 IL-13 GM-CSF (low)

14 Khosro Masjedi

CD8+ T lymphocytes Cytotoxic T lymphocytes (CTL) or T-cytotoxic cells (Tc) are lymphocytes with lytic

capability with surface expression of the CD8+CD3+ molecules. All nucleated cells express

class I MHC molecules and a Tc can therefore potentially recognize and eliminate almost any

self cells. Naïve Tc are incapable of killing target cells, and produce a limited amount of

cytokines (Tc0 phenotype). To gain lytic ability, such naïve Tc cells must undergo proper

antigen-specific activation, which consequently results in their differential into effector and

memory Tc cells. Generation of effector Tc from naïve Tc cells is dependent on a proper

cytokine milieu provided by Th1 cells together with antigen presentation by DC [23]. IL-2

and costimulation are important in the transformation of naïve Tc into effector and memory T

cells. In IL-2 knock out mice, the absence of IL-2 has been shown to abolish CTL-mediated

cytotoxicity [23]. Similar type 1 and 2 categorization as used for CD4+ T cell subsets has been

implied for CD8+ CTL cells. CD8+ T cells can differentiate upon antigenic stimulation into

Tc1 cells, characterized by high IFN-γ production but no IL-4; or into Tc2, characterized by

production of mainly IL-4, IL-5, IL-13 and IL-10 but no IFN-γ [24-27]. In a mouse model of

allergen induced airway hyper-responsiveness, it was shown that CD8-deficient mice develop

significantly lower airway hyper-responsiveness and IL-13 levels in bronchoalveolar lavage

fluid compared with wild-type mice, which could be restored by adoptive transfer of antigen-

primed CD8+ T cells [27]. This is an indication of production of IL-13 by CD8+ T cells and

the possible importance of IL-13 in developing inflammation using an allergy mice model.

Tc1 cells can efficiently migrate to inflamed tissue [28], and can, via secretion of IFN-γ, play

a major role in defense against tumor and virus-infected cells [28, 29]. Less is known about

Tc2 cells, but their engagement has been shown in human diseases such as viral infections

[26], cancer [30], and autoimmune diseases [31]. The presence of Tc2 has been correlated

with disease severity and progression [30, 32].

There are two different ways whereby Tc effector cells mediate target cell killing; both

leading to target cell death by apoptosis. The process starts when the TCR on a Tc recognizes

antigen in association with class I MHC molecules on a target cell with additional assistance

of CD8-MHC class I interaction. This chain of events is followed by interaction between the

LFA-1 receptor on CTL and its ligand ICAMs on the target cell. Because of the formation of

this LFA-1-ICAM complex, LFA-1 converts from a low-affinity to a high-affinity state,

which last just for few minutes. These events are followed by the release of granules from Tc

Immune responses to metal and organic haptens 15

cells containing effector molecules such as perforin and several serine proteases called

granzymes. The release of these granules in the vicinity of the target cells sets off a cascade of

reactions eventually leading to DNA fragmentation and apoptosis of the target cells within

minutes. The second way of killing involves a membrane interaction between the Fas ligand

(on CTL) with the Fas receptor (on the surface of target cells). The Fas-Fas ligand interaction

also leads to apoptosis of the target cell, albeit more slowly.

T-regulatory subsets A further subset of T cells termed T-regulatory (TReg) cells has been identified. The cells have

an immunosuppressive function and a cytokine profile distinct from that of either Th1 or Th2

cells [33]. CD4+ TReg cells can be divided into at least three different subsets; type 1 TReg

(Tr1), CD4+CD25+ TReg, and Th3 cells, which are not necessarily different cell types, but

perhaps overlapping phenotypes. Yet an additional subset of CD8+ TReg have been suggested

to play a role in oral tolerance [34].

Inducible TReg cells, or type 1 regulatory (Tr1) cells, are defined by their ability to produce

high levels of IL-10 and transforming growth factor (TGF)-β [35, 36]. In the presence of IL-

10 and IFN-α, or a combination of IL-4 and IL-10, antigen-specific Tr1 cells may be

generated from naive CD4+ T cells in vitro [33, 35, 36]. It is known that during the early

course of allergy, Tr1 cells are generated and, by regulating allergen-specific Th1 and Th2

responses, may play a role in down-regulating the allergic response [37]. Therefore, Tr1 cells

might be considered as a potential target for modulation of peripheral tolerance as a therapy

against allergies and autoimmunity [38, 39].

Constitutive TReg cells, or CD4+CD25+ TReg cells, are immunosuppressive cells, and consist of

5-10% of the CD4+ T cells pool in peripheral blood. These cells have been shown to inhibit

the activation of effector T cells in the periphery. The suppressive mechanisms of

CD4+CD25+ TReg cells is via the inhibition of the IL-2 receptor α chain in target T cells [40].

Different autoimmune diseases such as arthritis, and diabetes, may develop spontaneously in

the absence of the appropriate frequency of functional CD4+CD25+ TReg cells [41]. Also

CD4+CD25+ TReg cells can produce IL-10 and TGF-β, therefore these cells might be

overlapping with the Tr1 cells.

16 Khosro Masjedi

Th3 cells, are suppressive T cells which produce TGF-β and variable amounts of IL-4 and IL-

10 [42, 43]. Many functions described for these cells overlap with the Tr1 phenotype. These

cells are engaged in immunosuppression in health and diseases. The family of T-regulatory

cells (TReg) is growing. Recently, a population of CD8low TReg immunosupressive cells with

production of TGF-β in female mice, was described to prevent rejection of male skin grafts

[44, 45].

Memory T cells Our current understanding of the generation, trafficking and function of memory T cells has

originated mainly from studying models with acute infections or immunization with strong

adjuvants. The early events leading to the generation of memory from naïve T cells is decided

upon availability of antigen, proper presentation through APC, and proper co-stimulation

[46]. Upon availability of the antigen to the immune system, local DC take up the antigen,

process it, while moving towards the regional lymph nodes [1, 47]. Naïve T cells constantly

scan DC in lymph nodes for the availability and presentation of foreign antigens. Memory T

cells are generated from naïve T cells with a lag time of one to a few weeks after proper

antigen exposure. This lag time depends on several parameters including the nature of

antigen, type of immunization, quality and duration of antigen presentation, and antigen

duration and availability to the system [46]. Memory T cells may be generated from CD4+ or

CD8+ T lymphocytes [48]. Based on the expression of CD45RA, CD62L and especially

CCR7, CD4+ or CD8+ lymphocytes may be categorized as ‘central memory’ (TCM) or

‘effector memory’ T cells (TEM) [48-50]. CCR7 is a molecule that mediates homing to lymph

nodes through high endothelial venules [49]. TCM are CCR7+ and present in lymph nodes

organs, spleen and blood. TEM are CCR7- and present in the blood, spleen, and non-lymphoid

tissues [49, 50]. It has been postulated that TCM cells located in secondary lymphoid organs

after stimulation with antigen would decrease CCR7 expression, migrate to peripheral organs,

and become TEM cells [48].

CD45RA and CD45RO, two isoforms of CD45, are expressed by many populations of naïve

and effector and memory T cells. These two isoforms are produced by alternative splicing of

the RNA transcript of the CD45 gene [51, 52]. Both isoforms of these membrane molecules

mediate TCR signal transduction. Memory T cells express both isoforms with predomination

of CD45RO. Effector T cells express mainly CD45RO. The CD45RO isoform associates

Immune responses to metal and organic haptens 17

more effectively with the TCR complex and the CD4 and CD8 coreceptors. CD45RA is

mainly expressed by naïve T cells.

The CD45RO memory T cells are capable of patrolling the skin by expressing a special

adhesion molecule called cutaneous lymphocyte-associated antigen (CLA) [53, 54]. The CLA

antigen is a homing receptor for T cells with tropism for the skin. CLA has a carbohydrate

structure, similar to sialyl Lewis X antigen, and is a part of bigger protein called PSGL-1 [55].

Its ligand E-selectin, is an adhesion molecule, which is induced on endothelial cells upon

inflammation. CLA is expressed by more than 90% of infiltrating T cells present in cutaneous

inflammatory sites, and is about 20% of the T cells in other non-cutaneous sites, and is

expressed on about 15% of peripheral blood T cells.

18 Khosro Masjedi

Allergies Allergy is a condition in which the body has an exaggerated immune response to substances

that most of the population does not react to. This exaggerated response is also known as

hypersensitivity.

Classifications of allergies The types I to IV reactions of Coombs and Gell are the classical way of categorizing

hypersensitivity reactions (Table 2) [56]. In immediate-type (type I) reactions, soluble

antigens interact with allergen-specific IgE antibody molecules bound to high-affinity

receptors (FcεRI) on mast cells or basophils. The allergen-IgE and FcεRI interaction induce

the release of inflammatory mediators as well as several cytokines from mast cells or

basophils. Type II reactions are defined by production of IgG antibodies against either cell

surface-bound antigens (type IIa) or cell-surface receptors (type IIb). In type IIa, activation of

complement can cause lysis of target cells and in type IIb reactions between antibodies and

cell-surface receptors can cause altered cell function or signaling. Type III hypersensitivity is

based on the formation of antigen/antibody complex, mediated by soluble antigens and IgG

that activate the complement system. Finally, type IV hypersensitivity reaction is T-cell

mediated and can be subdivided into three groups. In type IVa1, Th1 cells are engaged while

in type IVa2, Th2 type cells are involved. In Type IVb reactions, CTL cells are interacting

with antigen-associated molecules leading to cell destruction. A typical example of type IVb

reaction is contact dermatitis [56, 57]. However, in contrast to the original classification

which considered contact dermatitis mainly as Th1-type reaction (Table 2), more recent

results point to the involvement also of Th2-type reactions in ACD (see below).

Immune responses to metal and organic haptens 19

Table 2: Four types of hypersensitivity reactions

Type I Type II Type III Type IV Immune

reaction

IgE IgG IgG Th1 Th2 CTL

Effector

mechanism

Ag induces

cross linking

of IgE bound

to mast cells

ADCC; Ab

directed

against cell

surface Ag

Ab-Ag

complex

deposition

Macrophage

activation

Eosinophil

activation

through

cytokines

Cytotoxicity

Example of

hypersensitivity

reaction

Allergic

rhinitis,

asthma

Drug

allergies

(e.g.

penicillin)

Serum

sickness

Contact

dermatitis,

tuberculin

reaction

Chronic

asthma,

chronic

allergic

rhinitis

Contact

dermatitis

Modified from [56]

An abnormality called atopy involves the development of immediate hypersensitivity

reactions against common environmental antigens. One feature of atopy is production of IgE

against non-parasitic antigens, leading to IgE-mediated destruction as seen in type I

hypersensitivity. Typical allergic symptoms include asthma, rhino-conjunctivitis,

gastrointestinal symptoms and characteristics skin lesions [58]. The abnormal IgE response in

atopic individuals is genetically linked to a region encoding a variety of cytokines including

IL-3, IL-4, IL-5, IL-9 and IL-13. IgE, produced against allergens, binds to the high affinity

receptor FcεRI on the surface of mast cells or basophils [59, 60]. Cross-linkage of bound IgE

on mast cells or basophils induces degranulation with the release of a number of mediators,

which cause the inflammatory responses that result in the clinical symptoms seen in type I

hypersensitivity reactions. Histamine, leukotrienes, prostaglandins, and cytokines (IL-3, IL-4,

IL-5, IL-9, IL-13, GM-CSF and TNF-α) are among the mediators causing symptoms. Besides

bound IgE, other factors such as anaphylatoxins (C3a and C5a), and various drugs may trigger

degranulation. Although the majority of allergens causing IgE-mediated reactions are proteins

produced in nature, e.g., pollen, furry-animals, moulds, house dust mites and foodstuffs;

certain chemicals have the potential to induce IgE-mediated allergic reactions. Atopic

allergies is related to Th2 memory cell activity which may cause an increased number of

eosinophils [61].

Based on a recent proposal the classification of hypersensitivity and eczema reactions were

re-defined as below [58, 62]. Hypersensitivity is a reaction initiated by exposure to a defined

20 Khosro Masjedi

stimulus at a dose tolerated by normal subjects. Allergy is a hypersensitivity reaction initiated

by immunologic mechanisms which can be antibody (IgE) or cell-origin mediated. Atopy is a

personal or familial tendency to produce IgE antibodies in response to low doses of allergens.

A local inflammation of the skin is defined as dermatitis.

Hypersensitivity reaction type IV and delayed-type hypersensitivity Contact dermatitis is an altered state of skin reactivity induced by exposure to an external

chemical agent. According to the mechanism of elicitation, the following types of contact

reactions may be distinguished: (1) ACD; (2) irritant contact dermatitis (ICD); (3) phototoxic

and photo ACD, and (4) immediate type contact reactions [63]. Clinical manifestation in

humans may be accompanied by erythema, infiltration, possible papules, or possible

coalescing vesicles.

ACD is a disease with economical and psychological impact on society. The incidence of

ACD varies from clinic to clinic and the results are influenced by sex, age and occupation. It

has been estimated that 15-20% of the adult population in Europe is sensitized to one or more

contact allergens [64]. More than 3000 chemical compounds have been described as potential

contact allergens [65]. Such compounds have the capacity to induce cell-mediated contact

allergy, a DTH reaction. ACD is the clinical manifestation of skin reactions to contact

allergens. In most patients with ACD, a limited number of compounds are responsible for

sensitization and symptoms. It has been estimated that 15-20% of the adult population in

Europe is sensitized to one or more contact allergens [64].

Metals, especially nickel (Ni), but also chromium and cobalt, are common causes of contact

allergy [66]. Most contact allergens are low molecular weight organic substances, some of

which are present in plants and other natural materials but many are components of man-made

products [67]. Rubber additives, as well as monomers and oligomers used in the manufacture

of plastics (acrylates, epoxy resins, phenol-formaldehyde resins) frequently cause ACD [68].

Another wide-spread problem is sensitization caused by preservatives and fragrances present

in cosmetics and hygiene products [69]. The information network of departments of

dermatology (IVDK) is a multicenter project, based in Germany, which connects more than

30 dermatological centers and the information provided has been used for epidemiological

Immune responses to metal and organic haptens 21

studies [70]. Based on information gathered from these centers the incidence for some of the

main contact sensitizers is shown in Table 3.

Table 3. Percentage of incidences of most frequent contact allergens in eczematous dermatitis patients [70] Allergen 1992 1993 1994 1995 1996 (Number of patients) (6700) (8750) (10150) (10050) (9600) Nickel sulfate 16.7* 17.0 17.1 17.1 16.3 Fragrance mix 7.4 11.2 13.2 9.7 10.3 Thiomersal 4.2 4.8 6.2 6.4 7.5 Balsam of Peru 5.5 6.3 7.0 6.3 7.0 Cobalt chloride 4.4 4.8 5.0 4.8 4.9 p-Phenylenediamine 4.8 5.0 5.4 4.6 4.3 Wool wax alcohols 2.2 2.1 2.5 2.6 3.8 Potassium dichromate 5.9 4.3 4.4 4.1 3.8 Colophony 2.8 3.4 3.6 3.2 3.6 Thiuram Mix 2.7 2.5 3.2 2.6 2.6 Hg (II)-amide-chloride 2.2 2.3 3.2 2.1 2.5 MCI/MI 3.0 2.5 2.4 2.0 2.5 Neomycin sulfate 2.0 2.5 2.8 2.2 2.3 Formaldehyde 2.2 2.1 2.4 1.9 1.9 Turpentine 0.5 0.5 0.3 0.5 1.7 MDGBPE** 1.2 1.9 2.0 1.7 1.7 Paraben Mix 1.5 1.4 1.8 1.1 1.6 Benzocaine 1.3 1.6 1.6 1.6 1.5 Epoxy resin 1.2 1.3 1.1 1.1 1.1 *Percentage is shown **Methyldibromo-glutaronitrile phenoxyethanol Mechanistic cellular events in allergic contact dermatitis Contact allergic reaction could be divided into two phases; the induction (sensitization) phase

is initiated by contact with the hapten which initiates a chain of events that leads to the person

becoming sensitized. The effector (elicitation) phase refers to the reactions set off upon

secondary exposure with the hapten leading to clinical manifestation of ACD [71-73]. Upon

binding of hapten to the carrier molecule through covalent or ionic bonds, the hapten-

modified protein is taken up and processed by cutaneous DC including epidermal LC and

dermal DC. The activated DC subsequently migrates to the paracortical zone of local draining

lymph nodes via the afferent lymphatic vessels. Upon repeated exposure to the sensitizer, the

memory/effector arm of the immune system is augmented, and the overall process leads to

visible local DTH responses within 18-48 h. A variety of different APC are involved in DTH

response. LC, macrophages and the vascular endothelial cells (that express MHC class II

22 Khosro Masjedi

molecules) may function as APC. Infiltration of not only the memory/effector T cells but also

a variety of different types of cells into the exposed skin occurs. In the epidermis, such

infiltrates consist primarily of lymphocytes and, occasionally, of polymorphonuclear

neutrophils and eosinophils. In the case of the dermis, dense cell infiltrates are composed

almost exclusively of mononuclear cells [74].

Irritant and allergic contact dermatitis Contact dermatitis is a common disease including both ICD and ACD. While ACD is

accounting for only 20% of the contact dermatitis cases in most dermatology units, ICD is

more common, accounting for approximately 80% of those with contact dermatitis. Unlike

ACD the development of ICD does not require prior sensitization [75]. The clinical findings

of ICD and ACD can be very similar and make the diagnosis difficult but the patch test may

be helpful for a correct diagnosis. Clinically, ICD may show demarcated scaly erythematous

eruption that can be distinguished from ACD. An irritant reaction is often seen in individuals

who are involved in wet work, hairdressers, or bar workers, just to name a few. It is believed

that ICD is caused mainly by a local toxic effect of a chemical. For this to occur, a high dose

of the chemical is required [76].

The pathogenesis of irritancy was originally considered mainly as non-immunological.

However, it is now clear that the immune cells and cytokines play an important role in the

pathogenesis of the symptoms. In response to irritants, keratinocytes undergo structural

changes and this may vary according to the type of irritant used [77, 78]. TNF-α appears to be

one of the key cytokines induced in ICD [79]. TNF-α increases MHC class II expression and

adhesion molecules on keratinocytes, thereby helping to initiate and maintain irritant reaction.

Other proinflammatory cytokines such as IL-1, IL-6, IL-8, and IL-10, are relevant cytokines

induced in ICD. Although no immunological memory is involved in irritancy, it has been

shown that CD4+ T cells infiltrate the skin upon stimulation with irritants producing type 1

cytokines mainly IFN-γ and IL-2 [80-82]. However, IFN-γ inducible chemokines (CXCL9

(MIG), CXCL10 (IP-10) and CXCL11 are expresses by hapten-specific T cells in ACD, but

not in ICD [83].

Immune responses to metal and organic haptens 23

Subsets of T cells and their role in contact allergy Due to the general conception that contact hypersensitivity (CHS) in mice is a DTH-mediated

response it was originally believed that CHS was mediated by CD4+ T cells [84, 85].

However, based on more recent studies it has been suggested that effector cells of CHS in

mice, are CD8+ T cells whereas CD4+ T cells down-regulate and dampen the response [86-

89].

Most of our current knowledge about contact allergy, the involvement of T-cell subsets and

their role in the CHS, has been obtained from studies in mice employing highly potent

haptens (strong haptens). Compared to ordinary haptens (weak haptens), strong haptens

display strong irritancy due to their toxicity. The toxicity of strong haptens triggers, to a larger

extent than weak haptens, the innate immune cells to produce proinflammatory cytokines that

are required for the initiation of the CHS response. Moreover, strong haptens are usually more

reactive chemicals, which make them able to bind to many cellular components and hence

increase the chances of antigen processing and presentation through MHC molecules on APC.

However, it was recently shown that the cellular mechanisms behind induction of CHS with

some weak haptens may be also similar to strong haptens [86].

Different strategies have been used to study the CHS reaction in mice. These include

depletion of CD4+ and CD8+ T cells using antibodies in vivo, transfer of haptenated DC/LC,

transfer of CD4+ or CD8+ T cells to unsensitized naive mice, or transfer of CD4+ or CD8+ T

cells to MHC class I- or class II-deficient mice. It was shown that MHC class I-deficient mice

(which do not have any CD8+ T cells, but normal CD4+ T cells) did not develop CHS to

dinitrofluorobenzene (DNFB), whereas class II-deficient mice (which do not have any CD4+

T cells, but normal CD8+ T cells) developed an enhanced CHS [90]. In vivo depletion of

CD4+ or CD8+ T cells supported these data [91]. Similar results were obtained with other

haptens such as oxazolone and trinitrophenyl (TNP) [92, 93]. The conclusion from these

studies was that while hapten-specific CD8+ T cells are mandatory for development of CHS

whereas hapten-specific CD4+ T cells are mainly engaged in down-regulating the response.

This concept was confirmed in mice depleted of CD4+ T cells that developed an exaggerated

CHS reaction, an indication that CD4+ T cells play an important role in down regulating CHS

[71, 94].

24 Khosro Masjedi

It has been shown, based on studies in mice, that hapten-specific CD8+ T cells produce mainly

type 1 cytokines, while hapten-specific CD4+ T cells produce mainly type 2 cytokines,

including IL-4, IL-5 and IL-10 [92]. These data was confirmed for CD8+ T cells using the

enzyme-linked immunospot assay (ELISpot) [89]. Moreover, it was shown that hapten

presentation to the T cells is MHC restricted [93, 95]. MHC class I-expressing DC induced a

normal CHS reaction whereas passive transfer of DC lacking MHC class I (obtained from

MHC class I deficient mice) did not [96]. Finally, it was postulated that MHC-restricted

hapten-peptide presentation to the hapten-specific T cells may not require processing of the

hapten (see below) [97].

It is believed that mechanisms of ACD in human display similarities with CHS in mice, and

that the effector and down-regulatory T cells are similar [87]. However, a majority of these

studies are based on information obtained from the generation of T-cell clones (TCC), which

may suffer from some conceptual drawbacks (see ‘Results & Discussion’).

Involvement of type 1 and/or type 2 cytokines were proven in ACD, mainly based on

generation of TCC obtained from ACD subjects. Using the lipophilic hapten model, urushiol,

it was shown that hapten-specific CD8+ T cells, with the profile of type 1 cytokines (IL-2 and

IFN-γ), represent the majority of TCC; and that urushiol presentation to T cells is MHC class

I and class II dependent although, for some TCC are independent of processing [98, 99].

Based on information obtained from TCC generated from peripheral blood mononuclear cells

(PBMC) or skin lesions, it was shown that, overall, CD4+ T cells, with a mixed type 1 and

type 2 cytokine profile, are dominant cells in mediating ACD to Ni [100, 101] and that Ni

may directly bind to MHC class I and class II for presentation without the need for hapten

processing [102]. A recent study in humans with the strong hapten DNFB revealed the

presence of both hapten-specific CD4+ and CD8+ T cells with mainly type 1 profile (IFN-γ,

little IL-5) [103].

Skin biology The skin is the largest ‘organ’ of the body with a surface area of approximately 1.8 m2,

constituting almost one sixth of the total body weight. It is the barrier, which regulate

important body functions such as water balance, thermoregulation, dynamic involvement in

immunological defense and protection, UV-protection, synthesis of substances such as

vitamin D and detoxification. The skin forms the continuous external surface of the body, and

Immune responses to metal and organic haptens 25

in different regions of the body the skin varies in thickness, color, and the presence of hairs,

glands and nails. Despite these variations which reflect different functional demands, all types

of skin have the same basic structure. Skin is the interface between the external and internal

environment, and consist of three layers called epidermis, dermis and subcutis. As an

immunological barrier, the skin is engaged in inflammatory responses. Different cells

including keratinocytes, LC, DC, vascular endothelial cells, lymphocytes, mast cells,

macrophages, fibroblasts, and eosinophils in the skin are involved in the innate and adaptive

immunity and immune-surveillance.

The external surface of the skin consists of a keratinised squamous epithelium called the

epidermis with a thickness of ∼50-100 µm. Epidermis consists of cells including keratinocytes

(∼95%), LC (∼2-3%) and melanocytes (∼2-3%) and has four layers, from outside-in including

stratum corneum, stratum granulosum, stratum spinosum and stratum basal. The epidermis is

supported and nourished by a thick layer of dense, fibro-elastic connective tissue called the

dermis (1-4 mm in thickness), which is highly vascular and contains many sensory receptors.

The dermis consist mainly of connective tissue (collagen, matrix, and fibroblast cells), vessels

(endothelia cells), cells (mast cells, macrophages, eosinophils), and skin appendages (hair,

sweat and sebaceous glands). The dermis is attached to underlying tissues by a layer of loose

connective tissue called the subcutis or subcutaneous layer which contains variable amounts

of adipose tissue and the thickness may vary by several centimeters. There is continuous

immune-surveillance of the skin by T lymphocytes.

Skin in healthy individuals usually is an efficient, almost perfect barrier which restricts

penetration. Along with the skin properties as a barrier, the physicochemistry of substances

may rule the penetration. Easy penetration may be limited to low molecular weight molecules

(<500 Dalton); and is enhanced by a moderate lipophilicity for the organic molecules.

Diffusion of ions is much harder and slower through the normal skin. Any alterations in the

stratum corneum of the skin may impact the penetration of chemicals and pathogens [104-

106]. The outer layer of the skin, stratum corneum, is the most important barrier for blocking

the entry of substances. This layer consists mainly of dead cells called corneocyte, generated

from keratinocytes, which are surrounded by a lipid matrix (brick and mortar model).

26 Khosro Masjedi

Related & specific background Haptens

In 1935 Karl Landsteiner introduced the term hapten for low molecular weight antigens [107].

In classical immunology, haptens have been defined as chemicals which by themselves

cannot elicit immune responses. Instead, the chemical coupling of haptens to larger

immunogenic proteins (carrier molecules), termed haptenation, is required for them to be

capable of inducing an immune response. As is the case for other antigens, several factors

may influence the immune responsiveness to a hapten. Molecular size, chemical composition,

heterogeneity and recognition of hapten-carrier complex by T and/or B cells are requirements

for a good recognition. Moreover, route and frequency of exposure play a pivotal role in the

fate of final recognition and reaction [108].

In the field of contact allergy, haptens (contact allergens) are defined as chemicals that can

bind to carrier molecules to initiate a cellular immune response, and the response is initiated

through MHC-TCR interactions which can activate hapten-specific T cells. T cells are

thought to recognize haptens as modified epitopes on carrier molecules or even directly bound

hapten to MHC molecules (see below). Based on their chemical nature, and the degree of

exposure and reactivity, the frequency of ACD to different haptens may differ between

populations (Table 3, see topics below for details).

Chemical and biological properties of contact allergens Normal skin is exposed to many exogenous (xenobiotic) compounds including natural or

industrial-made substances. The immune system normally does not react to the xenobiotics,

but those that act as contact allergens can eventually elicit ACD. Based on the murine local

lymph node assay (LLNA), and the skin dose threshold for elicitation of a proliferative

reaction, chemicals can be ranked in terms of their allergenic potency [109]. In LLNA, mice

receive a topical application of the chemicals on the dorsum of both ears. Later, the mice

receive an injection of [3H]thymidine into their tail. The mice are sacrificed after five hours

and proliferation is measured in cells obtained from draining lymph nodes. Moreover, as has

been shown in LLNA, the magnitude of the proliferative response is dose dependent.

Immune responses to metal and organic haptens 27

To elicit a response, contact allergens have to pass through the skin barrier. It is known, for

many haptens, that a moderate lipophilicity facilitates passing through the skin barrier.

Physicochemical properties of the hapten, along with skin-related biological factors influence

the penetration of hapten through the skin barrier [110]. Skin-related biological factors

include the number of cell layers in the skin, age, and degree of hydration of the skin [110-

113]. The importance of lipophilicity of haptens for elicitation of a reaction was supported by

experiments in which different degrees of sensitization potency was observed when mice

were subjected to contact allergens in various vehicles [114].

Once the contact allergen has passed through the skin barrier, its chemical nature may

determine the final type of immune responsiveness. Some chemicals are unstable, and further

metabolism and chemical reactions drives them to become more stable. Moreover, the skin is

able to metabolize potentially dangerous chemicals and convert them into ‘non-toxic’

materials. But metabolization may also convert a potentially harmless material (prohapten) to

a reactive hapten which may promote an immune reaction. Pentadecylcatechol, one of the

allergens in poison ivy, is one example of a prohapten that is thought to be activated by

metabolic oxidation [115]. Furthermore, non-enzymatic processes, involving atmospheric

oxygen or ultraviolet radiation, can also activate molecules. For instance, limonene is not

allergenic in itself, but is oxidized to different allergenic forms upon exposure to air [116,

117].

Contact allergens interact with macromolecules by covalent or non-covalent bonds. To form a

covalent bond, electron deficient (electrophilic) centers of contact allergens attack electron

rich centers (nucleophiles) of the target molecules. The outcome of such interactions may lead

to the formation of a covalent bond, as is seen when reactive organic compounds interact with

the amino acids on proteins (Figure 1) [118]. Not all haptens bind covalently to

macromolecules. Transition metals such as Ni and chromium for instance, form coordination

complexes with nucleophilic macromolecules (Figure 2) [119].

28 Khosro Masjedi

Figure 1. Michael addition at the C-5 position of methylchloroisothiazolinone followed by

chlorine elimination; nucleophile (Nu:), protein (P)

Figure 2. Structure of coordination complexes formed between nickel (Ni) and ligand (L)

General properties and clinical relevance of haptens included in this study To compare the immune reactivity induced by haptens of disparate chemical nature, three

different haptens were included in these studies. Ni was included as a representative of

metals. Metals react with the carrier molecules and form recognizable antigens through

coordination bonds. From the group of organic haptens, the water-soluble hydrophilic

chemicals MCI/MI (mixture of methylchloroisothiazolinone (MCI) and methylisothiazolinone

(MI) MCI/MI), and a lipophilic chemical, parthenolide, were included. Organic haptens react

with the carrier molecules and form recognizable antigens through covalent bonds.

Nickel Ni is the most common cause of contact allergy in Europe. In Europe, 10-20% of women and

approximately 2% of the men are sensitized to Ni [120]. The higher percentage of

sensitization in women compared to men could be explained with the fact that women usually

Immune responses to metal and organic haptens 29

wear more Ni-containing material such as jewellery and ear piercing. Sensitization requires

direct and prolonged skin exposure to Ni. Ni may dissolve in sweat which may explain how it

can pass through the skin barrier [121]. Ni is a reactive molecule, it can bind to extra- or

intracellular compounds and can be presented to Ni-specific T cells through either MHC class

I, or MHC class II through normal antigen processing or alternatively, it can bind directly to

grooves of MHC molecules. Upon MHC-Ni-TCR interactions between APC and T cells, Ni-

specific T cells have been reported to produce mainly type 1, but also type 2 cytokines in

ACD individuals based on studies performed (see below for detailed discussion) [100, 122].

Methylchloroisothiazolinone MCI/MI are ingredients in many biocides and preservatives [123] (Figure 3). For example,

Kathon CG™ which consists of a ratio of 3:1 of MCI/MI is widely used in a range of hygiene

products, especially cosmetics [124]. These compounds are very effective preservatives at low

concentrations, but can also sensitize at these low concentrations in particular MCI. In an

unselected Danish population, 0.65% were found to be contact allergic to MCI/MI [120].

Among patients with dermatitis, the prevalence of sensitization vary from 0-10% [123]. In a

multicenter study performed in Germany, 2.5% of the women and 2.8% of the men suspected

of having ACD were shown to be sensitized to MCI/MI [69]. A possible scenario for the

formation of MCI-protein conjugates is shown in Figure 1.

SN

Cl

O

CH3 S

N

O

CH3

Figure 3. The chemical structures of methylchloroisothiazolinone (MCI) and

methylisothiazolinone (MI).

MCI MI

30 Khosro Masjedi

Parthenolide Sesquiterpene lactones (SL) are extracted from the Compositae plant family. SL mix is a

mixture of C-15 terpenoids, consists of an equal mixture of alantolactone, costunolide and

dehydrocostus lactone, with anti-inflammatory activities but is often also reported to cause

ACD [125]. The incidence of Compositae-induced dermatitis is 0.7-1.4% in the general

population in Europe [126, 127]. The SL parthenolide (Figure 4) from the plant Feverfew

(Tanacetum parthenium) has been shown to cause contact allergy in man [128, 129].

Parthenolide yields a positive patch test in approximately 75% of patients positive for SL mix

[130].

Figure 4. The chemical structure of parthenolide. Patch testing

The gold standard for the diagnosis of ACD is patch testing [76, 131]. The patch testing

protocol involves the application of a selection of contact allergens on patches that are placed

on the upper back of patients (day 0). After 48 h (day 2), the patches are removed. On day 3,

and preferably also on day 6-7, the upper back is examined for the presence of a reaction to

the suspected contact sensitizers, and the results are scored based on the recommendations of

the International Contact Dermatitis Research Group (Table 4) [132]. Haptens in the standard

series are applied epicutaneously in appropriate vehicles in a small volume. Haptens included

in the standard patch test series are selected to represent the haptens most commonly causing

contact allergy and are to a large extent listed in Table 3 [70, 126]. The standard series used,

however, differ between regions and clinics and is constantly updated. One of the most

commonly used patch test systems is the thin-layer rapid use epicutaneous test (TRUE test).

Immune responses to metal and organic haptens 31

In the TRUE test, the allergen is incorporated into a thin layer gel that upon contact with the

skin adsorbs moisture which results in the release of the haptens.

Table 4. The scoring system for patch test reactions recommended by the International Contact Dermatitis Research Group[132] Score 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 reaction Discrete patchy erythema or homogonous erythema without

infiltration ? doubtful reaction Faint macular or homogenous erythema, no filtration

32 Khosro Masjedi

Aims of the study

The overall objective of the present studies was to define the in vitro reactivity of haptens

with disparate chemical character (metal ion, lipophilic and hydrophilic organic haptens), in

terms of cytokine production by PBMC from ACD patients or controls upon hapten

stimulations. The studies had the following specific objectives:

• To determine and compare the immune responses induced by contact allergens of

disparate chemical nature including metals (represented by nickel), hydrophilic

organic haptens (represented by MCI/MI), and lipophilic organic haptens (represented

by parthenolide) with regard to the production of type 1 and type 2 cytokines in

PBMC from allergic and non-allergic subjects.

• To investigate the T-cell phenotypes involved in the production of type 1 and type 2

cytokines in contact allergies induced by the lipophilic hapten parthenolide versus the

water soluble metal ion Ni.

• To evaluate the possibility of diagnosing contact allergy to nickel, MCI/MI and

parthenolide using the enzyme-linked immunospot (ELISpot) assay, that allows a

sensitive ex vivo analysis of cytokine production at the single-cell level.

• To investigate to what extent the variation in the patch test reactivity (in vivo) over

time is influenced by fluctuations in the systemic immune reactivity (in vitro), using

reactivity to Ni as a model.

Immune responses to metal and organic haptens 33

Subjects and methods

Original papers in this study are referred to in the text by their Roman numerals

Subjects [ I-IV] Contact allergic [ I-IV] individuals were selected to be included in these studies based on a

historical positive patch test reactivity. To assess current patch test reactivity, subjects were

subjected to repeated patch tests [I-IV]. Eleven [I] and 15 [IV] female patients with a history

of Ni allergy, 10 patients [II] with a history of contact allergy to MCI/MI (6 females and 4

males) and 10 parthenolide-allergic subjects (9 females and 1 male) [IV] participated in these

studies. Nine [I], 9 [II] and 5 [III] sex and age-matched non-allergic volunteers with negative

patch test reactivity to the relevant haptens participated as controls. The Ni-allergic

individuals and their matched controls in study [I] were recruited from the Department of

Dermatology and Venereology at Karolinska Hospital in Stockholm; the MCI/MI- [II],

parthenolide- [III] and Ni-allergic individuals [IV] as well as the corresponding control

subjects were recruited from the Department of Occupational and Environmental

Dermatology at Malmö University Hospital. The studies were approved by the Ethics

Committee at the Karolinska Hospital [I] and by the Ethics Committee of the Medical Faculty

of Lund University, Lund, Sweden [II-IV]. All subjects gave their informed consent prior to

participation.

Isolation of peripheral blood mononuclear cells (PBMC) [I-IV] Blood samples from allergic patients and non-allergic controls were obtained by venepuncture

and collected in sterile heparinized glass vials. Following separation by density gradient

centrifugation over Ficoll-Paque, freshly prepared PBMC were tested in the proliferation or

ELISpot assays [I]. The remaining cells were frozen in heat-inactivated fetal calf serum (FCS)

containing 10% dimethylsulfoxide (DMSO) and maintained in liquid nitrogen until used for

ELISpot [I and II] or enzyme-linked immunosorbent assay (ELISA) [II] analysis (see below).

Alternatively, PBMC were frozen in cell culture medium containing 20% heat-inactivated

FCS and 10% DMSO [III & IV].

34 Khosro Masjedi

Patch testing [I-IV] The allergic status of the subjects was confirmed by assessing their patch test reactivity to

NiSO4 (5% in petrolatum) [I], MCI/MI (200 ppm in water) [II] and to parthenolide 0.1% w/w

in petrolatum [III]. In study IV, 10 different concentrations of NiSO4 (range: 12.5 - 0.0032 %

in water with a dilution factor of 2.5) were used, The patches were removed after 48 h and

reading was performed on day 3 [II-IV] or 7 [II & III]. The patch test results were scored

based on the scoring system recommended by the International Contact Dermatitis Research

Group [132] (Table 4). In studies I-IV, patch tests were performed after taking blood samples

from ACD individuals for in vitro analysis.

As MCI/MI is highly reactive it could potentially sensitize individuals. On the other hand,

using a lower concentration of this substance in the patches may not detect reactive

individuals (false negative). Literature reports recommend 100 ppm MCI/MI for routine patch

testing. However, the Swedish Contact Dermatitis Group (Gruvberger B, personal

communication) recommends testing with MCI/MI at a concentration of 200 ppm, since some

patients with true contact allergy to MCI/MI do not react to 100 ppm [133]. Kathon CG which

contains the two active ingredients MCI (1.125%) and MI (0.375%) (MCI/MI) in water was

used in the patches at 200 ppm according to recommendations by the Swedish Contact

Dermatitis Group.

The lymphocyte proliferation assay [I-II] Freshly isolated PBMC (2 x 105 /well) in identical triplicates were cultured for 6 [I] or 5 [II]

days on 96-well U-bottomed plates and in culture medium containing 10% autologous plasma

(not heat inactivated) with and without the addition of NiCl2 [I] or MCI/MI [II]. DNA

synthesis was determined on the basis of [3H]thymidine incorporation; the results are

presented as mean counts per min (cpm) of triplicates. The results of the proliferation assay

are expressed as stimulation index (SI), which refers to mean cpm of Ni- or MCI/MI-

stimulated PBMC divided by the corresponding values for unstimulated cells.

Quantification of cytokine-producing cells by ELISpot [I-IV] For the ELISpot assay, fresh [I] or frozen and thawed [I-IV] PBMC were cultured in medium

supplemented with 10% heat-inactivated FCS, seeded in triplicates into polyvinylidene

difluoride (PVDF). The ELISpot plates were wrapped in aluminum foil [II-IV] and incubated

Immune responses to metal and organic haptens 35

at 37°C under 5% CO2 in humidified air in the absence or presence of NiCl2, MCI/MI or

parthenolide for 20-48 hours depending on the cytokine analyzed [I-IV]. The ELISpot plates

were coated with monoclonal antibodies (mAb) directed towards human IL-1β, IL-2, IL-4,

IL-5, IL-10, IL-12, IL-13, IFN-γ or TNF-α and the cytokines captured were detected

employing matching secondary antibodies. Stimulation with phytohaemagglutinin (PHA) [I-

IV] was used as a control for cell viability. In study IV, Ni, tetanus toxoid (TT) and PHA

were used for the stimulation of PBMC.

ELISpot data are presented either as the mean number of spots induced by antigen stimulation

per number of cells plated [I-IV] or as the cytokine ratio (CR) [II, III], obtained by dividing

the mean number of spots obtained with stimulated cells by the number of spots produced by

cells cultured in the absence of allergen. CR values of >2 were considered positive, provided

that stimulated cells gave rise to at least 5 [II, III] more spots than the unstimulated cells.

Analysis of cytokine production by enzyme-linked immunosorbent assay (ELISA) [II] PBMC (2 x 106/ml) were cultured in the absence or presence of MCI/MI for 20-48 h

(depending on the cytokine analyzed), supernatants were collected, centrifuged and thereafter

stored at -20°C until use [II]. To perform ELISA, plates were coated with antibodies directed

towards human IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IFN-γ or TNF-α, and the

captured cytokine was detected with matched biotinylated detection antibodies. All samples

were tested in duplicates, and the mean results were reported as pg/ml or CR, analogous to

those described for ELISpot. CR values of >2 were considered positive if the difference

between the level of the cytokine released by stimulated and unstimulated cells exceeded 5

pg/ml. The lower detection limit in these ELISA assays was set to 5 pg/ml.

Depletion of CD4+ or CD8+ cells and flow cytometry [III] In order to determine the phenotype of the IL-13- and IFN-γ-producing cells, CD4+ or CD8+

cells were depleted from PBMC from 2 subjects with parthenolide allergy and 6 subjects with

Ni allergy (see below) and the different PBMC fractions stimulated with parthenolide.

Cytokine production was measured by ELISpot. The depletion procedure was performed

using BDTM IMag anti-human CD4 or CD8 magnetic nanoparticles according to the

manufacturer’s instructions, and the CD4+ and CD8+ depleted cell fractions were adjusted to

36 Khosro Masjedi

3.0 x 106 cells/ml. The purity of the different PBMC fractions was analyzed with a FACScan

using anti-CD4 or CD8 mAb according to the manufacturer’s instructions.

Statistical methods [I-IV] The statistical significance of the differences between the values for the allergic and non-

allergic groups was analyzed with the non-parametric Mann-Whitney U-test [I & II], based on

ranking of data. Evaluation of possible relationships between the various parameters was

monitored based on Spearman rank-order correlation coefficients [I-IV]. A p value of <0.05

was considered to be statistically significant. Statistical analyses including ELISpot or ELISA

data were calculated based on values obtained by subtraction of spontaneous cytokine

production from following PBMC stimulation with hapten [I-IV]. Negative values were set to

zero. ELISA values below the lower limit of detection were set to 5 pg/ml for these

calculations [II].

In study [IV], the differences in the patch test reactivity and the cytokine reactivity between

three time points at the group level was computed using Friedman rank test. To determine

how the variations in cytokine production compared to the variation in patch test reactivity

within individuals, the changes in the patch test reactivity obtained between time points and

from the corresponding analysis of Ni-induced cytokine production in in vitro test were

calculated and analysed using Spearman rank-order correlation coefficients.

Immune responses to metal and organic haptens 37

Results and Discussion

Proliferation of PBMC induced by nickel and MCI/MI [I-II] In the studies I-II, as a first step in characterizing the immune responses to the contact

allergens Ni and MCI/MI, and because previous studies were mostly based on proliferation

assays, we performed proliferation assays for comparison. PBMC were isolated from allergic

subjects and stimulated with the relevant hapten and the responses obtained compared to that

of PBMC from age-matched non-allergic controls. Results were expressed as SI, defined as

mean counts per min (cpm) of Ni- or MCI/MI-stimulated PBMC divided by mean cpm of

unstimulated cells.

Nickel [I]

Eleven Ni-allergic and 3 non-allergic subjects showed a positive proliferative response to 50

µM Ni (SI>3). The response was significantly higher in the Ni-allergic group as compared to

the non-allergic group. This finding was in agreement with previous studies [66, 134, 135]. A

concentration of 100 µM of Ni generally provided a higher stimulatory response in allergic

but also in non-allergic groups. Although many studies of proliferative response to Ni have

suggested proliferation assays to be a valuable tool in discriminating between allergic and

non-allergic individuals, the sensitivity and specificity of the proliferation assay, also called

lymphocyte transforming test (LTT) in the diagnosis of Ni contact allergy has not always

been satisfactory [136]. Nevertheless, a version of LTT called MELISA (memory lymphocyte

immunostimulation assay) is currently commercially available for analysis of T-cell reactivity

to several haptens [134]. In the case of metal haptens such as gold, palladium and Ni, the

sensitivity of LTT has been reported to be between 55-95% and the specificity between 17-

79% [134]. Further optimization may be needed for LTT in order to improve its diagnostic

value.

MCI/MI [II]

In PBMC from subjects with or without patch test reactivity to MCI/MI [II], using multiple

ranges of concentrations of MCI/MI, the optimal concentrations of the hapten mix that

induced lymphocyte proliferation was determined. The response was significantly higher in

the MCI/MI-allergic group as compared to the non-allergic control group. All allergic

individuals with moderate or strong (++ or +++) patch test reactions responded positively

38 Khosro Masjedi

(SI>3) to at least one concentration of MCI/MI but none of the two individuals with weak (+)

patch test reactivity responded. Except for one positive response (SI>3) to a single

concentration of MCI/MI in a control subject, non-allergic individuals did not respond. Prior

to our study only one publication has investigated the proliferative response of PBMC to

MCI/MI [137]. In that study, none of the non-allergic individuals and 50% of the MCI/MI

allergic individuals with strong patch test reactions exhibited a positive proliferative response.

Production of type 1 and type 2 cytokines induced in PBMC by nickel, MCI/MI and parthenolide [I-III] Nickel [I]

Upon Ni stimulation of PBMC from allergic and non-allergic (control) individuals, a higher

number of cells that produced cytokines in the allergic group compared to that in the control

group was observed for IL-4 (p<0.01), IL-5 (p<0.05), IL-13 (p<0.05), and IFN-γ (p<0.05) but

not for IL-12. The number of IL-4- and IL-5-producing cells correlated well with the number

of IL-13-producing cells. The increased number of IFN-γ-producing cells in the Ni-allergic

group, in addition to the production of IL-4, IL-5 and IL-13, indicated a mixed Th1- and Th2-

cytokine response. However, while a strong correlation was observed between all Th2

cytokines (IL-4, IL-5 and IL-13) and between IL-13 and IFN-γ, proliferation induced by 50

µM Ni did not correlate with any of the cytokines tested.

Early studies in humans considered the DTH response to Ni mainly as a reflection of a

predominating T-cell type 1 response [138, 139]. However, later studies have shown that the

DTH response to Ni is associated both with production of type 1 and 2 cytokines. Using

ELISA it was shown that PBMC from allergic individuals, upon Ni stimulation produced

significantly higher IL-4 [140, 141], IL-5 [140] and IFN-γ [142] compared to non-allergic

controls. These findings are in agreement with our findings in study I that strongly suggested

the involvement also of Th2 type cytokines in the immunological reaction to Ni. In study I we

did not analyze the production of IL-2. Others, however, have demonstrated a significant

production of this cytokine in PBMC from Ni-allergic individuals compared to non-allergic

individuals [141, 143].

The lack of production of type 2 cytokines and strong orientation towards type 1 cytokines in

ACD, reported in early studies of PBMC, could be primarily due to the use of other

experimental protocols and less sensitive methods. ELISA was used in the previous studies

Immune responses to metal and organic haptens 39

for investigating Th2-type production in ACD. There are methodological limitations in the

sensitivity of ELISA to measure Th2-type cytokines. For example, IL-4 produced upon

antigen stimulation is consumed by cellular receptors and hence depleted from the culture

medium and may thus not be detectable by conventional ELISA [144] and could thus

incorrectly be found to be negative. Due to the immediate capture of the target cytokine in

ELISpot, the ELISpot results are less affected by the cytokine consumption [144].

In PBMC cultures, triggering the production of cytokines upon antigen stimulation to obtain a

measurable response could be difficult. To overcome this limitation and increase the detection

sensitivity, various strategies have been employed. Long-term culture of cells in the presence

of contact allergens may increase the accumulation of cytokines in the culture medium [145].

Another attempt has been to stimulate PBMC with haptens in the presence of a polyclonal T-

cell activator such as PHA, assuming that Ni-specific T cells from allergic subjects may

produce more cytokines compared to non-allergic [140]. Yet another strategy to increase the

sensitivity has been to add co-stimulatory cytokine cocktails in the cell cultures, with the aim

to improve the environment so that hapten-reactive T cells respond more optimally [145,

146]. These modifications used by others, with the aim of enhancing the response, may

eventually alter the measurable cytokine reactivity in vitro in an artificial manner [147].

Hence, in the present study [I], a short-term cell culture was used without any enhancement

strategy for the measurement of type 1 and 2 cytokines induced by Ni in ACD and control

subjects. The Ni-induced cytokine production was measured using ELISpot, which identifies

cytokine production at the single cell level with high sensitivity with no further need for

additional enhancement strategies.

MCI/MI [II]

Employing ELISpot and ELISA, the ability of MCI/MI [II] to stimulate the production of

eight cytokines including IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-13 and TNF-α by PBMC

from allergic and non-allergic subjects was assessed. When comparing allergic to non-allergic

subjects, stimulation of PBMC with MCI/MI with predefined optimal concentrations of

MCI/MI resulted in significantly higher number of cytokine-producing cells for IL-13

(p<0.05) and IL-2 (p<0.05) and production of significantly higher levels of IL-13 (p<0.05).

Upon stimulation of PBMC by MCI/MI, no significant difference was observed between

allergic compared to non-allergic individuals for the production of IL-1β and TNF-α.

40 Khosro Masjedi

Significantly positive correlations were observed between MCI/MI-induced production of IL-

2, IL-4, IL-5 and IL-13 and the proliferation assay.

By applying cut off definitions for positive responses in PBMC from 8 individuals with

allergy to MCI/MI, with moderate or strong patch test reactivity (++ or +++), 7/8 individuals

gave a positive response for IL-13 using either ELISpot or ELISA. Four of these strongly

reactive subjects were also positive for IL-2 by ELISpot and ELISA assay and in addition, 3/7

of them were positive for IL-4 and IL-5 in ELISpot. Two allergic subjects with weak patch

reaction (+) and one with moderate reaction (++) to MCI/MI did not respond in the in vitro

assays.

In study II, the production of both type 1 and type 2 cytokines induced by a hydrophilic

organic hapten, represented as MCI/MI, was thoroughly analyzed for eight different cytokines

employing the ELISpot method. Strong induction of IL-13 upon stimulation by MCI/MI in

allergic but not non-allergic subjects, as well as induction of IL-2 indicated the involvement

of both type 1 and type 2 cytokines in contact allergy to MCI/MI. A similar profile was found

in response to Ni [I] suggesting that the mixed cytokine production upon hapten stimulation

may be a general feature in contact allergies to different haptens in ACD patients (see below).

Parthenolide [III]

We next aimed to investigate the nature of the immunological responses to an organic hapten

of lipophilic nature. Upon PBMC stimulation by parthenolide and using ELISpot for analysis

of cytokine responses, the number of cells producing IFN-γ, IL-2, IL-4, IL-5 and IL-13 was

assessed and compared between PBMC from allergic and non-allergic groups [III]. First, to

ensure the proper solubilization of parthenolide and its availability in the water-based cell

culture medium containing 0.1% DMSO, a pilot study was conducted and the cytokine

production was measured for IL-4, IL-13 and IFN-γ production using a broad range of

parthenolide concentrations. Based on the results of the pilot tests, three optimal parthenolide

concentrations were chosen for the main study. We found that the number of cytokine-

producing cells induced by the lipophilic hapten parthenolide was significantly higher in the

allergic compared to the non-allergic group for IFN-γ, IL-2, IL-4, IL-5 and IL-13 (p<0.05).

The number of cytokine-producing cells in the allergic group was correlated between all

cytokines. Next, we were interested in identifying the phenotype of cells responsible for IFN-

γ and IL-13 production in PBMC from the parthenolide-allergic patients. CD4+ or CD8+ cells

Immune responses to metal and organic haptens 41

were depleted from PBMC of two parthenolide-allergic subjects using magnetic beads. We

found that depletion of CD8+ cells from PBMC abrogated the cytokine response, while the

whole PBMC or CD4+ depleted PBMC from the same subjects maintained high production of

IFN-γ and IL-13 after stimulation. In Ni-reactive subjects, included for comparison, the

production of IFN-γ and IL-13 upon stimulation with Ni was devoid in the PBMC fraction

depleted of CD4+ cells whereas CD8+ depleted PBMC and whole PBMC displayed full

reactivity with Ni.

By use of cut-off definitions for positive and negative responses, PBMC from all 8 moderate

and strong (++ and +++), but not the 2 weak (+), parthenolide-patch test reactive patients

were positive for IFN-γ production, and 7 of them (out of 8) were also positive for IL-13

production upon stimulation with parthenolide. Five (out of 10) individuals were positive for

all analyzed cytokines for at least two parthenolide concentrations.

General discussion [I-III]

The knowledge on the cytokine patterns induced by organic haptens in PBMC is limited in

comparison to metal ions where in particular Ni has been extensively studied. Moreover,

despite the many studies on Ni, reports on the cytokine profile induced by Ni in allergic

individuals have been controversial. Therefore, in this thesis we determined and compared the

immune responses induced by metal (Ni), organic hydrophilic (MCI/MI) and lipophilic

(parthenolide) haptens in PBMC from individuals suffering from ACD to the corresponding

haptens. We observed a significant production of T-cell type 1 as well as type 2 cytokines in

the allergic individuals compared to the non-allergic controls upon stimulation with Ni,

MCI/MI and parthenolide. Thus, the results from this thesis [I-III] support that both T cell

type 1 and type 2 cytokines are involved in the response to different haptens, irrespective of

their chemical nature. A similar mixed cytokine pattern has been shown for other metals (e.g.

cobalt and palladium) causing contact allergy in humans [143].

The nature of haptens may have a profound impact on antigen formation and presentation.

Formation of haptenated macromolecules may occur in extra- or intra-cellular domains.

Alternatively, DC may present exogenous antigens on MHC I to elicit CTL responses, a

process that is referred to as cross-presentation [148]. Depending on their chemical reactivity

and lipophilicity, organic haptens may be processed and presented to the hapten-specific T

42 Khosro Masjedi

cells differently. Lipophilic antigens may directly penetrate the cell membranes, and be taken

up and presented via the endogenous pathway through MHC class I and primarily activate

CD8+ T cells. Hydrophilic haptens, on the other hand, may be taken up and presented to the

immune cells through the exogenous pathway on MHC class II and primarily activate CD4+ T

cells [72, 87]. Alternatively, haptens may, without the need for antigen processing, bind

directly to the groove of MHC class I and class II peptides and thereafter be presented to

hapten-specific T cells. The later model has been demonstrated for haptens of various

chemical nature. For instance, for organic haptens such as TNP, it was shown that hapten-

specific T cells react to MHC-associated TNP-peptides, but not to covalently TNP-modified

MHC molecules [149, 150]. In the case of urushiol, it was shown that hapten presentation is

both endogenous- (MHC class I) and exogenous- (MHC class II) [98] As an exception to this

general model, it was shown that Ni may also directly link TCR and MHC class II in a

peptide-independent manner and act as a superantigen [151], a process that may be

independent of antigen processing [152]. Finally, for larger molecules such as drugs, it has

been postulated that they may directly bridge between highly variable antigen-specific

memory T cells and MHC without the need for antigen processing [153]. This model may

explain the rapid immune reaction to certain drugs. In conclusion, the nature of the hapten,

including the chemical reactivity and the degree of lipophilicity, may thus have an impact on

the route of hapten presentation and subsequently on the differential activation of hapten-

specific CD4+ or CD8+ T-cells.

Although parthenolide elicited a cytokine pattern similar to that induced by Ni [101, 154] and

MCI/MI in allergic individuals, the T cells producing the type 1 and type 2 cytokines were

different from those reported for Ni. Ni-reactive T cells in PBMC proved to be of the CD4+ T

cell subset [101, 154], whereas parthenolide-reactive T cells in our study proved to be CD8+ T

cells [III]. This finding indicates that even though the general cytokine profile of different

haptens, in this study, show the same pattern of cytokine induction in allergic individuals

upon specific hapten stimulation, different mechanisms may lay behind the hapten

presentation. In studies conducted on MCI/MI [II], T-cell phenotypes involved in production

of such mixed cytokine profiles in allergic individuals were not determined. In addition to

defining the peripheral T cells recognizing MCI/MI, it would be of interest to further analyze

and compare, in PBMC and skin lesions, the phenotype of cells involved in allergic reactions

to Ni, MCI/MI and parthenolide.

Immune responses to metal and organic haptens 43

Much of the earlier information obtained regarding the involvement of functionally disparate

cytokines in contact allergy was achieved by generation and analysis of TCC obtained from

PBMC or skin lesions. In particular, contact allergy to Ni has been extensively studied as a

general ACD model in humans [100, 155]. Generation of TCC is based on long-term culture

of cells from a tissue, for example PBMC or skin cells, in the presence of Ni which eventually

will lead to death of most of the cells and selection of few hapten-specific T cells [156]. This

protocol has been used, in humans and mice, to study different immunological aspects of Ni

and other hapten-related CHS, such as MHC-hapten-TCR interactions and the cytokine

profiles induced by hapten-specific CD4+ or CD8+ T cells [100, 149, 157, 158]. In one study

250 TCC were generated in parallel from PBMC and skin lesions from 3 Ni-allergic

individuals, and it was shown that 2/3 of TCC were CD4+αβ TCR compared to 1/3 CD8+αβ

TCR (both in the PBMC and skin), and that PBMC-derived TCC produced mainly IFN-γ

(type 1) (and no or little IL-4) whereas skin-derived TCC showed mainly type 0 (IL-4 & IFN-

γ) or type 2 (IL-4) cytokine profiles [100]. When conducting studies based on TCC one has to

consider that generation and long-term maintenance of cells in the culture medium could

potentially lead to alterations of the selected cell e.g. the loss of chromosomes which may

affect the outcome [147, 159, 160]. Thus, one has to be cautious when comparing such data to

responses obtained after ex vivo stimulation of PBMC.

In these studies [I-III], for the first time, we demonstrated that IL-13 is produced by PBMC

from subjects with contact allergy to Ni, MCI/MI and, parthenolide but not from non-allergic

individuals. IL-13 is considered mainly a Th2 type cytokine, but its production has also been

reported in Th1, Th0 and CD8+ TCC [13, 161]. The physiological role of IL-13 in contact

allergy is currently unknown. However IL-13 shares many activities with IL-4. Therefore, it

would be of interest to further analyze the relevance and function of IL-13 in contact allergy.

Implications of the ELISpot assay for diagnosing contact allergy to nickel [I] Contact allergy is a disease composed of both irritant and allergic properties [162]. Although

both the patients’ history and physical examination are important in the diagnosis of ACD, it

was shown that only in 29-54% of cases, is history alone adequate for diagnosis [163].

Therefore there is a need for reliable diagnostic methods. The established method for

diagnosis of ACD is the patch test. The patch test can confirm that an eczematous reaction is

of allergic nature as well as define the causative reagent(s). However, there are advantages

44 Khosro Masjedi

and disadvantages to be considered with regard to this test. Ease of use and the possibility to

apply multiple contact allergens simultaneously on the back of patients are some of the main

advantages. Potential risk for patient sensitization, inconvenience for the patients, false

positive and false negative reactions, dependency of results on the observer’s experience, and

dependency of results on the skin condition (such as sensitive skin and sunburn) are among

the disadvantages [76, 164, 165]. Therefore there may be a need for alternative or

complimentary diagnostic tools.

Employing the ELISpot assay in the study [I], we could detect specific IL-4 production

following Ni stimulation only in PBMC from ACD patients and not in PBMC from non-

allergic controls. Later on this was further evaluated and confirmed by others [143, 166-168].

Based on this observation [I], we suggested that determination of IL-4 might be a potential

tool for future diagnosis of contact allergy to Ni. A limitation of the first study [I] was that

only a limited number of patients with moderate and strong patch test reactivity to Ni (++ and

+++ in historical patch test reactivity) and no patients with weak (+) reactions were included

in the study [I]. Therefore, we suggested that further studies may be required. In a later study,

it was shown that 100% of (+++), 80% of (++), 50% of (+) patch test positive patients and 0%

of controls displayed reactivity to Ni based on IL-4 and IL-13 in vitro assays [168].

The use of ELISpot in the diagnosis of ACD to Ni may have some advantages. The use of

frozen PBMC provides an opportunity for repetition of experiments in case of doubtful

results. Moreover, the use of the in vitro based methods eliminates the risk for patient

sensitization. Further simplification of the ELISpot test in diagnostics such as preadsorbed

capture antibodies, and use of one-step PBMC preparation tubes for simplicity was suggested

by us, and has to some extent been evaluated [167]. It may be noted that a limited amount of

information is available on the sensitivity, specificity and reliability of in vitro tests as a

diagnostical tool in ACD. Therefore, to draw more firm conclusions, more extensive studies

are needed. The need for well-trained technicians and a relatively high cost of the reagents are

among drawbacks of the in vitro test.

Immune responses to metal and organic haptens 45

Relationships between the variability over time in nickel patch test reactivity and systemic T-cell reactivity [IV] Although the patch test is the only widely accepted method for diagnosis of contact allergy,

used in conjunction with the clinical history, many studies have reported a limited

reproducibility. Simultaneous application of duplicate patches on both sides of the upper back

has resulted in a concordance rate, with double positive or negative patch test reactions,

ranging from 43 to 95% [164, 169-172]. Moreover, it has been shown that in patch tests

repeated over a period of time, the reactivity varies substantially [173-175]. One possible

explanation for such variation could be that the change of the patch test reactivity is due to an

actual change of the systemic immune responsiveness over time. Therefore, in study IV we

aimed to investigate if longitudinal changes in the patch test reactivity were correlated with

changes in the systemic immunological reactivity over time.

Employing 10 different serial dilutions of Ni in the patch test, and performing the patch test

three times over a period of six months, we found that all patch test reactive ACD patients

displayed variability over time in terms of the minimal Ni concentration that elicited a

positive reaction [IV]. This was in accordance with other studies [173, 174]. In parallel, the

systemic reactivity of PBMC from allergic individuals was evaluated at the same time points

as the patch test (i.e. three times in a period of six months). PBMC obtained at the three time

points were stimulated by Ni, and as an indication of the systemic reactivity the numbers of

IL-4- and IL-13-producing cells were measured by ELISpot. We also found that the systemic

reactivity to Ni within the individuals varied during the test period. No tendency for an

increase or decrease in the overall reactivity over time for either the in vivo or in the in vitro

assays was observed for the group. The overall (mean over time) magnitude of the patch test

reactivity was well correlated with the overall cytokine reactivity, i.e. patients with higher

patch test reactivity displayed a higher degree of cytokine production upon stimulation. This

observation was in accordance with a previous study, and demonstrates that the magnitude of

the patch test reactivity is closely related to the degree of systemic T-cell reactivity displayed

by an individual [168].

We were interested to analyze if, within individuals, the pattern of changes in the patch test

reactivity and the pattern of changes of the cytokine reactivity was concordant. We found that

the fluctuations in the patch test reactivity and the fluctuations in the systemic reactivity,

within individuals, did not correlate. However, while the pattern of the in vivo and in vitro

46 Khosro Masjedi

changes in most subjects appeared to be rather well associated, the pattern of changes was

highly diverged in some individuals. This may be an indication that changes of the patch test

reactivity is to a certain degree related to similar changes in the systemic reactivity but that

other causes for variation in the patch test overrides the impact of fluctuations in the systemic

reactivity. Methodological as well as biological factors may influence the results of the patch

test. Uneven distribution of allergens in the vehicle and dissimilar pressure supported by the

patch test system are among the methodological factors [176-178]. Among the biological

factors, regional responsiveness in the skin, and skin dryness are factors which may affect the

outcome of patch test results [164, 177, 178]. While it may be possible to limit the influence

of methodological-related factors using optimized procedure, less could be done to reduce the

influence of skin-related factors. In study IV, to limit methodological-related influence on the

reading, the patch test was performed with 10 different dilutions of Ni. Such protocols may

result in a more accurate reading due to the fact that if one patch did not detect a response

(false negative due to e.g. local skin condition) the other dilutions would, to a certain extent,

compensate for the overall reading. To reduce the risk for incorrect doses of Ni water was

used as a diluent and to avoid variation in the reading of tests, the patch test reading was done

by the same highly skilled dermatologist.

In conclusion, the overall reactivity in the patch test and the in vitro test was well correlated

confirming that the two methods measure reactivity to Ni in a similar manner. However,

fluctuations in the patch test reactivity over time was not well correlated with variations in the

systemic reactivity suggesting that other parameters than changes in systemic reactivity are of

high relevance for the variation in the patch test reactivity over time.

Immune responses to metal and organic haptens 47

Concluding remarks

• A similar mixed type 1 and type 2 cytokine profile is induced by contact allergens of

disparate chemical nature represented by nickel (a water-soluble metal ion), MCI/MI

(a hydrophilic organic hapten mix) and parthenolide (a lipophilic organic hapten) [I-

III].

• Parthenolide-stimulated PBMC, from parthenolide-allergic subjects, responsible for

the production of type 1 and type 2 cytokines were defined as CD8+ T cells. In

contrast, the response to nickel in PBMC from nickel-allergic subjects was manifested

by CD4+ cells thus suggesting that the chemical nature of a hapten has an impact on

the character of the resulting immune response [III].

• Measurement of hapten-induced IL-4 and IL-13 by ELISpot could be an alternative

way of discriminating between contact-allergic and non-allergic individuals in future

in vitro-based diagnostic applications [I].

• The high degree of correlation between the systemic immune response to nickel and

the nickel patch test reactivity suggests that the overall magnitude of the systemic

reactivity is highly associated with the degree of patch test reactivity. However,

fluctuations in patch test reactivity within individuals over time appears to be affected,

to a large extent, by other factors than variations in the systemic reactivity [IV].

48 Khosro Masjedi

Acknowledgment This thesis would not be possible without contributions from a number of different people. I

would therefore like to express my deepest gratitude to:

Niklas Ahlborg, my supervisor, for priceless support especially in difficult days, for

continuous patience and for accepting me as a student and sharing his vast knowledge.

Marita Troye-Blomberg, my supervisor, for support, excellent collaboration, fruitful

discussions and feedback.

My co-authors from Göteborg and Malmö Ann-Therese Karlberg, Magnus Bruze, Birgitta

Gruvberger, Monica Hindsén and Karin K. Olsson for excellent collaboration and support.

Thank you for initiate and continue this work with me.

My co-authors Annika Scheynius, Eva Jakobson, Jacob Minang, Helen Wahlkvist,

Bartek Zuber, and Lena Lundeberg, for fruitful discussions and support.

The seniors at the department of Immunology, Eva Sverremark-Ekström, Carmen

Fernandez, Manuchehr Abedi-Valugerdi, Klavs Berzins, and Eva Severinson for sharing

their wide knowledge of immunology with me.

My good friends and colleagues at Mabtech AB, Staffan Paulie, Alexandre Antoni, Anki

Höglind, Ann-Catrin Forssberg, Anja Mesenholl, Bartek Zuber, Bernt Axelsson,

Caroline Wigren, Cheryl Ng Shu-Ling, Christian Smedman, Dag Sehlin, Eva Gelius,

Gun Jönsson, Iréne Areström, Julia Salazar-Gustavsson, Jenny Fohlstedt, Jenny Huang,

Karin Svensson, Kjell Östrand, Kopek Nihlmark, Lars Björk, Madeleine Glennemark,

Maggan Karlsson, Mårten Andersson, Pelle (Per) Larsson, Sten Braesch-Andersen, and

Åsa Spolander.

And to all my Colleagues and friends at the Department of Immunology in Stockholm

University, National Institute for Working Life and Karolinska Institutet & Hospital.

Immune responses to metal and organic haptens 49

This work was supported by grants from Mabtech AB, the Swedish agency for innovation

systems (VINNOVA), the Swedish Research Council, the Swedish National Institute for

Working Life, the Vårdal Foundation for Health Care Sciences and Allergy Research

(Stockholm), the Swedish Asthma and Allergy Association’s Research Foundation, the

Swedish National Board for Laboratory Animals, the Swedish Medical Research Council

(project no 16X-7924), the Swedish Council for Work Life Sciences, and Karolinska

Institutet.

50 Khosro Masjedi

References

1. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y.J. Liu, B. Pulendran,

and K. Palucka, Immunobiology of dendritic cells. Annu Rev Immunol, 2000. 18: p. 767-811.

2. Herrington, C. and P.A. Hall, Molecular and cellular themes in inflammation and immunology. J Pathol, 2008. 214(2): p. 123-5.

3. Navi, D., J. Saegusa, and F.T. Liu, Mast cells and immunological skin diseases. Clin Rev Allergy Immunol, 2007. 33(1-2): p. 144-55.

4. Hogan, S.P., H.F. Rosenberg, R. Moqbel, S. Phipps, P.S. Foster, P. Lacy, A.B. Kay, and M.E. Rothenberg, Eosinophils: biological properties and role in health and disease. Clin Exp Allergy, 2008. 38(5): p. 709-50.

5. Schuurhuis, D.H., N. Fu, F. Ossendorp, and C.J. Melief, Ins and outs of dendritic cells. Int Arch Allergy Immunol, 2006. 140(1): p. 53-72.

6. Enk, A.H. and S.I. Katz, Contact sensitivity as a model for T-cell activation in skin. J Invest Dermatol, 1995. 105(1 Suppl): p. 80S-83S.

7. Rossi, M. and J.W. Young, Human dendritic cells: potent antigen-presenting cells at the crossroads of innate and adaptive immunity. J Immunol, 2005. 175(3): p. 1373-81.

8. Romani, N., D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, D. Niederwieser, and G. Schuler, Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods, 1996. 196(2): p. 137-51.

9. Martino, A. and F. Poccia, Gamma delta T cells and dendritic cells: close partners and biological adjuvants for new therapies. Curr Mol Med, 2007. 7(7): p. 658-73.

10. Marrack, P.C. and J.W. Kappler, Antigen-specific and nonspecific mediators of T cell/B cell cooperation. I. Evidence for their production by different T cells. J Immunol, 1975. 114(3): p. 1116-25.

11. Mosmann, T.R., H. Cherwinski, M.W. Bond, M.A. Giedlin, and R.L. Coffman, Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol, 1986. 136(7): p. 2348-57.

12. Mosmann, T.R. and R.L. Coffman, TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol, 1989. 7: p. 145-73.

13. Mosmann, T.R. and S. Sad, The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today, 1996. 17(3): p. 138-46.

14. O'Garra, A., Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity, 1998. 8(3): p. 275-83.

15. Dong, C. and R.A. Flavell, Cell fate decision: T-helper 1 and 2 subsets in immune responses. Arthritis Res, 2000. 2(3): p. 179-188.

16. Le Gros, G., S.Z. Ben-Sasson, R. Seder, F.D. Finkelman, and W.E. Paul, Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J Exp Med, 1990. 172(3): p. 921-9.

17. McKenzie, G.J., C.L. Emson, S.E. Bell, S. Anderson, P. Fallon, G. Zurawski, R. Murray, R. Grencis, and A.N. McKenzie, Impaired development of Th2 cells in IL-13-deficient mice. Immunity, 1998. 9(3): p. 423-32.

18. Hsieh, C.S., S.E. Macatonia, C.S. Tripp, S.F. Wolf, A. O'Garra, and K.M. Murphy, Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science, 1993. 260(5107): p. 547-9.

19. Robinson, D., K. Shibuya, A. Mui, F. Zonin, E. Murphy, T. Sana, S.B. Hartley, S. Menon, R. Kastelein, F. Bazan, and A. O'Garra, IGIF does not drive Th1 development

Immune responses to metal and organic haptens 51

but synergizes with IL-12 for interferon-gamma production and activates IRAK and NFkappaB. Immunity, 1997. 7(4): p. 571-81.

20. Fiorentino, D.F., M.W. Bond, and T.R. Mosmann, Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med, 1989. 170(6): p. 2081-95.

21. Mosmann, T.R. and R.L. Coffman, TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol, 1989. 7: p. 145-73.

22. Bettelli, E., M. Oukka, and V.K. Kuchroo, T(H)-17 cells in the circle of immunity and autoimmunity. Nat Immunol, 2007. 8(4): p. 345-50.

23. Woodland, D.L. and R.W. Dutton, Heterogeneity of CD4(+) and CD8(+) T cells. Curr Opin Immunol, 2003. 15(3): p. 336-42.

24. Croft, M., L. Carter, S.L. Swain, and R.W. Dutton, Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J Exp Med, 1994. 180(5): p. 1715-28.

25. Salgame, P., J.S. Abrams, C. Clayberger, H. Goldstein, J. Convit, R.L. Modlin, and B.R. Bloom, Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science, 1991. 254(5029): p. 279-82.

26. Maggi, E., M.G. Giudizi, R. Biagiotti, F. Annunziato, R. Manetti, M.P. Piccinni, P. Parronchi, S. Sampognaro, L. Giannarini, G. Zuccati, and S. Romagnani, Th2-like CD8+ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection. J Exp Med, 1994. 180(2): p. 489-95.

27. Miyahara, N., B.J. Swanson, K. Takeda, C. Taube, S. Miyahara, T. Kodama, A. Dakhama, V.L. Ott, and E.W. Gelfand, Effector CD8+ T cells mediate inflammation and airway hyper-responsiveness. Nat Med, 2004. 10(8): p. 865-9.

28. Cerwenka, A., T.M. Morgan, A.G. Harmsen, and R.W. Dutton, Migration kinetics and final destination of type 1 and type 2 CD8 effector cells predict protection against pulmonary virus infection. J Exp Med, 1999. 189(2): p. 423-34.

29. Kemp, R.A. and F. Ronchese, Tumor-specific Tc1, but not Tc2, cells deliver protective antitumor immunity. J Immunol, 2001. 167(11): p. 6497-502.

30. Ito, N., Y. Suzuki, Y. Taniguchi, K. Ishiguro, H. Nakamura, and S. Ohgi, Prognostic significance of T helper 1 and 2 and T cytotoxic 1 and 2 cells in patients with non-small cell lung cancer. Anticancer Res, 2005. 25(3B): p. 2027-31.

31. Inaoki, M., S. Sato, F. Shirasaki, N. Mukaida, and K. Takehara, The frequency of type 2 CD8+ T cells is increased in peripheral blood from patients with psoriasis vulgaris. J Clin Immunol, 2003. 23(4): p. 269-78.

32. Iezzi, G., A. Boni, E. Degl'Innocenti, M. Grioni, M.T. Bertilaccio, and M. Bellone, Type 2 cytotoxic T lymphocytes modulate the activity of dendritic cells toward type 2 immune responses. J Immunol, 2006. 177(4): p. 2131-7.

33. Taylor, A., J. Verhagen, K. Blaser, M. Akdis, and C.A. Akdis, Mechanisms of immune suppression by interleukin-10 and transforming growth factor-beta: the role of T regulatory cells. Immunology, 2006. 117(4): p. 433-42.

34. Weiner, H.L., Oral tolerance for the treatment of autoimmune diseases. Annu Rev Med, 1997. 48: p. 341-51.

35. Groux, H., A. O'Garra, M. Bigler, M. Rouleau, S. Antonenko, J.E. de Vries, and M.G. Roncarolo, A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature, 1997. 389(6652): p. 737-42.

52 Khosro Masjedi

36. Levings, M.K., R. Sangregorio, F. Galbiati, S. Squadrone, R. de Waal Malefyt, and M.G. Roncarolo, IFN-alpha and IL-10 induce the differentiation of human type 1 T regulatory cells. J Immunol, 2001. 166(9): p. 5530-9.

37. Akdis, M., J. Verhagen, A. Taylor, F. Karamloo, C. Karagiannidis, R. Crameri, S. Thunberg, G. Deniz, R. Valenta, H. Fiebig, C. Kegel, R. Disch, C.B. Schmidt-Weber, K. Blaser, and C.A. Akdis, 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, 2004. 199(11): p. 1567-75.

38. Akdis, C.A. and K. Blaser, IL-10-induced anergy in peripheral T cell and reactivation by microenvironmental cytokines: two key steps in specific immunotherapy. Faseb J, 1999. 13(6): p. 603-9.

39. Muller, U., C.A. Akdis, M. Fricker, M. Akdis, T. Blesken, F. Bettens, and K. Blaser, Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J Allergy Clin Immunol, 1998. 101(6 Pt 1): p. 747-54.

40. Annunziato, F., L. Cosmi, F. Liotta, E. Lazzeri, R. Manetti, V. Vanini, P. Romagnani, E. Maggi, and S. Romagnani, Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+) human thymocytes. J Exp Med, 2002. 196(3): p. 379-87.

41. Wildin, R.S., F. Ramsdell, J. Peake, F. Faravelli, J.L. Casanova, N. Buist, E. Levy-Lahad, M. Mazzella, O. Goulet, L. Perroni, F.D. Bricarelli, G. Byrne, M. McEuen, S. Proll, M. Appleby, and M.E. Brunkow, X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet, 2001. 27(1): p. 18-20.

42. Weiner, H.L., Induction and mechanism of action of transforming growth factor-beta-secreting Th3 regulatory cells. Immunol Rev, 2001. 182: p. 207-14.

43. Chen, Y., V.K. Kuchroo, J. Inobe, D.A. Hafler, and H.L. Weiner, Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science, 1994. 265(5176): p. 1237-40.

44. Hoglund, P., Induced peripheral regulatory T cells: the family grows larger. Eur J Immunol, 2006. 36(2): p. 264-6.

45. Maile, R., S.M. Pop, R. Tisch, E.J. Collins, B.A. Cairns, and J.A. Frelinger, Low-avidity CD8lo T cells induced by incomplete antigen stimulation in vivo regulate naive higher avidity CD8hi T cell responses to the same antigen. Eur J Immunol, 2006. 36(2): p. 397-410.

46. Lefrancois, L., Development, trafficking, and function of memory T-cell subsets. Immunol Rev, 2006. 211: p. 93-103.

47. Inaba, K. and M. Inaba, Antigen recognition and presentation by dendritic cells. Int J Hematol, 2005. 81(3): p. 181-7.

48. Seder, R.A. and R. Ahmed, Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nat Immunol, 2003. 4(9): p. 835-42.

49. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia, Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature, 1999. 401(6754): p. 708-12.

50. Lanzavecchia, A. and F. Sallusto, Understanding the generation and function of memory T cell subsets. Curr Opin Immunol, 2005. 17(3): p. 326-32.

51. Irie-Sasaki, J., T. Sasaki, and J.M. Penninger, CD45 regulated signaling pathways. Curr Top Med Chem, 2003. 3(7): p. 783-96.

52. Tchilian, E.Z. and P.C. Beverley, Altered CD45 expression and disease. Trends Immunol, 2006. 27(3): p. 146-53.

Immune responses to metal and organic haptens 53

53. Santamaria-Babi, L.F., CLA(+) T cells in cutaneous diseases. Eur J Dermatol, 2004. 14(1): p. 13-8.

54. Picker, L.J., S.A. Michie, L.S. Rott, and E.C. Butcher, A unique phenotype of skin-associated lymphocytes in humans. Preferential expression of the HECA-452 epitope by benign and malignant T cells at cutaneous sites. Am J Pathol, 1990. 136(5): p. 1053-68.

55. Fuhlbrigge, R.C., J.D. Kieffer, D. Armerding, and T.S. Kupper, Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells. Nature, 1997. 389(6654): p. 978-81.

56. Janeway, C.A., P. Travers, M. Walport, and M. Shlomchik, Immunobiology. The immune system in health and disease. 5 ed. 2001, New York Edinburgh: Garland publishing/Churchill Livingstone.

57. Kay, A.B., Allergy and Allergic Diseases, ed. A.B. Kay. 1997: Blackwell Science. 58. Johansson, S.G., J.O. Hourihane, J. Bousquet, C. Bruijnzeel-Koomen, S. Dreborg, T.

Haahtela, M.L. Kowalski, N. Mygind, J. Ring, P. van Cauwenberge, M. van Hage-Hamsten, and B. Wuthrich, A revised nomenclature for allergy. An EAACI position statement from the EAACI nomenclature task force. Allergy, 2001. 56(9): p. 813-24.

59. Mamessier, E. and A. Magnan, Cytokines in atopic diseases: revisiting the Th2 dogma. Eur J Dermatol, 2006. 16(2): p. 103-13.

60. Morar, N., S.A. Willis-Owen, M.F. Moffatt, and W.O. Cookson, The genetics of atopic dermatitis. J Allergy Clin Immunol, 2006. 118(1): p. 24-34; quiz 35-6.

61. Homey, B., M. Steinhoff, T. Ruzicka, and D.Y. Leung, Cytokines and chemokines orchestrate atopic skin inflammation. J Allergy Clin Immunol, 2006. 118(1): p. 178-89.

62. Johansson, S.G., T. Bieber, R. Dahl, P.S. Friedmann, B.Q. Lanier, R.F. Lockey, C. Motala, J.A. Ortega Martell, T.A. Platts-Mills, J. Ring, F. Thien, P. Van Cauwenberge, and H.C. Williams, Revised nomenclature for allergy for global use: Report of the Nomenclature Review Committee of the World Allergy Organization, October 2003. J Allergy Clin Immunol, 2004. 113(5): p. 832-6.

63. Krasteva, M., J. Kehren, M. Sayag, M.T. Ducluzeau, M. Dupuis, J. Kanitakis, and J.F. Nicolas, Contact dermatitis II. Clinical aspects and diagnosis. Eur J Dermatol, 1999. 9(2): p. 144-59.

64. Nielsen, N.H., A. Linneberg, T. Menne, F. Madsen, L. Frolund, A. Dirksen, and T. Jorgensen, Incidence of allergic contact sensitization in Danish adults between 1990 and 1998; the Copenhagen Allergy Study, Denmark. Br J Dermatol, 2002. 147(3): p. 487-92.

65. De Groot, A.C., Patch testing. Test concentrations and vehicles for 3700 chemcials. 2 ed. 1994, Baltimore: Williams & Wilkins.

66. Budinger, L. and M. Hertl, Immunologic mechanisms in hypersensitivity reactions to metal ions: an overview. Allergy, 2000. 55(2): p. 108-15.

67. Flyvholm, M.-A., Exposure assessment, in Textbook of Contact Dermatitis, R.J.G. Rycroft, et al., Editors. 2001, Springer-Verlag: Heidelbeg. p. 513-518.

68. Björkner, B., Plastic materials, in Textbook of Contact Dermatitis, R.J.G. Rycroft, et al., Editors. 2001, Springer-Verlag: Heidelbeg. p. 787-824.

69. Schnuch, A., J. Geier, W. Uter, and P.J. Frosch, Patch testing with preservatives, antimicrobials and industrial biocides. Results from a multicentre study. Br J Dermatol, 1998. 138(3): p. 467-76.

70. Uter, W., J.D. Johansen, D.I. Orton, P.J. Frosch, and A. Schnuch, Clinical update on contact allergy. Curr Opin Allergy Clin Immunol, 2005. 5(5): p. 429-36.

54 Khosro Masjedi

71. Grabbe, S. and T. Schwarz, Immunoregulatory mechanisms involved in elicitation of allergic contact hypersensitivity. Immunol Today, 1998. 19(1): p. 37-44.

72. Saint-Mezard, P., A. Rosieres, M. Krasteva, F. Berard, B. Dubois, D. Kaiserlian, and J.F. Nicolas, Allergic contact dermatitis. Eur J Dermatol, 2004. 14(5): p. 284-95.

73. Rustemeyer, T., I.M.W. van Hoogstraten, B.M.E. von Blomberg, and R.J. Scheper, Mechanisms in allergic contact dermatitis, in Textbook of Contact Dermatitis, R.J.G. Rycroft, et al., Editors. 2001, Springer-Verlag: Heidelberg. p. 15-58.

74. Lachapelle, J.-E., Histopathological and immunohistopathological features of irritant and allergic contact dermatitis, in Textbook of Contact Dermatitis, R.J.G. Rycroft, et al., Editors. 2001, Springer-Verlag: Heidelbeg. p. 161-63.

75. Malten, K.E., J.A. den Arend, and R.E. Wiggers, Delayed irritation: hexanediol diacrylate and butanediol diacrylate. Contact Dermatitis, 1979. 5(3): p. 178-84.

76. Mowad, C.M., Patch testing: pitfalls and performance. Curr Opin Allergy Clin Immunol, 2006. 6(5): p. 340-4.

77. Willis, C.M., C.J. Stephens, and J.D. Wilkinson, Epidermal damage induced by irritants in man: a light and electron microscopic study. J Invest Dermatol, 1989. 93(5): p. 695-9.

78. Smith, H.R., D.A. Basketter, and J.P. McFadden, Irritant dermatitis, irritancy and its role in allergic contact dermatitis. Clin Exp Dermatol, 2002. 27(2): p. 138-46.

79. Lisby, S., K.M. Muller, C.V. Jongeneel, J.H. Saurat, and C. Hauser, Nickel and skin irritants up-regulate tumor necrosis factor-alpha mRNA in keratinocytes by different but potentially synergistic mechanisms. Int Immunol, 1995. 7(3): p. 343-52.

80. Willis, C.M., C.J. Stephens, and J.D. Wilkinson, Differential patterns of epidermal leukocyte infiltration in patch test reactions to structurally unrelated chemical irritants. J Invest Dermatol, 1993. 101(3): p. 364-70.

81. Moed, H., D.M. Boorsma, C.P. Tensen, J. Flier, M.J. Jonker, T.J. Stoof, B.M. von Blomberg, D.P. Bruynzeel, R.J. Scheper, T. Rustemeyer, and S. Gibbs, Increased CCL27-CCR10 expression in allergic contact dermatitis: implications for local skin memory. J Pathol, 2004. 204(1): p. 39-46.

82. Hoefakker, S., M. Caubo, E.H. van 't Erve, M.J. Roggeveen, W.J. Boersma, T. van Joost, W.R. Notten, and E. Claassen, In vivo cytokine profiles in allergic and irritant contact dermatitis. Contact Dermatitis, 1995. 33(4): p. 258-66.

83. Flier, J., D.M. Boorsma, D.P. Bruynzeel, P.J. Van Beek, T.J. Stoof, R.J. Scheper, R. Willemze, and C.P. Tensen, The CXCR3 activating chemokines IP-10, Mig, and IP-9 are expressed in allergic but not in irritant patch test reactions. J Invest Dermatol, 1999. 113(4): p. 574-8.

84. Cher, D.J. and T.R. Mosmann, Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones. J Immunol, 1987. 138(11): p. 3688-94.

85. Diamantstein, T., R. Eckert, H.D. Volk, and J.W. Kupier-Weglinski, Reversal by interferon-gamma of inhibition of delayed-type hypersensitivity induction by anti-CD4 or anti-interleukin 2 receptor (CD25) monoclonal antibodies. Evidence for the physiological role of the CD4+ TH1+ subset in mice. Eur J Immunol, 1988. 18(12): p. 2101-3.

86. Vocanson, M., A. Hennino, M. Cluzel-Tailhardat, P. Saint-Mezard, J. Benetiere, C. Chavagnac, F. Berard, D. Kaiserlian, and J.F. Nicolas, CD8+ T cells are effector cells of contact dermatitis to common skin allergens in mice. J Invest Dermatol, 2006. 126(4): p. 815-20.

Immune responses to metal and organic haptens 55

87. Saint-Mezard, P., F. Berard, B. Dubois, D. Kaiserlian, and J.F. Nicolas, The role of CD4+ and CD8+ T cells in contact hypersensitivity and allergic contact dermatitis. Eur J Dermatol, 2004. 14(3): p. 131-8.

88. Martin, S., M.B. Lappin, J. Kohler, V. Delattre, C. Leicht, T. Preckel, J.C. Simon, and H.U. Weltzien, Peptide immunization indicates that CD8+ T cells are the dominant effector cells in trinitrophenyl-specific contact hypersensitivity. J Invest Dermatol, 2000. 115(2): p. 260-6.

89. Kehren, J., C. Desvignes, M. Krasteva, M.T. Ducluzeau, O. Assossou, F. Horand, M. Hahne, D. Kagi, D. Kaiserlian, and J.F. Nicolas, Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity. J Exp Med, 1999. 189(5): p. 779-86.

90. Bour, H., E. Peyron, M. Gaucherand, J.L. Garrigue, C. Desvignes, D. Kaiserlian, J.P. Revillard, and J.F. Nicolas, Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene. Eur J Immunol, 1995. 25(11): p. 3006-10.

91. Gocinski, B.L. and R.E. Tigelaar, Roles of CD4+ and CD8+ T cells in murine contact sensitivity revealed by in vivo monoclonal antibody depletion. J Immunol, 1990. 144(11): p. 4121-8.

92. Xu, H., N.A. DiIulio, and R.L. Fairchild, T cell populations primed by hapten sensitization in contact sensitivity are distinguished by polarized patterns of cytokine production: interferon gamma-producing (Tc1) effector CD8+ T cells and interleukin (Il) 4/Il-10-producing (Th2) negative regulatory CD4+ T cells. J Exp Med, 1996. 183(3): p. 1001-12.

93. Bouloc, A., A. Cavani, and S.I. Katz, Contact hypersensitivity in MHC class II-deficient mice depends on CD8 T lymphocytes primed by immunostimulating Langerhans cells. J Invest Dermatol, 1998. 111(1): p. 44-9.

94. Akiba, H., J. Kehren, M.T. Ducluzeau, M. Krasteva, F. Horand, D. Kaiserlian, F. Kaneko, and J.F. Nicolas, Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8+ T cytotoxic 1 cells inducing keratinocyte apoptosis. J Immunol, 2002. 168(6): p. 3079-87.

95. Kolesaric, A., G. Stingl, and A. Elbe-Burger, MHC class I+/II- dendritic cells induce hapten-specific immune responses in vitro and in vivo. J Invest Dermatol, 1997. 109(4): p. 580-5.

96. Krasteva, M., J. Kehren, F. Horand, H. Akiba, G. Choquet, M.T. Ducluzeau, R. Tedone, J.L. Garrigue, D. Kaiserlian, and J.F. Nicolas, Dual role of dendritic cells in the induction and down-regulation of antigen-specific cutaneous inflammation. J Immunol, 1998. 160(3): p. 1181-90.

97. Cavani, A., C. Albanesi, C. Traidl, S. Sebastiani, and G. Girolomoni, Effector and regulatory T cells in allergic contact dermatitis. Trends Immunol, 2001. 22(3): p. 118-20.

98. Kalish, R.S., J.A. Wood, and A. LaPorte, Processing of urushiol (poison ivy) hapten by both endogenous and exogenous pathways for presentation to T cells in vitro. J Clin Invest, 1994. 93(5): p. 2039-47.

99. Kalish, R.S. and C. Morimoto, The use of human T-lymphocyte clones to study T-cell function in allergic contact dermatitis to urushiol. J Invest Dermatol, 1990. 94(6 Suppl): p. 108S-111S.

100. Werfel, T., M. Hentschel, A. Kapp, and H. Renz, Dichotomy of blood- and skin-derived IL-4-producing allergen-specific T cells and restricted V beta repertoire in nickel-mediated contact dermatitis. J Immunol, 1997. 158(5): p. 2500-5.

101. Moed, H., D.M. Boorsma, T.J. Stoof, B.M. von Blomberg, D.P. Bruynzeel, R.J. Scheper, S. Gibbs, and T. Rustemeyer, Nickel-responding T cells are CD4+ CLA+

56 Khosro Masjedi

CD45RO+ and express chemokine receptors CXCR3, CCR4 and CCR10. Br J Dermatol, 2004. 151(1): p. 32-41.

102. Thierse, H.J., K. Gamerdinger, C. Junkes, N. Guerreiro, and H.U. Weltzien, T cell receptor (TCR) interaction with haptens: metal ions as non-classical haptens. Toxicology, 2005. 209(2): p. 101-7.

103. Pickard, C., A.M. Smith, H. Cooper, I. Strickland, J. Jackson, E. Healy, and P.S. Friedmann, Investigation of mechanisms underlying the T-cell response to the hapten 2,4-dinitrochlorobenzene. J Invest Dermatol, 2007. 127(3): p. 630-7.

104. Harding, C.R., The stratum corneum: structure and function in health and disease. Dermatol Ther, 2004. 17 Suppl 1: p. 6-15.

105. Barry, B.W., Breaching the skin's barrier to drugs. Nat Biotechnol, 2004. 22(2): p. 165-7.

106. Wickett, R. and M. Visscher, Structure and function of the epidermal barrier. Am J Infect Control., 2006. 34(10 Suppl): p. S98-S110.

107. Landsteiner, K. and J.L. Jacobs, Studies on the sensitization of animals with simple chemical compounds. J Exp Med, 1935. 61: p. 643.

108. Smith Pease, C.K., From xenobiotic chemistry and metabolism to better prediction and risk assessment of skin allergy. Toxicology, 2003. 192(1): p. 1-22.

109. Basketter, D.A., G.F. Gerberick, and I. Kimber, Measurement of allergenic potency using the local lymph node assay. Trends Pharmacol Sci, 2001. 22(6): p. 264-5.

110. Berard, F., J.P. Marty, and J.F. Nicolas, Allergen penetration through the skin. Eur J Dermatol, 2003. 13(4): p. 324-30.

111. Ya-Xian, Z., T. Suetake, and H. Tagami, Number of cell layers of the stratum corneum in normal skin - relationship to the anatomical location on the body, age, sex and physical parameters. Arch Dermatol Res, 1999. 291(10): p. 555-9.

112. Roskos, K.V. and H.I. Maibach, Percutaneous absorption and age. Implications for therapy. Drugs Aging, 1992. 2(5): p. 432-49.

113. Zhai, H. and H.I. Maibach, Effects of skin occlusion on percutaneous absorption: an overview. Skin Pharmacol Appl Skin Physiol, 2001. 14(1): p. 1-10.

114. Basketter, D.A., G.F. Gerberick, and I. Kimber, Skin sensitisation, vehicle effects and the local lymph node assay. Food Chem Toxicol, 2001. 39(6): p. 621-7.

115. Lepoittevin, J.-P. and V. Berl, Molecular interactions and chemical bonds, in Allergic Contact Dermatitis. The Molecular Basis, J.-P. Lepoittevin, et al., Editors. 1998, Springer-Verlag: Heidelberg. p. 41-42.

116. Karlberg, A.T., K. Magnusson, and U. Nilsson, Air oxidation of d-limonene (the citrus solvent) creates potent allergens. Contact Dermatitis, 1992. 26(5): p. 332-40.

117. Karlberg, A.T., L.P. Shao, U. Nilsson, E. Gafvert, and J.L. Nilsson, Hydroperoxides in oxidized d-limonene identified as potent contact allergens. Arch Dermatol Res, 1994. 286(2): p. 97-103.

118. Martin, S.F., I. Merfort, and H.J. Thierse, Interactions of chemicals and metal ions with proteins and role for immune responses. Mini Rev Med Chem, 2006. 6(3): p. 247-55.

119. Zhang, Y. and D.E. Wilcox, Thermodynamic and spectroscopic study of Cu(II) and Ni(II) binding to bovine serum albumin. J Biol Inorg Chem, 2002. 7(3): p. 327-37.

120. Nielsen, N.H., A. Linneberg, T. Menne, F. Madsen, L. Frolund, A. Dirksen, and T. Jorgensen, Allergic contact sensitization in an adult Danish population: two cross-sectional surveys eight years apart (the Copenhagen Allergy Study). Acta Derm Venereol, 2001. 81(1): p. 31-4.

121. Nestle, F.O., H. Speidel, and M.O. Speidel, Metallurgy: high nickel release from 1- and 2-euro coins. Nature, 2002. 419(6903): p. 132.

Immune responses to metal and organic haptens 57

122. Kalish, R.S. and P.W. Askenase, Molecular mechanisms of CD8+ T cell-mediated delayed hypersensitivity: implications for allergies, asthma, and autoimmunity. J Allergy Clin Immunol, 1999. 103(2 Pt 1): p. 192-9.

123. Gruvberger, B., Methylisothiazolinones. Diagnosis and prevention of allergic contact dermatitis. Acta Derm Venereol Suppl (Stockh), 1997. 200: p. 1-42.

124. Alexander, B.R., An assessment of the comparative sensitization potential of some common isothiazolinones. Contact Dermatitis, 2002. 46(4): p. 191-6.

125. Kanerva, L., T. Estlander, K. Alanko, and R. Jolanki, Patch test sensitization to Compositae mix, sesquiterpene-lactone mix, Compositae extracts, laurel leaf, Chlorophorin, Mansonone A, and dimethoxydalbergione. Am J Contact Dermat, 2001. 12(1): p. 18-24.

126. Bruynzeel, D.P., T.L. Diepgen, K.E. Andersen, F.M. Brandao, M. Bruze, P.J. Frosch, A. Goossens, A. Lahti, V. Mahler, H.I. Maibach, T. Menne, and J.D. Wilkinson, Monitoring the European standard series in 10 centres 1996-2000. Contact Dermatitis, 2005. 53(3): p. 146-9.

127. Jovanovic, M. and M. Poljacki, [Compositae dermatitis]. Med Pregl, 2003. 56(1-2): p. 43-9.

128. Paulsen, E., L.P. Christensen, and K.E. Andersen, Do monoterpenes released from feverfew (Tanacetum parthenium) plants cause airborne Compositae dermatitis? Contact Dermatitis, 2002. 47(1): p. 14-8.

129. Paulsen, E., L.P. Christensen, and K.E. Andersen, Compositae dermatitis from airborne parthenolide. Br J Dermatol, 2007. 156(3): p. 510-5.

130. Orion, E., E. Paulsen, K.E. Andersen, and T. Menne, Comparison of simultaneous patch testing with parthenolide and sesquiterpene lactone mix. Contact Dermatitis, 1998. 38(4): p. 207-8.

131. Belsito, D.V., Patch testing: after 100 years, still the gold standard in diagnosing cutaneous delayed-type hypersensitivity. Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M, 1997(91): p. 195-202.

132. Fregert, S., International Contact Dermatitis Research Group (ICDRG) Manual of contact dermatitis. 1981, Copenhagen: Munksgaard.

133. Bjorkner, B., M. Bruze, I. Dahlquist, S. Fregert, B. Gruvberger, and K. Persson, Contact allergy to the preservative Kathon CG. Contact Dermatitis, 1986. 14(2): p. 85-90.

134. Cederbrant, K., P. Hultman, J.A. Marcusson, and L. Tibbling, In vitro lymphocyte proliferation as compared to patch test using gold, palladium and nickel. Int Arch Allergy Immunol, 1997. 112(3): p. 212-7.

135. Lisby, S., L.H. Hansen, L. Skov, T. Menne, and O. Baadsgaard, Nickel-induced activation of T cells in individuals with negative patch test to nickel sulphate. Arch Dermatol Res, 1999. 291(5): p. 247-52.

136. Cederbrant, K., The Primary Lymphocyte Culture in the Diagnosis of Drug-and Metal-Induced Allergy, in Department of Health and Environment. 2000, Linkoping University: Linköping. p. 1-69.

137. Stejskal, V.D., M. Forsbeck, and R. Nilsson, Lymphocyte transformation test for diagnosis of isothiazolinone allergy in man. J Invest Dermatol, 1990. 94(6): p. 798-802.

138. Sinigaglia, F., D. Scheidegger, G. Garotta, R. Scheper, M. Pletscher, and A. Lanzavecchia, Isolation and characterization of Ni-specific T cell clones from patients with Ni-contact dermatitis. J Immunol, 1985. 135(6): p. 3929-32.

139. Kapsenberg, M.L., E.A. Wierenga, F.E. Stiekema, A.M. Tiggelman, and J.D. Bos, Th1 lymphokine production profiles of nickel-specific CD4+T-lymphocyte clones

58 Khosro Masjedi

from nickel contact allergic and non-allergic individuals. J Invest Dermatol, 1992. 98(1): p. 59-63.

140. Borg, L., J.M. Christensen, J. Kristiansen, N.H. Nielsen, T. Menne, and L.K. Poulsen, Nickel-induced cytokine production from mononuclear cells in nickel-sensitive individuals and controls. Cytokine profiles in nickel-sensitive individuals with nickel allergy-related hand eczema before and after nickel challenge. Arch Dermatol Res, 2000. 292(6): p. 285-91.

141. Falsafi-Amin, H., L. Lundeberg, M. Bakhiet, and K. Nordlind, Early DNA synthesis and cytokine expression in the nickel activation of peripheral blood mononuclear cells in nickel-allergic subjects. Int Arch Allergy Immunol, 2000. 123(2): p. 170-6.

142. Van Den Broeke, L.T., A. Graslund, P.H. Larsson, J.L. Nilsson, J.E. Wahlberg, A. Scheynius, and A.T. Karlberg, Free radicals as potential mediators of metal allergy: effect of ascorbic acid on lymphocyte proliferation and IFN-gamma production in contact allergy to Ni2+ and Co2+. Acta Derm Venereol, 1998. 78(2): p. 95-8.

143. Minang, J.T., I. Arestrom, M. Troye-Blomberg, L. Lundeberg, and N. Ahlborg, Nickel, cobalt, chromium, palladium and gold induce a mixed Th1- and Th2-type cytokine response in vitro in subjects with contact allergy to the respective metals. Clin Exp Immunol, 2006. 146(3): p. 417-26.

144. Ewen, C. and M.E. Baca-Estrada, Evaluation of interleukin-4 concentration by ELISA is influenced by the consumption of IL-4 by cultured cells. J Interferon Cytokine Res, 2001. 21(1): p. 39-43.

145. Moed, H., M. von Blomberg, D.P. Bruynzeel, R. Scheper, S. Gibbs, and T. Rustemeyer, Improved detection of allergen-specific T-cell responses in allergic contact dermatitis through the addition of 'cytokine cocktails'. Exp Dermatol, 2005. 14(8): p. 634-40.

146. Rustemeyer, T., S. De Ligter, B.M. Von Blomberg, P.J. Frosch, and R.J. Scheper, Human T lymphocyte priming in vitro by haptenated autologous dendritic cells. Clin Exp Immunol, 1999. 117(2): p. 209-16.

147. Karulin, A.Y., M.D. Hesse, M. Tary-Lehmann, and P.V. Lehmann, Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity. J Immunol, 2000. 164(4): p. 1862-72.

148. Basta, S. and A. Alatery, The cross-priming pathway: a portrait of an intricate immune system. Scand J Immunol, 2007. 65(4): p. 311-9.

149. Kohler, J., U. Hartmann, R. Grimm, U. Pflugfelder, and H.U. Weltzien, Carrier-independent hapten recognition and promiscuous MHC restriction by CD4 T cells induced by trinitrophenylated peptides. J Immunol, 1997. 158(2): p. 591-7.

150. Martin, S., B. Ortmann, U. Pflugfelder, U. Birsner, and H.U. Weltzien, Role of hapten-anchoring peptides in defining hapten-epitopes for MHC-restricted cytotoxic T cells. Cross-reactive TNP-determinants on different peptides. J Immunol, 1992. 149(8): p. 2569-75.

151. Gamerdinger, K., C. Moulon, D.R. Karp, J. Van Bergen, F. Koning, D. Wild, U. Pflugfelder, and H.U. Weltzien, A new type of metal recognition by human T cells: contact residues for peptide-independent bridging of T cell receptor and major histocompatibility complex by nickel. J Exp Med, 2003. 197(10): p. 1345-53.

152. Van Den Broeke, L.T., L.C. Heffler, M. Tengvall Linder, J.L. Nilsson, A.T. Karlberg, and A. Scheynius, Direct Ni2+ antigen formation on cultured human dendritic cells. Immunology, 1999. 96(4): p. 578-85.

153. Pichler, W.J., Direct T-cell stimulations by drugs--bypassing the innate immune system. Toxicology, 2005. 209(2): p. 95-100.

Immune responses to metal and organic haptens 59

154. Minang, J.T., I. Arestrom, B. Zuber, G. Jonsson, M. Troye-Blomberg, and N. Ahlborg, Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel. Clin Exp Immunol, 2006. 143(3): p. 494-502.

155. Cavani, A., D. Mei, E. Guerra, S. Corinti, M. Giani, L. Pirrotta, P. Puddu, and G. Girolomoni, 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, 1998. 111(4): p. 621-8.

156. Bour, H., J.F. Nicolas, J.L. Garrigue, A. Demidem, and D. Schmitt, Establishment of nickel-specific T cell lines from patients with allergic contact dermatitis: comparison of different protocols. Clin Immunol Immunopathol, 1994. 73(1): p. 142-5.

157. Sebastiani, S., C. Albanesi, F. Nasorri, G. Girolomoni, and A. Cavani, Nickel-specific CD4(+) and CD8(+) T cells display distinct migratory responses to chemokines produced during allergic contact dermatitis. J Invest Dermatol, 2002. 118(6): p. 1052-8.

158. Lu, L., J. Vollmer, C. Moulon, H.U. Weltzien, P. Marrack, and J. Kappler, Components of the ligand for a Ni++ reactive human T cell clone. J Exp Med, 2003. 197(5): p. 567-74.

159. Kaltoft, K., B.H. Hansen, C.B. Pedersen, S. Pedersen, and K. Thestrup-Pedersen, Common clonal chromosome aberrations in cytokine-dependent continuous human T-lymphocyte cell lines. Cancer Genet Cytogenet, 1995. 85(1): p. 68-71.

160. Kaltoft, K., C.B. Pedersen, B.H. Hansen, and K. Thestrup-Pedersen, Appearance of isochromosome 18q can be associated with in vitro immortalization of human T lymphocytes. Cancer Genet Cytogenet, 1995. 81(1): p. 13-6.

161. de Waal Malefyt, R., J.S. Abrams, S.M. Zurawski, J.C. Lecron, S. Mohan-Peterson, B. Sanjanwala, B. Bennett, J. Silver, J.E. de Vries, and H. Yssel, Differential regulation of IL-13 and IL-4 production by human CD8+ and CD4+ Th0, Th1 and Th2 T cell clones and EBV-transformed B cells. Int Immunol, 1995. 7(9): p. 1405-16.

162. Rietschel, R.L., Human and economic impact of allergic contact dermatitis and the role of patch testing. J Am Acad Dermatol, 1995. 33(5 Pt 1): p. 812-5.

163. Fleming, C.J., A.D. Burden, and A. Forsyth, Accuracy of questions related to allergic contact dermatitis. Am J Contact Dermat, 2000. 11(4): p. 218-21.

164. Ale, S.I. and H.I. Maibach, Reproducibility of patch test results: a concurrent right-versus-left study using TRUE Test. Contact Dermatitis, 2004. 50(5): p. 304-12.

165. Fischer, T. and I. Rystedt, False-positive, follicular and irritant patch test reactions to metal salts. Contact Dermatitis, 1985. 12(2): p. 93-8.

166. Lindemann, M., J. Bohmer, M. Zabel, and H. Grosse-Wilde, ELISpot: a new tool for the detection of nickel sensitization. Clin Exp Allergy, 2003. 33(7): p. 992-8.

167. Minang, J.T., N. Ahlborg, and M. Troye-Blomberg, A simplified ELISpot assay protocol used for detection of human interleukin-4, interleukin-13 and interferon-gamma production in response to the contact allergen nickel. Exogenous Dermatology, 2003. 2: p. 306-313.

168. Minang, J.T., M. Troye-Blomberg, L. Lundeberg, and N. Ahlborg, 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, 2005. 62(3): p. 289-96.

169. Bourke, J.F., K. Batta, L. Prais, A. Abdullah, and I.S. Foulds, The reproducibility of patch tests. Br J Dermatol, 1999. 140(1): p. 102-5.

170. Gollhausen, R., B. Przybilla, and J. Ring, Reproducibility of patch tests. J Am Acad Dermatol, 1989. 21(6): p. 1196-202.

60 Khosro Masjedi

171. Lachapelle, J.M. and J.L. Antoine, Problems raised by the simultaneous reproducibility of positive allergic patch test reactions in man. J Am Acad Dermatol, 1989. 21(4 Pt 2): p. 850-4.

172. Brasch, J., T. Henseler, W. Aberer, G. Bauerle, P.J. Frosch, T. Fuchs, V. Funfstuck, G. Kaiser, G.G. Lischka, B. Pilz, and et al., Reproducibility of patch tests. A multicenter study of synchronous left-versus right-sided patch tests by the German Contact Dermatitis Research Group. J Am Acad Dermatol, 1994. 31(4): p. 584-91.

173. Hindsen, M., M. Bruze, and O.B. Christensen, The significance of previous allergic contact dermatitis for elicitation of delayed hypersensitivity to nickel. Contact Dermatitis, 1997. 37(3): p. 101-6.

174. Hindsen, M., M. Bruze, and O.B. Christensen, Individual variation in nickel patch test reactivity. Am J Contact Dermat, 1999. 10(2): p. 62-7.

175. Jensen, C.D. and K.E. Andersen, Course of contact allergy in consecutive eczema patients patch tested with TRUE Test panels 1 and 2 at least twice over a 12-year period. Contact Dermatitis, 2005. 52(5): p. 242-6.

176. Vanneste, D., P. Martin, and J.M. Lachapelle, Comparative study of the density of particles in suspensions for patch testing. Contact Dermatitis, 1980. 6(3): p. 197-203.

177. Lindelof, B., Regional variations of patch test response in nickel-sensitive patients. Contact Dermatitis, 1992. 26(3): p. 202-3.

178. van Strien, G.A. and M.J. Korstanje, Site variations in patch test responses on the back. Contact Dermatitis, 1994. 31(2): p. 95-6.