Dendritic Cells Tools for Allergen Screening

Transcription

1 Dendritic Cells Tools for Allergen Screening ProefschriftMT.indd :12:11

2 The studies presented in this thesis were carried out at the Department of Dermatology and Pathology of the VU University Medical Center Amsterdam and were embedded within the inter-institutional research initiative V-ICI This publication was financially supported by RoC - BD Biosciences - Schering-Plough Medex Bio Sciences Cosmetics Designed by Léonie Sijtsma - Serendipity ontwerp; Printed by PrintPartners Ipskamp, Enschede, The Netherlands ISBN: ProefschriftMT.indd :12:12

3 VRIJE UNIVERSITEIT Dendritic Cells Tools for Allergen Screening ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. L.M. Bouter, in het openbaar te verdedigen ten overstaan van de promotiecommissie van de faculteit der Geneeskunde op maandag 15 december 2008 om uur in het auditorium van de universiteit, De Boelelaan 1105 door Mascha Jessica Toebak geboren te Puttershoek ProefschriftMT.indd :12:12

4 promotoren: copromotoren: prof.dr. R.J. Scheper prof.dr. D.P. Bruynzeel dr. T. Rustemeyer dr. S. Gibbs ProefschriftMT.indd :12:12

5 After all these implements and texts designed by intellects we re vexed to find evidently there s still so much that hides The Shins, Saint Simon, 2003 Ter nagedachtenis aan mijn moeder ProefschriftMT.indd :12:12

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7 7 Content Chapter I General introduction. Accepted for publication by Contact Dermatitis, 2008 Outline of the thesis Chapter II Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure. Experimental Dermatology, 2005: 14: Chapter III CXCL8 secretion by dendritic cells predicts contact allergens from irritants. Toxicology In Vitro, 2006: 20: Chapter IV Kinomic profiling of MUTZ-3 myeloid cells discriminates between allergens and irritant. Submitted for publication 81 Chapter V Intrinsic characteristics of contact and respiratory allergens determine p roduction of polarising cytokines by dendritic cells. Contact Dermatitis, 2006: 55: Chapter VI Dendritic cells from atopic individuals show predominant type-2-skewing cytokine production when exposed to Der p1 or Grass pollen atopens but not to small molecular weight allergens. Submitted for publication 117 Chapter VII Differential suppression of dendritic cell cytokine production by anti-inflammatory drugs. British Journal of Dermatology 2008: 158: ProefschriftMT.indd :12:12

8 Chapter VIII Summary and discussion 145 Nederlandse samenvatting 157 Curriculum Vitae 163 List of publications 165 Dankwoord 167 ProefschriftMT.indd :12:12

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10 10 Chapter 1 General introduction ProefschriftMT.indd :12:13

11 11 Chapter 1 General introduction ProefschriftMT.indd :12:13

12 12 Chapter 1 General introduction ProefschriftMT.indd :12:13

13 13 General introduction Abstract Allergic contact dermatitis results from a T-cell-mediated, delayed-type hypersensitivity immune response induced by allergens. Skin dendritic cells play a central role in the initiation of allergic skin responses. Following encounter with an allergen, dendritic cells become activated and undergo maturation and differentiate into immunostimulatory dendritic cells, and are able to present antigens effectively to T-cells. The frequency of allergic skin disorders has increased in the last decades. Therefore, the identification of potential sensitizing chemicals is important for skin safety. Traditionally, predictive testing for allergenicity has been conducted in animal models. For regulatory reasons, animal use for sensitisation testing of compounds for cosmetic purposes is shortly to be prohibited in Europe. Therefore, new non-animal based test methods need to be developed. Several dendritic cell-based assays have been described to discriminate allergens from irritants. Unfortunately, current in vitro methods are not sufficiently resilient to identify allergens and, therefore need refinement. Here, we review the immunobiology of skin dendritic cells (Langerhans cells and dermal dendritic cells) and their role in allergic and irritant contact dermatitis and then explore the possible use of dendritic cell-based models for discriminating between allergens and irritants. ProefschriftMT.indd :12:13

14 14 Chapter 1 General introduction Dendritic cells Dendritic cells (DCs) represent a heterogeneous cell population residing in most peripheral tissues, particularly at sites of interphase with the environment, e.g. skin and mucosa. DCs represent 1-3% of the total cell numbers of these tissues 1. DCs patrol through the blood, peripheral tissues, lymph and secondary lymphoid organs (Fig. 1.1). In the periphery, immature DCs (idcs) can capture and process antigens. Thereafter, they migrate towards T-cell rich areas of the secondary lymphoid organs via the afferent lymphatics. During this migration, DCs lose their capacity to internalize further antigens and acquire the capacity to present antigens to naïve T-cells, a process referred to as maturation of DCs. Antigens are presented in such a way that antigen-specific naïve T-cells get activated and start to proliferate ( T-cell priming ). Moreover, when DCs initiate a T-cell-mediated adaptive immune response, they also play an important role in polarisation of T-cell reactivity towards type-1 and/or type-2 responses. Fig The life cycle of dendritic cells. Circulating precursor DCs enter tissue as idcs upon uptake of antigens (e.g. bacteria, viruses), DCs induce secretion of cytokines and chemokines, which in turn can attract and activate eosinophils, macrophages and NK cells at the site of antigen entry. After antigen capture, idcs migrate to secondary lymphoid organs. DCs present peptide-major histocompatibility complexes (MHC), which allow selection of antigen-specific lymphocytes. These lymphocytes help DCs in final maturation, which allows T-cell expansion and differentation. Activated antigen-specific T-cells migrate to the injured tissue. T-helper cells secrete cytokines which permit activation of macrophages, eosinophils and NK-cells. Cytotoxic T-cells (CTLs) eventually lyse these cells. Inparallel, B- cells become activated after contact with T-cells and DCs and migrate to specialised areas where they mature into plasma cells. Plasma cells produce antibodies that neutrolise the initial antigen. Finally, after interaction with lymphocytes DCs die by apoptosis. ProefschriftMT.indd :12:15

15 15 Table 1.1: Characteristics of Langerhans cells and dermal dendritic cells Marker LC DDC Remark Reference Adhesion molecule CD11a +/- + Up-regulated upon migration 9 CD11b - + Up-regulated upon migration 10;11 CD11c + + Up-regulated upon migration 11;12 CD54 +/- + Up-regulated upon migration 9;12 CD58 +/- + Up-regulated upon migration 9;12 CD324 E-cadherin ; 14 CLA + +/- 15 Antigen presentation MHC-class I MHC-class II CD1a + +/- defines two subsets 17 CD1b ;18 CD1c +/- + 11;18 CD1d CD Birbeck granule Chemokine receptor CCR2 - + Down-regulated upon migration 22 CCR6 + + Down-regulated upon migration 22 CCR7 - - Up-regulated upon migration 22 CXCR1/2 - - Up-regulated upon migration 6;11 CXCR4 - nd Up-regulated upon migration 23 Cytokine receptor IFN-gamma R + + 6;11 IL-1R ;11 IL-1R ;11 IL-2Rα - + 6;9;11 IL-2Rβ - - 6;11 IL-2Rγ nd + 6;11 IL-3R nd + 6;11 IL-4R - + 6;11 IL-5R nd +/- 6;11 IL-6R +/- + 6;11 IL-7R - + 6;11 G-CSFR - - 6;11 GM-CSFRα + + 6;11 GM-CSFRβ +/- +/- 6;11 M-CSFR - - 6;11 TNFR MW - - 6;11 TNFR MW + + 6;11 ProefschriftMT.indd :12:15

16 16 Marker LC DDC Remark Reference Chapter 1 General introduction Fc receptor CD16 FcγRIII CD32 FcγRII + + 9;11 CD64 FcγRI Other CD14 - +/- defines two subsets 9 CD36 - +/- 9 CD205 DEC205 +/- + Up-regulated upon maturation 24 CD206 MMR CD207 Langerin + - Down-regulated upon maturation 26 CD208 DC-LAMP - - Up-regulated upon maturation 24 CD209 DC-SIGN ;27 Factor XIII nd: not described, -: negative, +/-: only a percentage positive, + positive. Cutaneous dendritic cells DCs in the epidermis and dermis participate in the recognition of invading pathogens. These cells are professional antigen presenting cells (APCs) and play a key role in sensing danger and initiating innate and adaptive responses. The two main populations of DCs present in normal human skin are epidermal Langerhans cells (LCs) and dermal dendritic cells (DDCs) (Table 1.1). Langerhans cells Paul Langerhans, as a medical student, identified DCs residing in the epidermis. These LCs represent approximately 3% of epidermal cells and form a constituent part of the skin immune system. They are found regularly spaced throughout the epidermis 2. LCs are typically characterised by the expression of CD1a and a unique cytoplasmic organelle named the Birbeck granule (BG). BGs constitute a sub domain of the endosomal-recycling compartment, perhaps being involved in antigen loading processing. The C-type lectin langerin (Lag, CD207) is responsible for BG formation and thus a key marker of the LC lineage 3. LC originate from bone marrow derived progenitors, as indicated by their CD45 expression 4. However, the process of LC differentiation and their migration into the epidermis is not clearly understood. It has been postulated that bone-marrow-derived myeloid cutaneous lymphocyte-associated antigen (CLA)-expressing LC precursors travel via peripheral blood through the dermis into the epidermis 5. Other studies suggested that skin-resident LC precursors exist in the dermis under normal non-inflammatory conditions. These LC precursors co-express Langerin and CD14 and their final migration into the suprabasal layer of the epidermis is most probably controlled by keratinocyte-derived CXCL14 6;7. Additionally, it is suggested that a combination of TGF-β1 and CCL20 is required for the attraction of human ProefschriftMT.indd :12:16

17 17 LC precursors through the dermal-epidermal barrier 8. The final differentiation of LC precursors depends on the cytokine environment of the epidermis 7. The cutaneous cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-15 and transforming growth factor (TGF)-β1 contribute to the establishment of immature LCs in the epidermis. During inflammation, however, circulating precursors might have an important role in replenishing the local pool of LCs 5. In vitro, LCs can be cultured from cord blood or bone-marrow-derived cells 31,32. These CD34 + -haemopoietic progenitors acquire LC features upon differentiation with a combination of cytokines, e.g. TGF-β1, GM-CSF and TNF-α. In addition, several blood-derived cell types can be differentiated into LC-like cells as depicted in Table 1.2. Undoubtedly, the presence of TGF-β1 in the cytokine milieu is pivotal in the differentiation to a LC phenotype. In the absence of TGF-β1, in vitro cultured LCs lack the development of Birbeck granules, whereas the other DC characteristics, e.g. CD1a and langerin expression, can still be presented. Table 1.2: Culture methods for human peripheral blood-derived LCs Characteristics progenitor cells Cytokine cocktail Reference CD34+ CLA+ cells GM-CSF, TNF-α 32 CD1a+ CD11c+ CD14- cells GM-CSF, TGF-β1, IL-4 33 CD14+ cells GM-CSF, TGF-β1, IL-4 34 CD14+ cells GM-CSF, TGF-β1, IL CD14+ cells GM-CSF, TGF-β1, IL Dermal dendritic cells The dermis consists of a large variety of cell types, including fibroblasts, macrophages, mast cells, T-cells and DDCs. These latter APCs are located at the level of the capillaries and in the higher part of the reticular human dermis. Two major subsets of DDCs are present in the dermis: CD1a high /CD4 + /CD14-/CD16-/CD206 high /CD207-/CD209 + ) and CD1a low /CD4 + /CD14 + / CD16-/CD206 low /CD207-/CD209 + cells 27. It has been suggested that the latter DC subset shows weaker allogeneic stimulatory capacities compared to the CD1a high subset 9. Some investigators claim that the CD1a low subset can be defined as macrophages rather than dendritic cells 36. Expression of CD36, CD209 and the coagulation factor XIII (FXIIIa) has been suggested to be specific markers for both populations of DDCs. However, macrophages also express these markers 27;37. Therefore, macrophages and DDCs may be hard to distinguish. In vitro, DDC can be generated from CD34 + -haemopoietic progenitor cells or CD14 + circulating monocytes with cytokine cocktails 38;39. All combinations of either IL-3, TNF-α, IL-13 or IL-4 together with GM-CSF stimulate the development of DDCs As is to be expected, the ProefschriftMT.indd :12:16

18 18 Chapter 1 General introduction presence of TGF-β1 in the cytokine milieu appears to inhibit DDC development and promote the differentiation of DC precursors to LCs. Dendritic cell migration Emigration of DCs from the skin is one of the first steps in the initiation phase of an immune response (Fig. 1.2). Skin contact with antigens is known to stimulate various epidermal cytokines, e.g. IL-6, TNF-α, GM-CSF and CXCL2/CXCL3 (MIP-2), among which IL-1β and TNF-α are essential in DC migration Fig Dendritic cell migration. Skin contact with a hapten triggers migration of dendritic cells via the afferent lymphatic vessels to the skin-draining lymph nodes. Production of cytokines particularly TNF-α, IL-1α and IL-1β facilitates migration of LCs out of the epidermis. At later stage, CCR7-CCL21 interaction plays a pivotal role in the migration of LCs into the T-cell areas of lymph nodes. Disentanglement of skin DC Cytokine signalling results in altered expression of adhesion molecules, which facilitates DC emigration out of the epidermis. The mobilisation of DCs is associated with loss of CD324 (E-cadherin), caused by TNF-α, IL-1β or IL-1α production 44;45. In brief, it is proposed that LCderived IL-1β performs two functions. Firstly, DC-derived IL-1β activates DCs in an autocrine loop via the IL-1 receptor 1 (IL-1RI). Secondly, IL-1β also stimulates epidermal keratinocytes (KCs) to secrete TNF-α, which acts in a paracrine manner to facilitate DC migration via TNF-RII 46. If the availability of either of these cytokines is compromised then allergen-lc ProefschriftMT.indd :12:24

19 19 migration and DC mobilisation in draining lymph nodes and the development of contact sensitisation are inhibited partially 47;48. Simultaneously, production of epidermal basement membrane degrading enzymes, such as matrix metalloproteinases (MMPs), is up-regulated in activated LCs 49. The MMP-2, MMP-3 and MMP-9 have been described to facilitate in the passage of LCs across the basement membrane. Subsequently, MMPs are also essential for migration of LCs and DDCs through the dermal tissue matrix, by cleavage of type IV collagen 50. The activity of MMP-2 and MMP-9 is under the control of naturally occurring proteinase inhibitors such as tissue inhibitor metalloproteinase (TIMP)-1 and TIMP-2, respectively. Several studies have demonstrated that inhibitors of MMPs prevented the emigration of LCs from the epidermis and DC accumulation to the afferent lymph node in the response to the epicutaneous application to sensitisers, such as nickel, oxazolone (OXA) and dinitrochlorobenzene (DNCB) These findings suggest that the development of contact hypersensitivity could be prevented by inhibition of the MMP activity. However, the development of contact hypersensitivity was not impaired in MMP-9-deficient mice, although LC migration was dramatically inhibited in these gene-deficient mice 50. Apparently, the few DDCs and LCs that still migrate in the absence of MMP-9 successfully induce contact hypersensitivity. In addition, contact hypersensitivity might be induced by the free diffusion of the contact allergen into the afferent lymph nodes where it can be presented by local DCs 53. Meanwhile, differential expression of adhesion molecules is induced to mediate interactions with the extracellular matrix, epidermal and dermal cells. Among the most important events is the reduced expression of CD324, which allows LCs to dissociate from surrounding keratinocytes 54. Additional roles are thought for CD 54 (intracellular adhesion molecule, I-CAM-1) and LFA-1, since blockage of these adhesion molecules inhibited migration of epidermal DCs to regional lymph nodes. The up-regulated expression of α6- and β1-integrin comprises very late antigen 6 (VLA-6) that confers on LC laminin-binding activity and thereby the ability to interact with basement membrane. Another adhesion molecule, junctional adhesion molecule 1 (JAM-1), which is expressed by DCs and therefore affects DC migration 55;56. Absence of JAM-1 facilitates DC trafficking to lymph nodes 57. Combined, these events result in disentanglement of LCs from surrounding KCs, thus allowing LC m i g r a t i o n. Chemokines and chemokine receptors LC migration is also associated with the expression of chemokine receptors on the surface of the cells. Immature DCs express chemokine receptors and these can guide them to inflammatory sites where antigen sampling occurs. Upon antigen sampling, DCs rapidly differentiate into a mature phenotype. This maturation process is associated with a switch in chemokine receptor profile. Skin homing chemokine receptors are down-regulated (CCR1, CCR2, CCR5 and CCR6), whereas receptors involved in homing to the local lymph node are up-regulated (CCR4, CXCR4 and CCR7) 58. Importantly, the mobilisation of DC from the periphery to the afferent lymph nodes is regulated by the gate-keeper chemokine receptor CCR7 31;59. Both lymphatic and high endothelial cells (HEVs) produce CCL21, a major CCR7 ProefschriftMT.indd :12:24

20 20 Chapter 1 General introduction ligand 60;61. Residing DCs in the paracortical areas of the lymph nodes produce CCL19, a second ligand of CCR7. Next to antigen-bearing LCs also naïve T-cells express CCR7. This orchestrates the encounter of DCs and naïve T-cells in the paracortical lymph node areas. The lipid mediators, cysteinyl leukotrienes (cyslts) and prostaglandin E 2 (PGE 2 ), all produced at sites of inflammation, are also required for LC migration 62;63. These lipid mediators promote, via up regulation of CCR7, optimal DC chemotaxis to the chemokine CCL19, but not to CCL21. In addition, the cysteinyl leukotriene transporter multi-drug resistance protein (MRP)-1 and lipid transporter p-glycoprotein (multi drug resistance, MDR-1) are also expressed by DCs and are necessary for their entry into afferent lymphatics 64;65. Negative regulation of DC migration In mice, regardless of the nature of the stimulus (e.g. skin contact to allergens, pathogens or trauma), only up to 30% of epidermal LCs leave the tissue 66;67. Possibly, these residential LCs have the function to guard the skin for subsequent exposures. LC retention might be caused by the rapid onset of the production of negative regulators of LC migration such as epidermal derived IL-4, IL-10, and TGF-β1. The former cytokine inhibits the migration of human LCs through down regulation of TNFRII expression 67. The two latter cytokines induce the expression of CCR6, also facilitating the retention of LCs in the epidermis 8. During cutaneous inflammation, IL-10 provides homeostatic counterbalance for IL-1β and TNF-α 42. TGF-β1 inhibits DC migration through inhibition of CCR7 expression and upregulation of CD324 expression 68. Differences in LC and DDC migration In mice, LCs and DDCs show different migratory capacities upon epicutaneous exposure to antigen sensitisers. Mouse studies showed that upon exposure of the skin to the fluorescent dye Tetramethyl Rhodamine Iso-Thiocyanate (TRITC), DDC migration clearly preceded LC migration 69. This might be due to the fact that LC take longer to reach afferent lymph nodes because they first need to detach from neighbouring KCs. Interestingly, within the T-cell zone DDCs are located in the outer paracortex just beneath the B-cell follicles, whereas LCs are located in the inner T-cell rich paracortex 69. These findings suggest that DDCs are involved in B-cell priming, whereas LCs are involved in T-cell activation. The differential migration capacity and lymph node destination between LCs and DDCs might result from a distinct chemokine or lipid mediator receptor expression on the LCs and DDCs 70. Recently, it was suggested that CCR7 expression plays an important role in this controversy. In vitro generated DDCs acquire a low CCR7 expression, whereas LCs acquire a high level of CCR7 expression 35. This finding might explain the migration of LCs, and not DDCs, to T-cell zones within the lymph node. Dendritic cell maturation DCs located in peripheral tissues are continuously monitoring their environment 71. Tissue injury, microbial and other changes of homeostasis provide danger signals, which activate ProefschriftMT.indd :12:25

21 21 DCs and trigger their transition from immature antigen-capturing cells to mature antigenpresenting DCs. Maturation process Once in contact with antigens, idcs use a broad range of pathways to facilitate antigen uptake. Many of these pathways, e.g. via α v β 5 -integrins or CD36, may also be used for uptake of self-antigens 72. Following antigen uptake, maturation of DCs is associated with several coordinated events: loss of endocytic/phagocytic receptors, up-regulation of costimulatory molecules (e.g. CD80 and CD86), change in morphology and up-regulation of class-ii MHC molecules 73. Functionally matured DCs accumulate in T-cell rich areas of lymphoid tissues. Within the paracortical areas, conditions are optimal for antigen bearing mature DCs (mdcs) to encounter naïve T-cells that specifically recognise the antigen-mhc molecule complexes. The dendritic morphology of these APCs strongly facilitates multiple cell contacts, leading to binding and activation of antigen-specific T-cells. Antigen-specific T-cells now expand abundantly and generate effector and memory T-cells, which are released via the efferent lymphatics into the circulation. In conclusion, a variety of danger signals induce DC maturation, ultimately resulting in generation of antigen-specific T-cells which in turn facilitates immunity. Intracellular pathways DC maturation can be initiated in vitro by inflammatory stimuli, such as proinflammatory cytokines (e.g. TNF-α, IL-1β), lipopolysaccharide (LPS), CD40 ligation and contact allergens 74;75. These maturation stimuli induce phosphorylation of mitogen-activated protein kinases (MAPKs), such as extracellar signal-regulated kinases (ERKs), c-jun N-terminal kinases (JNKs), and p38 MAPK. These three MAPK signalling-pathways have distinct roles in the maturation process. Stress stimuli and inflammatory cytokines can strongly activate the JNK and p38 MAPK pathways, whereas the ERK pathway is strongly activated by growth factors through receptors for tyrosine kinases 76. In the maturation process of DCs, the expression of surface molecules such as CD40, CD80, CD83, CD86 and MHCII and production of inflammatory cytokines is up-regulated. Upregulation of these membrane molecules can be prevented by specific kinase inhibitors, such as p38 MAPK inhibitor (SB203580) 77. Stimulators of DC maturation In vivo, DCs may encounter many different pathogens, for example prokaryote-derived lipoproteins, flagellin, CpG and LPS 78. Immature DCs express a large array of receptors that can specifically recognise specific motives of pathogens; pathogen associated molecular patterns (PAMPs). These PAMPs are ligands for Toll-like receptors (TLRs) on antigen presenting cells. Basically, bacterial components can be recognised by DCs via TLR1, TLR2, TLR4, TLR5 and TLR6, and viral RNA via TLR3 and TLR7 79;80. In addition, idc express C-type lectins, like CD209, recognising carbohydrate structures on pathogens 23. Contact sensitisers may activate the innate immune system in a similar way as PAMPs of bacteria and viruses. Like PAMPs, contact sensitisers can induce DC activation by signalling ProefschriftMT.indd :12:25

22 22 Chapter 1 General introduction cascades, upregulation of costimulatory molecules and cytokine production 81;82. p38 MAPK is considered to be a critical player in the initiation of contact hypersensitivity. In vitro exposure of DCs to the allergens DNCB and nickel sulphate (NiSO 4 ), leads to activation and translocation of p38 MAPK and ultimately to the up-regulation of CD80, CD83 and CD86 77;83;84. However, it is unclear whether or not TLRs are involved in allergen-induced DC activation. In contrast to inducing immune responses against invading pathogens, skin DCs can also play a role in induction of tolerance. By expression of inducible co-stimulatory molecule ligand (ICOS-L; B7-H2) or the immunoregulatory enzyme indoleamine 2,3-dioxygenase, LCs are capable of maintaining a state of tolerance 85. In the absence of the appropriate co-stimulatory co-stimulatory signals, naïve T-cells may be anergized, frequently associated with T-cell receptor (TCR)/CD4 or CD8 downregulation 86. T-cells may turn into an unresponsive state and might also eventually die via apoptosis. Dendritic cell polarisation Once DCs enter the regional lymph node, T-cells that possess TCRs complementary to the MHC molecule-antigen complex on the surface of the LC can become activated. The site of interaction between the APCs and the naïve T-cell is called the immunological synapse and sufficient stimulation by a set of three distinct groups of signals is pivotal for T-cell activation 87. Signal 1 is provided by interaction between the peptide-bearing MHC molecule on the DC and the TCR on the naïve T-cell ( antigen-specific signal) 88. Signal 2 consists of contact-dependent interactions, of which the co-stimulatory molecules CD80 or CD86 expressed on DC interact with CD28 expressed on T-cells this is the most potent signal ( co-stimulatory signal) 89. In addition, interactions of CD54 and CD102 (ICAM-2) and CD252 (OX40 ligand) with their T-cell counterparts can contribute to signal The combination of signal 1 and 2 results in antigen-specific activation and proliferation of naïve T-cells and the development of effector and/of memory T-cells. Additional factors (signal 3) are needed for polarisation of T-cells towards type-1 or type-2 helper T cells (for CD4 + T-cells) and type-1 or type-2 cytotoxic T cells (for CD8 + T-cells) 91. Type-1 T-cells produce high levels of interferon (IFN)-γ, TNF-α and TNF-β. These cytokines are important in the induction of cell-mediated immunity against intracellular pathogens. Type-2 T-cells produce high levels of IL-4, IL-5 and IL-13. These cytokines are important in production of antibodies by B-cells. Next to type-1 and type-2 T-cells a less-polarised T-cell type (type-0) can develop which secretes a broad range of cytokines including IL-2, IFN-γ, IL-4 and IL-10. It has been suggested that during the first antigen contact the majority of emerging effector/memory T-cells belong to this group of less polarised cells. Upon repeated antigen stimulation they can be further polarised into the more pronounced type-1 or type-2 cytokine secreting cell populations 92. Polarisation of DC DCs play an important role in the polarisation of T-cells (Fig. 1.3). Type-1 DC-derived cytokines strongly stimulate IFN-γ production in naïve T-cells and thereby promote type-1 ProefschriftMT.indd :12:25

23 23 T cell responses 93. Among the type-1 T-cell polarising cytokines of DCs, IL-12 is the most important member of the IL-12 cytokine family, which includes IL-12, IL-18, IL-23 and IL-27 94;95. Immature DCs are imprinted to secrete IL-12 upon stimulation by various pathogen types (e.g. bacteria, viruses, protozoa). After migration to the draining lymph nodes, these pathogen-exposed DCs will produce even higher levels of IL-12 upon CD40-CD40L interactions 96. Mature DCs express the membrane molecule CD40 which responds to their counter structure CD40L on naïve T-cells to stimulate type-1 T-cell proliferation. In addition to an IL-12 conditioned cytokine environment, also intracellular interactions between CD54 and CD11a can promote the induction of type-1 T-cell responses 98. a a Il-1b Fig Dendritic cell polarisation. Schematic representation of factors that influence dendritic cell polarisation and ultimately polarisation of naïve T-cells as proposed by De Jong et al. (2005) 98. Type-2 DC-derived cytokines potently stimulate IL-4, IL-5 and IL-13 production in naïve T-cells and thereby promote generation of type-2 T-cells 93. Whereas type-1 T-cell-inducing factors are well-studied, little is known about mediators promoting type-2 T-cell responses. It has been suggested that type-2 T-cell polarisation is only induced in the absence of type-1 DC cytokines 99. DC- or T-cell-derived IL-10 is able to down-regulate DC-derived IL-12 production, facilitating type-2 responses 100. As to specific type-2 polarising surface interactions, presence of the co-stimulatory molecules CD275 (ICOSL), CD252 (OX40L), Notch-ligand Jagged on DCs, all have been implicated in this regard 101;102. ProefschriftMT.indd :12:27

24 24 Chapter 1 General introduction Type-0 DC /Regulatory idc DCs can not only induce immune activation, but also control the level of activation by regulatory loops. Thus, next to type-1 and type-2 polarisation, DCs also play a role in the induction of peripheral tolerance. Two subsets of regulatory T-cells (Tr) are described: naturally occurring Tr and adaptive Tr 103. In contrast to natural Tr, adaptive Tr originate from uncommitted peripheral naïve or central memory T-cells. Via the selective induction of adaptive Tr 104, regulatory idcs can induce peripheral tolerance after repetitive stimulation of T-cells in the absence of sufficient co-stimulatory signals 105. In vitro, regulatory idcs can be induced after culturing idcs with the anti-inflammatory cytokines TGF-β1, IL-10 or anti-inflammatory drugs like corticosteroids 104;105. These regulatory idcs express several factors, e.g. CD274 (PD-L1), CD273 (PD-L2) and can secrete high levels of IL-10, as a negative signal for T-cell responses 108;109. Inducers of DC polarisation Different compounds have been described as typical inducers of type-1 or type-2 immune responses in DCs. Certain viruses, (myco)bacteria and protozoa will induce a type-1 immune response, whereas helmints can induce a type-2 response 98. For example, the synthetic double-stranded RNA, polyriboinosinic polyribocytidylic acid (poly I:C) is known for its type-1 polarisation. Direct exposure of DCs with poly I:C results in type-1 DCs which subsequently trigger type-1 T-cell responses 101. As to skin sensitisation, it has been reported that different allergens can also provoke qualitatively distinct immune responses 110. Topical exposure of contact allergens (e.g. DNCB) in rodents will induce a prototypical type-1 response in allergen activated lymph node cells, with high levels of type-1 and low levels of type-2 cytokines. In contrast, skin contact with respiratory allergens (e.g. trimellitic anhydride, TMA) results in the opposite. However, to what extent this translates to the human situation is largely unclear. Role of microenvironment on DC polarisation The plasticity of DCs to act as type-0, type-1 or type-2 polarising APCs is influenced by many factors, e.g. by epithelial cells (route of entry of the antigen), antigen dose, molecular nature of the antigen. By receiving danger signals from antigen-affected cells and tissues, DCs can also indirectly obtain information about the molecular identity of the antigen. Considering the fact that DC reside in close proximity of epithelial cells, such as skin KCs or airway epithelial cells, these cells most likely influence and enhance the local polarisation of type-1 or type-2 APCs, respectively. As mentioned above, direct exposure of DCs with the synthetic double-stranded RNA poly I:C results in type-1 DCs which subsequently trigger type-1 T-cell responses 101. Indirectly, poly I:C can also modulate DC function via interactions with KCs, the epithelial cells of the skin 111. In that study, keratinocyte-derived type-1 IFNs and IL-18 were identified as soluble factors driving the type-1 phenotype of DCs and T-cells. On the other hand, exposure of keratinocytes to a well-known non-polarising cytokine cocktail, IL-1β and TNF-α, did not result in type-1 IFNs and IL-18 production and subsequently induced both type-1 and type-2 ProefschriftMT.indd :12:27

25 25 immunity. Apparently, keratinocyte-derived factors can modulate DC functions. In contrast to type-1 skewing cytokines, thymic stromal lymphopoietin (TSLP) and PGE 2 are important tissue-derived inducers of type-2 immunity 112;113. Under homeostatic conditions, TSLP is produced in epithelia of mucosal tissues and in the thymus 114;115. TLSP can also be found in atopic dermatitis skin or in bronchial epithelium and submucosa in allergic asthma 116. Recently, it has been proposed that normal levels of TSLP in mucosa are important for the maintenance of tolerance-inducing type-2 T-cells, whereas high levels of TSLP result in the development of pro-inflammatory type-2 T-cells as found in allergic astma 117;118. As to skin exposure to contact allergens, keratinocyte-derived IL-18 is involved in the cutaneous type-1 immune response 112. Exposure of human keratinocytes to the contact sensitiser DNCB results in the secretion of IL-18 which subsequently triggers type-1 T-cell responses. In contrast, after mucosal contact with contact allergens, type-2 T-cell responses are most prominent. In the mucosal cytokine environment, DCs release only small quantities of IL-12, whereas IL-4 and IL-6 production of e.g. macrophages and NK cells is relatively high, favouring type-2 responses 98. As described earlier, the skin contains a network of CD1a + DCs, which are responsible for defence against pathogens. Compared to the skin, the situation of the gut appears to be more complex. Mucosal DCs allow on one hand the continuous uptake of nutrients and fluids through the epithelial layers, whereas on the other hand mucosal DCs should prevent entry of harmful pathogens. In response to commensal bacterial signals, murine DCs produce mainly anti-inflammatory cytokines like IL-6, IL-10 and TGF-β, prime T-cells towards a type-2, regulatory T-cells response, and induce B-cell to produce IgA 119. In contrast, pathogenic bacterial signals induce production of inflammatory cytokines and chemokines, prime type-1 T-cells and induce IgG and IgA production by B-cells 120. ProefschriftMT.indd :12:28

26 26 Chapter 1 General introduction The role of DCs in allergic and irritant contact dermatitis Allergic contact dermatitis (ACD) is a result of a T-cell-mediated inflammatory reaction occurring in sensitised individuals upon allergen challenge (Fig. 1.4). In the sensitisation phase, allergens that have penetrated into the skin are internalised by skin DCs which subsequently migrate to the draining lymph nodes and present the hapten-peptide-mhc complexes to naïve allergen-specific T-cells. This process results in clonal expansion of specific T-cells, which subsequently can be recruited, from the circulation into the skin. Re-exposure to the relevant allergen can initiate the elicitation phase and thereby the clinical manifestation of the disease. As a consequence of the release of cytokines and chemokines, circulating allergen-specific T-cells and APCs meet at the site of allergen challenge. At this stage, next to DCs also non-professional APCs such as KCs present the allergen to T-cells. This results in the release of pro-inflammatory cytokines and chemokines of epidermal cells. These mediators attract more T-cells, mainly allergen-non-specific, and other inflammatory cells, which further amplify the local mediator release. Finally, the skin site clinically develops an eczematous reaction. After several days to weeks, allergen removal and down-regulatory factors (e.g. IL-10, PGE 2, TGF-β) are locally released, which results in clinically healed skin. Fig Allergic contact dermatitis. Epidermal LCs reside in normal skin in a functional resting state, characterised by high antigen-processing capacity. Topical allergen application triggers migration of LCs via the afferent lymphatics towards regional skin draining lymph nodes. In the T-cell rich paracortical area of the lymph node, LCs encounter naïve T-cells and activate these via interaction between MHC-peptide complex and TCR. Hapten-specific T-cells expand and generate effector or memory function and will go into the circulation. Upon re-exposure to the allergen, epidermal cells release pro-inflammatory cytokines and chemokines, which will recruit hapten-specific T-cells. Infiltrating ProefschriftMT.indd :12:29

27 27 T-cells release inflammatory cytokines which stimulate KCs to produce chemokines amplifying cellular infiltration. These infiltrating cells migrate into dermal and epidermal compartments. Ultimately, inflammation characterised by e.g. oedema and spongiosis, reaches its maximum at hours after allergen exposure. Allergic contact dermatitis: sensitisation phase Sensitisation is critically dependent on association of allergens with epidermal LC-bound MHC molecules, or peptides present in the groove of these molecules. Formation of hapten-protein complexes Allergens are typically chemically reactive, low molecular weight compounds that are unable themselves to stimulate directly the immune response 121. Some allergens are reactive, but the vast majority of allergens interact with soluble or cell-associated skin proteins to form a covalent binding and to become immunogenic. Contact allergens that have to be converted before becoming reactive are referred to as pro-haptens. This binding is dependent on their chemical reactivity. Many chemicals have electrophilic properties and are able to react with various nucleophiles within the skin. The human skin contains a very large array of biological structures, e.g. hydroxyl, amino and thiol functionalities on proteins, containing electron-rich groups. As electrophiles, contact allergens are capable of reacting with these electron-rich groups to form a covalent and therefore irreversible bond. For example, the hapten trinitrophenyl (TNP) binds covalently to the ε-amino group of the side chain of lysine residues 122. For sesquiterpene lactones, binding to thiol groups is needed to become a hapten 123. It has been suggested that typically 30-40% of sensitising potential of allergens is associated with their electrophilicity 124. However, covalent binding is not necessary for all sensitisers. Metals react with skin proteins and form coordinate bonds. For nickel, different modes of coordinative binding to MHC and/or MHC bound peptides and the TCR have been shown to be relevant in the activation of Ni-specific T-cells 125. Recently, a large number of heat shock proteins and chaperones were described as Ni-interacting proteins 126. Other chemicals may directly connect MHC and TCR without binding 127. Ultimately, each approach results in the activation of chemical-reactive T-cells that can cause ACD. The hapten-carrier protein complexes form the complete antigen and are processed by LCs and DDCs 128. Both MHC class I and II may be altered in this way, and thereby both CD8 + and CD4 + T-cells, respectively, can be involved in ACD. Lipophilic haptens, e.g. DNCB, can directly penetrate into the LCs, conjugate with cytoplasmic proteins and be processed along the endogenous processing route, which results in MHC class I presentation. In contrast, hydrophilic allergens such as nickel ions may rather be presented via the exogenous processing route and thus favour the generation of MHC class II molecules 129. Hapten-induced activation and migration of dendritic cells Cutaneous DCs continuously migrate out of the skin at a low rate, which is dramatically increased after allergen exposure. The emigration of skin DCs is one of the first events in ACD and is regulated by various factors. Within fifteen minutes after allergen painting, ProefschriftMT.indd :12:30

28 28 Chapter 1 General introduction LCs start to produce and express IL-1β 42. As discussed before, IL-1β stimulates the production of the cytokines TNF-α and GM-CSF and differential expression of adhesion molecules and MMPs, which facilitates the migration of LCs. Following encounter with an allergen, LCs become activated and undergo maturation and differentiate from antigen-capture and processing cells into potent immunostimulatory DCs, able to present antigen effectively to effector T-cells. Among the changes reported in LCs as a result of exposure to the chemical allergens are internalisation of surface MHC class II molecules via endocytosis, induction of tyrosine phosphorylation, the modulation of the co-stimulatory molecules CD80 and CD86 and increased expression of IL-1β 42;130. During this maturation the shape of mdcs changes from a round to a more ruffling appearance. This maximizes the chance to encounter with naïve T-cells in the local lymph nodes and results in optimal antigen presentation. Allergic contact dermatitis: elicitation phase Allergen re-exposure at former challenged site The effector phase of ACD is initiated upon reexposure to a contact allergen. Allergen reexposure results in production of pro-inflammatory cytokines by epidermal cells. After 4-8 hrs, hapten-specific T-cells are recruited by inflammatory cytokines and chemokines. Infiltrating T-cells release inflammatory cytokines, such as IFN-γ and IL-4, which stimulate keratinocytes to produce chemokines (CXCL9, CXCL10, and CXCL11). This in turn results in the attraction of CXCR3 + T-cells 131. In humans, approximately 70% of the infiltrating CD4 + and CD8 + T cells express the receptor CXCR Most of these co-express the cutaneous lymphocyte antigen CLA 132;133. Subsequently infiltrating cells, including monocytes, DCs and non-antigen-specific T-cells migrate into dermal and epidermal compartments of the skin. These activated cells are an additional source of chemokines and cytokines, which further amplifying the inflammatory response. CCL5 and CCL22 appear after 12 hrs concomitantly with the infiltration of mononuclear cells into the dermis and epidermis 134. The expression of CXCL9, CXCL10, CXCL11, CCL17, CCL18, CCL21 expression begins at 12 hrs and peaks at 72 hrs, paralleling the strong infiltration of lymphocytes 135. Ultimately, inflammation reaches its maximum, characterised by (epi)dermal infiltrates, oedema and spongiosis. In conclusion, the variety of chemokines expressed during ACD determines a robust and rapid recruitment of leukocytes into the skin. The inflammatory infiltrate is composed mainly of CD4 + and CD8 + T-cells, monocytes and DCs, with an early and transient presence of neutrophils. Murine contact hypersensitivity mainly correlates with the activity of hapten-specific CD8 + T-cells, exerting their effector function through cytotoxic activity as well as the release of cytokines. Whereas allergenspecific CD8 + T-cells may contribute to local release of cytokines, allergen-specific CD4 + T- cells are major sources of pro-inflammatory cytokines in human allergic contact dermatitis. CD4 + T-cells play a more complex role, with both type-1 and type-2 subsets contributing to disease expression, and regulatory T-cells primarily involved in the down regulation of the clinical manifestation of the disease 136. ProefschriftMT.indd :12:30

29 29 Down-regulation of inflammation Gradually, the inflammatory reaction decreases and inflammation disappears. This down regulatory process is mediated by secretion of anti-inflammatory mediators, such as IL-10, TGF-β and PGE 2 and cell-cell contact dependent mechanisms. In these processes, adaptive and naturally occurring regulatory T-cells play a central role 137. Firstly, the adaptive allergen-reactive T regulatory-1 (Tr 1 ) T-cells block, in an IL-10 dependent manner, the maturation of DCs and thereby inhibit IL-12 release, thus impairing their capacity to activate type-1 effector T-cells 138. During the late phase of ACD, CCL1, ligand for CCR8, is produced by keratinocytes and activated T-cells 139. Subsequently, CCR8 + - bearing Tr 1 cells are attracted to the site of allergen contact. Secondly, another group of regulatory T-cells prevent T-cell responses in a cell-cell contact-dependent manner. These naturally occurring CD4 + CD25 + regulatory T-cells are anergic and represent approximately 10 percent of CD4 + T-cells in peripheral blood 140. Suppression of both CD4 + and CD8 + T-cells reactivity may occur via the regulatory markers CTLA-4 and glucocorticoid-induced tumour necrosis factor receptor (GITR) 141. Next to T-cells, keratinocytes and macrophages are also capable of producing IL In addition, keratinocytes also release the immunosuppressive cytokines TGF-β and PGE 2, thus contributing to the down regulation of ACD 143;144. TGF-β silences activated T-cells and inhibits further infiltration by down-regulating the expression of adhesion molecules of both endothelial cells and skin cells 91. PGE 2 inhibits the production of pro-inflammatory cytokines, thus contributing to dampening of the skin immune system. In resting skin, few skin-homing T-cells are present. The so-called local skin memory at former contact eczematous sites can be explained by the persistent production of CCL27 by local keratinocytes. It has been shown that the interaction of CCL27 with its receptor CCR10 is critical in the retention of CD4 + T-cells but not CD8 + T-cells at previously challenged ACD sites 145. The persistence of CD4 + CCR10 + T-cells seems to be crucial for local skin memory, as in vitro results show that these cells, and not CD8 + T-cells from allergic patients show proliferative responses and produce cytokines in response to allergen 145. Immune tolerance One of the mechanism by which specific suppression can be mediated is by induction of immune tolerance. Oral ingestion of antigen might modify systemic immune responses by inducing a state of hypo-responsiveness or oral tolerance. For example, individuals having had oral contacts with nickel releasing dental braces prior to ear piercing, showed a reduced frequency of nickel hypersensitivity, whereas hypersensitivity to other potential allergens were unaffected 146. Individuals who had dental braces after ear piercing did not show a decreased frequency but a slight increase of nickel ACD. These data show that oral administration of antigen prior to potential sensitisation can induce systemic oral tolerance. Characterisation of cytokine profiles produced by T-cells from these tolerized individuals demonstrated an increased TGF-β and IL-10 production 147. These T-cells act as suppressor cells by secreting anti-inflammatory cytokines. ProefschriftMT.indd :12:31

30 30 Chapter 1 General introduction Differences between allergic and irritant contact dermatitis The immunological mechanisms underlying allergic and irritant contact dermatitis reactions are different. However, both phenomena result in vasodilatation, up regulation of endothelial adhesion molecules 148, mast cell degranulation 149, keratinocyte cytokine and chemokine production 150, influx of leukocytes 151;152, and LC migration into the dermis 153;154. The similarity of these events can be explained by the irritant properties of allergens, which strongly contribute to their allergenicity 155. Differences between allergen or irritant exposure have been studied in murine models. MHC class II, IL-1α, IL-1β, CXCL10 and CXCL2/CXCL3 (MIP- 2) were found to be only up regulated after allergen painting 42. In humans, expression of CXCL9, CXCL10 and CXCL11 has been reported to be specifically up-regulated in ACD and not in ICD 156. Of course, true differences between an allergic and irritant reaction depend on whether or not specific T-cells become involved. Only those chemicals with sufficient affinity for MHC molecules, and for which matching T-cell receptors exist can ultimately cause ACD. As DC maturation is required for proper T-cell activation, one can speculate that allergens and not irritants are able to sufficiently activate maturation of skin DCs. Indeed, in vitro studies showed that potential contact sensitisers can be distinguished from irritants by their different ability to initiate LC/DDC migration and maturation 81;82;157. ProefschriftMT.indd :12:31

31 31 Predictive assays for skin sensitising testing The frequency of allergic skin disorders has increased in the last decades. Therefore, the identification of potential sensitizing chemicals is important for skin safety. Traditionally, predictive testing for allergenicity has been conducted in guinea pigs 158. In recent years, the murine local lymph node assay (LLNA) has become the preferred test method for assessing skin sensitisation potential 159. For regulatory reasons, animal use for sensitisation testing of compounds for cosmetic purposes is shortly to be prohibited in Europe. Therefore, new non-animal based test methods need to be developed. The following events/properties in skin sensitisation are crucial in skin sensitisation and might, therefore, be explored by various in silico, in vitro and ex vivo models: 1. The ability of the chemical to penetrate through the stratum corneum. In order to induce skin sensitisation the physicochemical properties of a compound must enable it to penetrate into the epidermis. Low molecular size, net charge and octanol-water partition coefficient (logp) contribute to the epidermal bioavailability. LogP represent the relative affinities of the allergen in an octanol water mixture and is a descriptor of hydrophobicity 160. Overall, small, non-polar and moderately lipophilic substances penetrate the stratum corneum best, whereas highly polar, water-soluble compounds with high molecular weight penetrate poorly. Different in silico models have been developed that aim to predict skin penetration and sensitisation potential of chemicals by structure-activity relationships (SAR) 161;162. Unfortunately, these methods are not suitable for identification of all allergens. For example, metal allergens, such as Ni 2+, are not identified as sensitising compounds. 2. The potential of the chemical to form stable conjugates to create an immunogenic complex. For immunogenicity, the hapten must form a stable association with a macromolecule (e.g. protein). Either skin sensitising chemicals are naturally protein reactive or can be metabolically transformed within the skin into a protein-reactive species, the so-called prohapten 163. Sensitising organic compounds bind covalently to protein nucleophilic groups, such as thiol, amino and hydroxyl groups, as is the case 3. with poison oak/ivy allergen 164. In contrast, metal ions, e.g. nickel cations, form stable metal-protein chelate complexes by coordination bonds 165. The availability of skin-derived cytokines that facilitate chemical-induced LC migra- tion. Migration of LC is stimulated by e.g. TNF-α, IL-1β and IL These cytokines are important for LC migration and the development of skin sensitisation in mice. 4. The ability of a chemical to induce maturation of DCs. In this paragraph, we will discuss human in vitro models for allergen screening based on phenotypical changes in DCs. 5. The ability of a chemical to provoke T-cell response via DCs. The activation and clonal expansion of allergen-specific memory and effector T-cells are pivotal in the acquisition of allergic contact dermatitis. Sensitisation is dependent on direct association of haptens with MHC-molecules or peptides in the groove of these molecules of epidermal ProefschriftMT.indd :12:31

32 32 Chapter 1 General introduction LCs. For a number of chemical allergens, e.g. 2,4,6-trinitrophenyl (TNP) or sesquiterpene lactones, it has been shown that they generate hapten-modified peptides that are presented and recognised by CD8 + or CD4 + T-cells on MHC class I and class II molecules, respectively. For metal chemicals, coordinative binding to MHC molecules and/ or MHC bound peptides and the TCR have been shown to be relevant in the activation of allergen-specific T-cells 125. Other chemicals, seem to directly connect MHC and TCR without binding 127. Ultimately, each approach results in the activation of chemicalreactive T-cells, which can cause ACD. Only those chemicals with sufficient affinity for MHC molecules and for which matching T-cell receptors exist can ultimately cause an adapted allergic reaction. DCs play a crucial role in the initiation phase of contact hypersensitivity reactions in the skin. Given the importance of skin dendritic cells in the initiation of skin sensitisation, various models (ex vivo and in vitro) have been proposed for screening of potential sensitising chemicals and will be discussed below. Ex vivo: human skin explant cultures Models have been developed for predictive testing of the sensitising capacity of allergens in human skin explant cultures. The group of Pranab Das used short-term human skin organ cultures to examine the time course effects of either allergens or irritants on human LCs 167. The advantage of this approach is the involvement of the additional danger signals provided by skin residential cells e.g. KCs and fibroblasts, which can enhance the allergenic effects on LC. In unchallenged skin, LCs preserved their characteristics and distribution within the epidermis of these cultures. Epicutaneous applications of the contact allergens, e.g. DNCB, oxazolone (OXA), NiSO 4 or diphencyprone (DPC), showed reduction of LCs in the epidermis, whereas remaining LC were localised along the epidermal-dermal junction after 24 hours. Furthermore, the assay showed that decrease of LC in the epidermis was dependent on the concentration of the contact allergens and the sensitising capacities of the test compounds 157. In contrast, the irritants, e.g. sodium dodecyl sulfate (SDS), croton oil (CO), nonanoic acid (NON) or benzalkonium chloride (BC) did not induce these changes. Although the LC migration assay using human skin explants seems effective in identifying allergens, broader introduction of this assay might be hampered by its time-consuming and costly features as well as its dependence of a ready supply of freshly excised skin from surgery. In vitro: Peripheral CD14 + blood or CD34 + progenitor derived DCs Since Sallusto and Lanzavecchia introduced a method to generate large numbers of dendritic cells in vitro from modcs, numerous studies have been conducted on the effects of allergens on DCs 38. Aiba et al. (1997) was the first to show that allergens can induce phenotypic alterations of immature modc 74. They have shown that the allergens nickel and DNCB can significantly up-regulate the surface expression of the markers CD54, CD86, ProefschriftMT.indd :12:32

33 33 HLA-DR and IL-1β production, while the irritants SDS, BC and zinc chloride (ZnCl 2 ) could not augment these markers. IL-1β gene expression in modc is up regulated within 30 minutes after allergen exposure 168. However, allergen-induced up regulation of IL-1β mrna seems to be donor-dependent. Exposure to dinitrofluorobenzene (DNFB) resulted in up regulation of IL-1β mrna (two- to threefold) in cells derived from only three out of eight donors. MoDC from donors that had increased IL-1β mrna to DNFB, also induced gene expression of IL- 1β in response to the other allergens, e.g. paraphenylenediamine (PPDA) and methylchloroisothiazolinone/ methylisothiazolinone (MCI/MI) 169. Likewise, modcs from donors, which failed to respond to DNFB also did not respond to treatment with either PPDA or MCI/MI. Responder and non-responder donors are stable over time, thus these observations are not due on inter-experimental differences. We observed that DC from atopic individuals seem to be more susceptible to allergen-induced DC polarisation than DC from healthy individuals 170. Likewise, it might be possible that DC derived from atopic individuals in the study of Pichowski reflect the responder donors. Collectively, several investigators reported alterations in expression of CD40, CD54, CD83, CD86, HLA-DR 74, CXCR4, CCR5 150, CCR7 171 or production of CXCL8 82, intracellular I L-1 β 172 or mrna expression of CCL2, CCL3 and CCL4 173 after allergen exposure in either monocyte or CD34 + -derived DCs. Importantly, gene expression profiles of allergen or irritant exposed DCs were investigated in two different studies 81;174. First, nickel induced significant changes of 283 genes in CD34 + -derived DC 81. The following interesting genes were affected: cytokine(receptor) (e.g. IL-1β, IL-6, IL-1RA, IL-7R, CXCL1, CXCR4), membrane markers (CD1c, CD83, CD86), adhesion molecules (E-Cad, MMPs), signalling pathways (MAPK, NF-κβ, STAT). Unfortunately in these studies, only nickel is used as an allergen and no irritants were included. Gildea et al. (2006) reported that a group of eleven genes are strongly associated with allergen-induced DC activation 174. However, some of these genes (e.g. ABCA6, CCL23, SLAM) were also affected by one of the four irritants. Strikingly, only one gene, HML2, was able to discriminate the eleven allergens from the four irritants. In vitro: Dendritic cell lines During the last decade, human cell lines have been explored as potential surrogates for DC for screening potential sensitisers. The great advantage of using cell lines is the avoidance of donor variability as discussed above. Cytokine treatment of the human acute myeloid leukaemia cell line, KG-1 has been shown to induce a DC-like phenotype and morphology 175;176. Exposure to chemical allergens had little or no effect on the expression of HLA-DR, CD54, CD80 and CD86 in these cells 177. The THP-1 cell line, derived from human monocytic leukaemia cells, has been examined for its potential to replace modcs in the development of an in vitro predictive test for contact sensitisers 177,178. Exposure to a number of skin sensitisers has been shown to enhance surface expression of CD86 and CD54. In addition, THP-1 cells demonstrated an increased internalization of MHC class II molecules in response to allergen exposure 171. Most recently, the human myeloid leukaemia-derived MUTZ-3 cell line has been proposed as a dendritic cell line 179;180. Azam et al. (2006) tested MUTZ-3 against a wide panel ProefschriftMT.indd :12:32

34 34 Chapter 1 General introduction of sensitisers and irritants on the cell surface marker expression of HLA-DR, CD40, CD54, CD80, B7-H1, B7-H2, and B7-DC 181. In general, some of the DC cell lines, such as MUTZ-3 and THP-1, show promising tools for sensitisation testing. However, it should be noted that, the parameters of activation examined to date have shown relatively less expression changes in DC cell lines as compared to blood-derived DCs. Identification of novel markers or development of new culture methods might enhance the sensitivity of the cell lines. Concluding remarks At present, surface expression of CD86 74;181, CXCL8 production 82 and gene expression of HML2 174 seem to be the most reliable markers for screening potential sensitisers in both blood-derived DCs and DC lines. In the future, the most robust method to identify the sensitising capacity of all groups of allergens might benefit from a broad panel of markers, which are restricted to allergen-induced DC maturation. Additionally, it may be possible to exploit DC responses in vitro not only for identification of allergens but also for predicting whether type-1 or type-2 immune responses will be induced. Notwithstanding, a more detailed understanding of the ways in which allergens interact with DCs will result in robust tools for identification of allergens. ProefschriftMT.indd :12:33

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42 42 Chapter 1 General introduction 131. Flier J, Boorsma DM, Bruynzeel DP, Van Beek PJ, Stoof TJ, Scheper RJ, Willemze R, Tensen CP. 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 Oct;113(4): Moed H, Boorsma DM, Stoof TJ et al. Nickel-responding T cells are CD4+ CLA+ CD45RO+ and express chemokine receptors CXCR3, CCR4 and CCR10. Br J Dermatol 2004;151(1): Clark RA, Chong B, Mirchandani N et al. The vast majority of CLA+ T cells are resident in normal skin. J Immunol 2006;176(7): Goebeler M, Trautmann A, Voss A et al. Differential and sequential expression of multiple chemokines during elicitation of allergic contact hypersensitivity. Am J Pathol 2001;158(2): Serra HM, Eberhard Y, Martin AP et al. Secondary lymphoid tissue chemokine (CCL21) is upregulated in allergic contact dermatitis. Int Arch Allergy Immunol 2004;133(1): Cavani A, Albanesi C, Traidl C et al. Effector and regulatory T cells in allergic contact dermatitis. Trends Immunol 2001;22(3): Foussat A, Cottrez F, Brun V et al. A comparative study between T regulatory type-1 and CD4+CD25+ T cells in the control of inflammation. J Immunol 2003;171(10): Cavani A, Nasorri F, Prezzi C et al. Human CD4+ T lymphocytes with remarkable regulatory functions on dendritic cells and nickel-specific Th1 immune responses. J Invest Dermatol 2000;114(2): Sebastiani S, Albanesi C, Nasorri F et al. Nickel-specific CD4(+) and CD8(+) T cells display distinct migratory responses to chemokines produced during allergic contact dermatitis. J Invest Dermatol 2002;118(6): Jonuleit H, Schmitt E, Stassen M et al. Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood. J Exp Med 2001;193(11): McHugh RS, Shevach EM. The role of suppressor T cells in regulation of immune responses. J Allergy Clin Immunol 2002;110(5): Schwarz A, Grabbe S, Riemann H et al. In vivo effects of interleukin-10 on contact hypersensitivity and delayed-type hypersensitivity reactions. J Invest Dermatol 1994;103(2): Curiel-Lewandrowski C, Venna SS, Eller MS et al. Inhibition of the elicitation phase of contact hypersensitivity by thymidine dinucleotides is in part mediated by increased expression of interleukin-10 in human keratinocytes. Exp Dermatol 2003;12(2): Kanda N, Mitsui H, Watanabe S. Prostaglandin E(2) suppresses CCL27 production through EP2 and EP3 receptors in human keratinocytes. J Allergy Clin Immunol 2004;114(6): Moed H, Boorsma DM, Tensen CP et al. Increased CCL27-CCR10 expression in allergic contact dermatitis: implications for local skin memory. J Pathol. 2004;204(1): Van Hoogstraten IM, Andersen KE, Von Blomberg BM et al. Reduced frequency of nickel allergy upon oral nickel contact at an early age. Clin Exp Immunol 1991;85(3): Rustemeyer T, von Blomberg BM, van Hoogstraten IM et al. Analysis of effector and regulatory immune reactivity to nickel. Clin Exp Allergy 2004;34(9): Goebeler M, Roth J, Brocker EB et al. Activation of nuclear factor-kappa B and gene expression in human endothelial cells by the common haptens nickel and cobalt. J Immunol 1995;155(5): ProefschriftMT.indd :12:36

43 Walsh LJ, Lavker RM, Murphy GF. Determinants of immune cell trafficking in the skin. Lab Invest 1990;63(5): Spiekstra SW, Toebak MJ, Sampat-Sardjoepersad S et al. Induction of cytokine (interleukin-1alpha and tumor necrosis factor-alpha) and chemokine (CCL20, CCL27, and CXCL8) alarm signals after allergen and irritant exposure. Exp Dermatol 2005;14(2): Silberberg I, Baer RL, Rosenthal SA. The role of Langerhans cells in allergic contact hypersensitivity. A review of findings in man and guinea pigs J Invest Dermatol 1989;92(4 Suppl):160S; discussion 161S-163S Brasch J, Burgard J, Sterry W. Common pathogenetic pathways in allergic and irritant contact dermatitis. J Invest Dermatol 1992;98(2): Silberberg-Sinakin I, Thorbecke GJ et al. Antigen-bearing langerhans cells in skin, dermal lymphatics and in lymph nodes. Cell Immunol 1976;25(2): Hill S, Edwards AJ, Kimber I, Knight SC. Systemic migration of dendritic cells during contact sensitization. Immunol 1990;71(2): Willis CM, Young E, Brandon DR, Wilkinson JD. Immunopathological and ultrastructural findings in human allergic and irritant contact dermatitis. Br J Dermatol 1986;115(3): Flier J, Boorsma DM, van Beek PJ et al. Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. J Pathol 2001;194(4): Rustemeyer T, Preuss M, von Blomberg BM et al. Comparison of two in vitro dendritic cell maturation models for screening contact sensitizers using a panel of methacrylates. Exp Dermatol 2003;12(5): Buehler EV. Delayed contact hypersensitivity in the guinea pig. Arch Dermatol 1965;91: Kimber I, Weisenberger C. A murine local lymph node assay for the identification of contact allergens. Assay development and results of an initial validation study. Arch Toxicol 1989;63(4): Potts RO, Guy RH. Predicting skin permeability. Pharm Res. 1992;9(5): Patlewicz G, Basketter DA, Smith CK et al. Skin-sensitization structure-activity relationships for aldehydes. Contact Dermatitis 2001;44(6): Aptula AO, Patlewicz G, Roberts DW. Skin sensitization: reaction mechanistic applicability domains for structure-activity relationships. Chem Res Toxicol 2005;18(9): Smith CK, Moore CA, Elahi EN et al. Human skin absorption and metabolism of the contact allergens, cinnamic aldehyde, and cinnamic alcohol. Toxicol Appl Pharmacol 2000;168(3): Becker D, Valk E, Zahn S et al. Coupling of contact sensitizers to thiol groups is a key event for the activation of monocytes and monocyte-derived dendritic cells. J Invest Dermatol 2003;120(2): Thierse HJ, Moulon C, Allespach Y et al. Metal-protein complex-mediated transport and delivery of Ni2+ to TCR/MHC contact sites in nickel-specific human T cell activation. J Immunol 2004;172(3): Cumberbatch M, Dearman RJ, Antonopoulos C et al. Interleukin (IL)-18 induces Langerhans cell migration by a tumour necrosis factor-alpha- and IL-1beta-dependent mechanism. Immunol 2001;102(3): Pistoor FH, Rambukkana A, Kroezen M et al. Novel predictive assay for contact allergens using human skin explant cultures. Am J Pathol 1996;149(1): ProefschriftMT.indd :12:37

44 44 Chapter 1 General introduction 168. Reutter K, Jager D, Degwert J, Hoppe U. In vitro model for contact sensitisation: II Induction of IL-1β in human blood-derived dendritic cells by contact sensitisers. Toxicol In Vitro 1997;11: Pichowski JS, Cumberbatch M, Dearman et al. Investigation of induced changes in interleukin 1beta mrna expression by cultured human dendritic cells as an in vitro approach to skin sensitization testing. Toxicol In Vitro 2000;14(4): Toebak MJ, Moed H, Gibbs S et al. Dendritic cells from atopic individuals show predominant type-2-skewing cytokine production when exposed to Der p1 or Grass pollen atopens but not to small molecular weight allergens. Submitted for publication Jugde F, Boissier C, Rougier-Larzat N et al. Regulation by allergens of chemokine receptor expression on in vitro-generated dendritic cells. Toxicol 2005;212(2-3): Tuschl H, Kovac R. Langerhans cells and immature dendritic cells as model systems for screening of skin sensitizers. Toxicol In Vitro 2001;15(4-5): Verheyen GR, Schoeters E, Nuijten JM et al. Cytokine transcript profiling in CD34+-progenitor derived dendritic cells exposed to contact allergens and irritants. Toxicol Lett 2005;155(1): Gildea LA, Ryan CA, Foertsch LM et al. Identification of gene expression changes induced by chemical allergens in dendritic cells: opportunities for skin sensitization testing. J Invest Dermatol 2006;126(8): St Louis DC, Woodcock JB, Franzoso G et al. Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model. J Immunol 1999;162(6): Hulette BC, Rowden G, Ryan CA et al. Cytokine induction of a human acute myelogenous leukemia cell line (KG-1) to a CD1a+ dendritic cell phenotype. Arch Dermatol Res 2001;293(3): Yoshida Y, Sakaguchi H, Ito Y et al. Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line. Toxicol In Vitro 2003;17(2): Ashikaga T, Hoya M, Itagaki H et al. Evaluation of CD86 expression and MHC class II molecule internalization in THP-1 human monocyte cells as predictive endpoints for contact sensitizers. Toxicol In Vitro 2002;16(6): Masterson AJ, Sombroek CC, De Gruijl TD et al. MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+ precursors. Blood. 2002;100(2): Santegoets SJ, Masterson AJ, van der Sluis PC et al. A CD34(+) human cell line model of myeloid dendritic cell differentiation: evidence for a CD14(+)CD11b(+) Langerhans cell precursor. J Leukoc Biol 2006;80(6): Azam P, Peiffer JL, Chamousset D et al. The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers. Toxicol Appl Pharmacol 2006;212(1): ProefschriftMT.indd :12:37

45 ProefschriftMT.indd :12:37 45

46 46 Chapter 1 Outline of the thesis ProefschriftMT.indd :12:37

47 47 Outline of the thesis The immunological mechanisms underlying allergic and irritant contact dermatitis are different. However, allergic and irritant reactions are histopathologically hard to distinguish. In this thesis, our first goal was to investigate the role of skin residential cells, e.g. fibroblasts and keratinocytes, in the initiation phase of allergic and irritant responses. Therefore, chapter 2 describes the early production of cytokines and chemokines after allergen and irritant exposure of human keratinocytes and fibroblasts. As demonstrated in chapter 2, both contact allergens and irritants have irritant properties in skin cells. However, only the first trigger specific T-cells leading to allergic responses in sensitised individuals. Since ethical and legislative regulations banned animal use for sensitisation testing, reliable in vitro assays for the identification of contact allergens are needed. Until now, several dendritic cell-based assays have been described to discriminate allergens from irritants. Unfortunately, current in vitro methods are not sufficiently resilient to identify allergens and, therefore, these methods need refinement. To this end, we developed an in vitro assay by using monocyte-derived dendritic cells (DCs) and determined allergen-specific chemokine production (Chapter 3). In line with chapter 3, a new methology to identify allergens and irritants was explored in chapter 4. For the first time, kinomic profiling was explored to identify new parameters for the detection of potential allergens. DCs play an important role in the polarisation process of T-cells. Type-1 DC-derived cytokines strongly promote type-1 T-cell responses. On the other hand, type-2 DC-derived cytokines potently stimulate generation of type-2 T-cells. Allergic contact dermatitis is mainly mediated by type-1 T-cells, whereas in respiratory allergy type-2 T-cells are the key players. In both type of diseases, little is known about the role of DC-derived polarising cytokines. Hereto, chapter 5 describes whether contact and respiratory allergens posses intrinsic capacities to polarise DCs towards secretion of type-1 or type-2 cytokines. Allergen-specific type-2 T-cells, and their cytokines, are important in the acute phase of atopic diseases, e.g. atopic dermatitis. In atopic individuals, preferentially type-2 T-cells are generated in the sensitisation phase. Until now, it is unknown whether DCs from atopic individuals preferentially develop into type-2 cytokine producing DCs upon activation by contact allergens. Therefore, we compared DC cytokine profiles of atopic individuals and healthy controls in response to a set of allergens (chapter 6). The most effective way of preventing an allergic reaction is to avoid allergen contact. Unfortunately, consequent avoidance is sometimes not possible and therefore the use of anti-inflammatory drugs is needed for treatment of skin inflammation. The mechanisms by which these drugs affect T-cells and their capacity to influence T-cell functions have been investigated. However, suppression of immune responses does not only concern T- cell functions, but also a priori antigen-presenting properties of DCs. Therefore, in chapter 7 the modulatory effects of anti-inflammatory drugs on DC maturation and polarisation have been studied. In chapter 8, we discussed the results obtained in the chapters 2-7 in the context of relevant and new literature, with emphasis on the role of DCs. ProefschriftMT.indd :12:38

48 48 Chapter 2 Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure ProefschriftMT.indd :12:38

49 49 Chapter 2 Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure ProefschriftMT.indd :12:38

50 50 Chapter 2 Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure ProefschriftMT.indd :12:38

51 51 Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure Abstract The immune system is called into action by alarm signals generated from injured tissues. We examined the nature of these alarm signals after exposure of skin residential cells to contact allergens (nickel sulphate and potassium dichromate) and a contact irritant (sodium dodecyl sulphate: SDS). Nickel sulphate, potassium dichromate and SDS were applied topically to the stratum corneum of human skin equivalents. A similar concentration dependent increase in chemokine (CCL20, CCL27 and CXCL8) secretion was observed for all three chemicals. Exposure to nickel sulphate and SDS were investigated in more detail: similar to chemokine secretion no difference was observed in the time- and concentration dependent increase in proinflammatory cytokine (IL-1α and TNF-α) secretion. Maximal increase in IL-1α secretion occurred within 2 hours after exposure to both nickel sulphate and SDS and prior to increased chemokine secretion. TNF-α secretion was detectable 8 hours after chemical exposure. After allergen or irritant exposure increased CCL20 and CXCL8 but not CCL27 secretion was inhibited by neutralizing human antibodies to either IL-1α or TNF-α. Our data show that alarm signals consist of primary and secondary signals. IL-1α and TNF-α are released as primary alarm signals, which trigger the release of secondary chemokine (CCL20, CXCL8) alarm signals. However, some chemokines e.g.: CCL27 can be secreted in an IL-1α and TNF-α independent manner. Our data suggest that skin residential cells respond to both allergen and irritant exposure by releasing mediators that initiate infiltration of immune responsive cells into the skin. ProefschriftMT.indd :12:38

52 52 Introduction Chapter 2 Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure Allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) are two immunologically distinct forms of contact dermatitis the former being a specific immune reaction against an allergen whereas the latter is a general reaction to an irritant compound. However, these two types of dermatitis can be clinically and histologically difficult to distinguish from each other 1-3. Clinical observations show a clear role for irritancy in ACD: many allergens have irritant properties and irritated skin is easier to sensitise than non-irritated skin 4. During both an ACD and an ICD reaction, alarm signals in the form of skin barrier disruption, epidermal cellular changes and cytokine/chemokine release stimulate the initial trafficking of immune cells to the site under attack. Clearly, allergens as well as irritants give rise to alarm signals but the nature of these signals and also whether or not common alarm signals are involved is still largely unknown. In ACD as well as ICD, increased mrna levels of IL-1α and TNF-α have been detected 1;5. An increased release of biologically active IL-1α has also been shown in psoriatic keratinocytes and in chronically inflamed skin 6;7. In keratinocytes, IL-1α is stored intracellularly and can be released quickly upon epidermal injury 8. Furthermore, in keratinocyte and fibroblast cultures these cytokines have been shown to stimulate the production of chemokines CCL20 (MIP-3α) and CXCL8 (IL-8) These chemokines in addition to CCL27 (a chemokine only expressed in the epidermis) are up-regulated in ACD These findings suggest that IL-1α and TNF-α may be released as general alarm signals which trigger chemokine signals in response to danger from the environment. In this publication we investigate whether or not exposure of skin residential cells (keratinocytes and fibroblasts) to allergens and irritants results in cytokine (IL-1α and TNF-α) dependent chemokine (CCL20, CXCL8 and CCL27) secretion and also whether differences occur between contact allergens and irritants. For this study we have used a human skin equivalent (HSE) model as contact allergens and irritants can be topically applied to the stratum corneum of the cultures in a similar manner to that done in vivo 16. However, in contrast to in vivo studies the secretion of cytokines and chemokines can easily be investigated after chemical exposure. Furthermore, as only keratinocytes and fibroblasts are present, this model provides the opportunity to investigate the effect of allergens and irritants in the absence of infiltrating, immune cells including Langerhans cells. The HSE consists of a reconstructed epidermis grown on a dermal matrix (fibroblast populated collagen gel) Nickel sulphate, potassium dichromate and SDS were applied topically to the stratum corneum in order to investigate alarm signals released by the skin residential cells. Our results show that exposure to all three chemicals results in a similar increase in CCL20, CCL27 and CXCL8 secretion. Allergen and irritant induced CCL20 and CXCL8 secretion is dependent on both IL-1α and TNF-α CCL27 secretion however, is regulated in an IL-1α/ TNF-α independent manner. ProefschriftMT.indd :12:39

53 53 Materials and Methods Cell Culture Fibroblast culture : Dermal fibroblasts were isolated from neonatal foreskins and cultured as described by Ponec et al. (1977) in Dulbecco s modified Eagle medium (DMEM) (ICN biomedicals, Irvine, CA) containing 5% fetal calf serum (Hyclone) 19. Fibroblasts were incorporated into collagen gels (1 x 10 5 cells/ml) essentially as described by Smola et al. (1993) 20. Keratinocyte culture: Epidermal keratinocytes were isolated from neonatal foreskins essentially as described earlier 19;21. Keratinocytes were seeded and cultured in DMEM / Hams F12 (ICN biomedicals, Irvine, CA) (3:1), supplemented with 1 % ultroserg (BipSepra S.A, Cergy-Saint-Christophe, France), 1 µm hydrocortisone, 1 µm isoproterenol, 0.1 µm insulin and 2 ng/ml KGF. Sub-confluent cultures were then used to construct HSE. Human skin equivalent (HSE) culture: Secondary keratinocyte cultures were seeded onto fibroblastpopulated collagen gels and incubated overnight in medium containing DMEM / Hams F12 (3:1), 1% ultroserg, 1 µm hydrocortisone, 1 µm isoproterenol, 0.1 µm insulin. Cultures were then lifted to the air-liquid interface and cultured for a further 4 days in Standard Keratinocyte Culture Medium (DMEM / Hams F12 (3:1), 1 µm hydrocortisone, 1 µm isoproterenol, 0.1 µm insulin, 1.0 x 10-5 M L-carnitine, 1.0 x 10-2 M L-serine, 1 µm DL-α-tocopherol acetate and enriched with a lipid supplement containing 25 µm palmitic acid, 15 µm linoleic acid, 7 µm arachidonic acid and 24 µm BSA) supplemented with 0.25% ultroser G. Hereafter, HSEs were cultured in Standard Keratinocyte Culture Medium supplemented with 50 µg/ml ascorbic acid for an additional 14 days. Hydrocortisone was omitted from the culture medium 48 hours before exposure to chemicals or cytokines. Unless otherwise stated, all additives were purchased from Sigma (St. Louis, MO). For all cultures the medium was renewed two times per week and all experiments were performed in triplicate unless otherwise stated. All culture supernatants were stored at -20 C for further analysis by ELISA. Exposure to chemicals, cytokines and blocking antibodies Chemical exposure: Finn Chamber filter paper discs 11 mm (Epitest LTD Oy, Finland) were impregnated with 30 µl of vehicle (water), allergen (nickel sulphate [NiSO 4.6H 2 0], potassium dichromate) or irritant (SDS) dissolved in water. Chemicals ranging from 0% to 5% were applied topically to the stratum corneum of the cultures and the cultures were further incubated at 37 C and 5% CO 2 until harvested. Chemicals were supplied by Sigma. Cytokine exposure: Human recombinant IL-1α or TNF-α (Sigma) was added at a concentration of 2000 U/ml to the culture medium for 24 hours prior to harvesting. Blocking antibodies: IL-1α or TNF-α blocking antibodies (100 ng/ml; R&D Systems Inc., Minneapolis, Minnesota) were added to the culture medium for 36 hours prior to exposure and also during a 24 hour topical exposure of HSE to 1.0% nickel sulphate or 0.5% SDS. ProefschriftMT.indd :12:39

54 54 Chapter 2 Morphology and cytotoxicity For morphological determinations samples were fixed in 4% paraformaldehyde and processed for conventional paraffin embedment. Sections (5 µm) were cut and stained with hematoxylin and eosin for light microscopic examination. Epidermal cytotoxicity, measured as a decrease in keratinocyte RNA, was assessed by pyronine Y staining. 5 µm sections were deparaffinized and then incubated for 20 minutes at room temperature in a fresh pyronine Y staining solution (0.1% pyrinine Y) ( Fluka chemie GMBH, Buchs, Switserland) in 0.2 M sodium acetate buffer, ph 4.0). The sections were washed 3 times in water, air dried and embedded in Depex mounting medium (Gurr BDH lab. Supplies, Poole, UK). Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure ELISA CCL20, CCL27, CXCL8, IL-1α and TNF-α levels in culture supernatants were determined by ELISA. For CXCL8 quantification, a Pelipair reagent set (CLB, Amsterdam, The Netherlands) was used essentially as described by the suppliers. For IL-1α quantification, a Duoset IL- 1α kit (DY200) was used and for TNF-α quantification, a TNF-α Quantikine high sensitivity kit (HSTAOOC) was used (detection limit: 0.5 pg/ml). For CCL20 and CCL27 quantification, ELISA plates (Nalge Nunc international, Roskilde, Denmark) were coated overnight with capture antibody: 1 µg/ml mouse anti-human CCL20 (MAB360), or 1 µg/ml CCL27 (MAB376). Subsequently, the plates were blocked (1 hr PBS/BSA 0.5%) and then chemokine standards 2.7 pg/ml to 1600 pg/ml rhccl20 (360-mp-025) or 15.6 pg/ml to 2000 pg/ml rhccl27 (376- mp-025) or samples were applied to the plate. After incubation (1 hr room temperature) and washing (PBS/0.05% Tween-20), detecting antibody was added: 50 ng/ml biotinylated goat anti-human CCL20 (BAF360) or 50 ng/ml biotinylated goat anti-humanccl27 (BAF376). After incubation (1 hr room temperature) and washing (PBS/0.05% Tween-20) 1:10000 diluted streptavidin-hrp (CLB, Amsterdam, The Netherlands) was added. The enzyme reaction was initiated by adding 0.2 mg/ml orthophenylene diamine in 0.11 M acetate ph 5.5 and 0.03% hydrogen peroxide and stopped with 2 M H 2 SO 4. Absorbance was determined at 490 nm. All ELISA kits, antibodies and standards were purchased from R&D Systems Inc., Minneapolis, Minnesota unless otherwise stated. Results Topical application of the allergens nickel sulphate, potassium dichromate and the irritant SDS increase CCL20, CXCL8 and CCL27 secretion in HSE As alarm signals arise from injured tissues before cytotoxicity occurs it was important first to determine the maximal non-toxic dose of the chemicals to be studied. Therefore a dose response study was performed in which nickel sulphate, potassium dichromate and SDS were applied topically to the stratum corneum of HSE for 24 hours. The concentration of the chemicals was considered toxic when clear deleterious changes in the tissue architecture of the cultures was observed (e.g.: increased number of vacuoles, necrosis, loss of a distinguishable stratum granulosum) and when cytoplasmic RNA levels decreased. Staining ProefschriftMT.indd :12:39

55 55 of RNA with pyronine Y showed that as a chemical penetrated and became cytotoxic, the amount of RNA staining in the upper epidermal layers decreased first (Fig. 2.1; see 1% SDS). In this way, non-toxic chemical concentrations were identified as 2.5% nickel sulphate, 0.75% potassium dichromate and 0.5% SDS. Control 2.5% nickel sulfate 0.75% potassium dichromate 0.5% SDS Water 5% nickel sulfate 1% potassium dichromate 1% SDS Fig. 2.1: Determination of non-cytotoxic concentrations of nickel sulphate, potassium dichromate and SDS. The chemicals were topically applied to HSE for 24 hours. Cytoplasmic RNA levels were detected by pyronine Y staining of RNA on 5 µm paraffin embedded sections. The ability of the allergens nickel sulphate and potassium dichromate, and the irritant SDS to increase CCL20, CXCL8 and CCL27 secretion was determined by applying the chemicals topically to the stratum corneum of HSE in a dose dependent manner for 24 hours. The amount of CCL20, CXCL8 and CCL27 secreted into the culture medium was measured by ELISA. Non-cytotoxic concentrations of all 3 chemicals resulted in a dose dependent increase in chemokine secretion when compared to water (vehicle) exposed cultures. The peak of the dose response curve occurred at or below the maximum non-cytotoxic concentration of each chemical with the maximum induction factor for CCL20 (6 30 fold) being greater than for CCL27 (approx. 3 fold) or CXCL8 (approx. 3 fold) (Fig. 2.2). Exposure to water did not result in an increase in chemokine secretion above that of unexposed cultures (data not shown). Our results showed that secretion of all three chemokines is increased irrespective to whether the tissue injury is caused by allergen or irritant exposure. ProefschriftMT.indd :12:40

56 56 Chapter 2 Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure CCL20 secretion (ng/ml) CCL27 secretion (ng/ml) CXCL8 secretion (ug/ml) ** * Nickel sulfate concentration % ** ** Fig. 2.2: CCL20, CCL27 and CXCL8 are induced after exposure to nickel sulphate, potassium dichromate and SDS. Supernatants of HSE were harvested 24 hours after topical exposure to increasing concentrations of aqueous nickel sulphate, potassium dichromate or SDS. Supernatants were analysed by means of a CCL20, CCL27 and CXCL8-specific ELISA. Mean +/- SEM is shown, n = 4. Students t-test: * = p < 0.10; ** = p<0.05; *** = p< Rate of chemokine secretion is similar between nickel sulphate and SDS exposed HSE As both allergen and irritant exposure results in similar increases in the total amount of chemokine secreted after 24 hours, the question was asked whether the rate of chemokine secretion during this period was also similar. HSEs were exposed to 1% nickel sulphate and 0.5% SDS as at these chemical concentrations an increase in all three chemokines was observed (Fig. 2.2). As shown in Fig. 2.3, increased CCL20 secretion was already observed 2-4 hours after exposure to both nickel sulphate and SDS. The rate of CCL20 secretion continued to increase until the 6-8 hour time period and then decreased. CCL27 secretion began to increase 2-4 hours after exposure to SDS and 4-6 hours after exposure to nickel sulphate. A maximum rate of CCL27 secretion was observed for both nickel sulphate and SDS after 6-8 hours which, in contrast to CCL20, remained constant for the remaining time periods studied. An increasing trend in CXCL8 secretion was observed 2-4 hours after exposure *** Potassium dichromate concentration % ** ** ** SDS concentration % ProefschriftMT.indd :12:41

57 57 to nickel sulphate and 6-8 hours after exposure to SDS and similarly to CCL27, the rate of secretion remained higher than in unexposed cultures for the duration of the experiment. Our results showed that no significant difference in chemokine secretion occurs between nickel sulphate and SDS exposed cultures. Fig. 2.3: Rate of chemokine secretion after exposure of HSE to nickel sulphate or SDS. The culture medium was removed and replaced during the given time periods. The rate of chemokine secretion during each time period is shown. Supernatants were analysed by means of a CCL20, CCL27 and CXCL8-specific ELISA. Water (vehicle) = open bar; 1.0% nickel sulphate = grey bar; 0.5% SDS = solid bar. Mean +/- SEM is shown, n = 3. Dependence of increased chemokine secretion on IL-1α and TNF-α It has previously been reported in fibroblast and keratinocyte cultures that CCL20, and CXCL8 are secreted upon stimulation with the proinflammatory cytokines IL-1α and TNF-α 9;10. In order to determine whether IL-1α and TNF-α could play a role in allergen and irritant mediated CCL20, and CXCL8 secretion and also CCL27 secretion we first determined whether these cytokines were secreted after exposure of HSE to nickel sulphate or SDS. A dose response curve for nickel sulphate and SDS shows that both IL-1α and TNF-α can be detected at increased levels in culture supernatants (maximum secretion approximately 100 pg/ml for both nickel sulphate and SDS) (Fig. 2.4A). Kinetic studies showed that the maximum increase in IL-1α secretion occurs within 2 hours after exposure to either nickel sulphate or SDS (Fig. 2.4B). Noticeable was that maximum IL-1α secretion occurred before the increase in chemokine secretion was observed (compare Fig. 2.3 with Fig. 2.4B). The slightly increased rate of secretion observed in water exposed cultures within 2 hours compared to later time ProefschriftMT.indd :12:42

58 58 Chapter 2 periods was most probably due to renewal of the culture medium at the beginning of exposure as a similar effect was observed in unexposed cultures (data not shown). In contrast to IL-1α, TNF-α secretion could only be detected at the later time-periods 8-14 hours and hours at a low rate of 0.1 pg/ml/hr (data not shown). Again, no difference between allergen or irritant stimulated production could be found. In order to verify whether IL-1α and TNF-α could increase chemokine secretion in HSE, cultures were supplemented with recombinant IL-1α or TNF-α (Fig. 2.5). Indeed, increased CCL20, CCL27 and CXCL8 expression was observed in response to both IL-1α and TNF-α. However, the increase in CCL27 secretion was only marginal in response to TNF-α. Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure Fig Nickel sulphate and SDS increase I L-1 α and TNF-α secretion. HSEs were exposed to increasing concentrations of nickel sulphate or SDS. For IL-1α determination, supernatants were harvested after 2 hours and for TNF-α determination, supernatants were harvested after 24 hours. Mean +/- SEM is shown, n=3. The rate of IL-1α secretion after exposure of HSE to nickel sulphate or SDS is shown. The culture medium (supernatant) was removed and replaced during the given time periods. Water (vehicle) = open bar; 1.0% nickel sulphate = grey bar; 0.5% SDS = solid bar. Bars show the average secretion of two independent experiments. In order to determine the dependency of increased CCL20, CCL27 and CXCL8 secretion on IL-1α and TNF-α, IL-1α or TNF-α neutralizing antibodies were incubated for 36 hours prior to exposure and also during exposure of HSE to nickel sulphate or SDS. As shown in figure 2.6, both IL-1α and TNF-α neutralizing antibodies were capable of totally blocking the induced secretion of CCL20 and CXCL8 after exposure to nickel sulphate and they were capable of partially blocking the increase in secretion of CCL20 and CXCL8 after exposure to SDS. Basal chemokine expression was not altered by incubation with anti-il-1α or anti-tnf-α. Surprisingly, incubation with anti-il-1α or anti-tnf-α was not able to block the increased ProefschriftMT.indd :12:43

59 59 CCL27 secretion after exposure to SDS or nickel sulphate. Our results indicate that allergen and irritant increased CCL20 and CXCL8 secretion is dependent on IL-1α and TNF-α, whereas increased CCL27 secretion occurs in an IL-1α and TNF-α independent manner. CCL20 secretion (ng/ml) ** ** CCL20 secretion (ng/ml) ** * * Nickel sulfate SDS CCL27 secretion (ng/ml) CXCL8 secretion (ng/ml) IL-1α TNF-α IL-1α TNF-α ** ** CCL27 secretion (ng/ml) CXCL8 secretion (ng/ml) Nickel sulfate Nickel sulfate SDS SDS 0 IL-1α TNF-α Fig IL-1α and TNF-α increase CCL20, CCL27 and CXCL8 secretion. HSE cultures were grown in the presence or absence of recombinant IL-1α or TNF-α (2000 U/ml). Supernatants were harvested 24 hours later and analysed by means of a CCL20, CCL27 and CXCL8-specific ELISA. Mean +/- SEM is shown, n=3. Fig Increased chemokine secretion is dependent on IL-1α and TNF-α. IL-1α or TNF-α blocking antibodies (100 ng/ml) were added to the culture medium 36 hours prior to exposure and also during a 24 hour topical exposure of HSE to 1.0% nickel sulphate or 0.5% SDS. Open bar = isotype IgG; grey bar = anti-il-1α; black bar = anti-tnf-α. No difference was observed between water (vehicle) exposed cultures incubated with isotype IgG, anti-il-1α or anti-tnf-α, the average value is shown by a dotted line. Culture supernatants were analysed by means of a CCL20, CCL27 and CXCL8-specific ELISA. Mean +/- SEM is shown, n=3. ProefschriftMT.indd :12:44

60 60 Discussion Chapter 2 Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure In this publication we show that exposure to either an allergen or irritant results in IL-1α and TNF-α dependent increases in CCL20 and CXCL8 secretion. Allergen and irritant mediated CCL27 secretion however occurs in an IL-1α and TNF-α independent manner even though recombinant IL-1α (but not TNF-α) is able to slightly increase CCL27 secretion. No distinction can be made between the chemicals tested with regards to chemokine and cytokine secretion. Our results suggest that injured skin residential cells, such as keratinocytes and fibroblasts, release general alarm signals when activating the immune system. This is in contrast to skin derived Langerhans cells (LCs) which create an allergenic signal in the form of LC maturation and migration in response to an allergen but not in response to an irritant 22;23. We show that IL-1α and TNF-α act as primary alarm signals, which can trigger secondary chemokine alarm signals. IL-1α and TNF-α dependent (CCL20, CXCL8) and also independent (CCL27) chemokine signals are part of an intricate chemokine cascade which is responsible for the trafficking of immune cells to the site of inflammation. CCL20, CXCL8 and CCL27 are chemoattractant cytokines for a variety of immune cells eg: immature dendritic cells, T cells, B cells and neutrophils The rapid secretion of these chemokines after exposure of the skin to danger will result in an influx of immune cells which may in turn facilitate an ACD or ICD reaction. Our finding that CCL20, CXCL8 and CCL27 secretion is increased after allergen or irritant exposure is in agreement with other researchers. Schmuth et al. (2002) described an increase in CCL20 mrna upon exposure of the skin to danger, in this case after acute disruption of the epidermal permeability barrier by tape stripping 15. CXCL8 has been shown to be induced during ICD 1;2 and our submitted data show that CCL27 is already increased six hours after initiating both ACD and ICD reactions. A common irritant alarm signal has been proposed to be involved in both ACD and ICD reactions 1. This is supported by a number of in vivo and in vitro studies in which both allergen and irritant exposure results in increased cytokine levels in keratinocytes and fibroblasts 1;5;28;29. The question remains as to which is the initiating cytokine and whether it is stored or whether de novo synthesis occurs. Enk and Katz (1992) found maximal increases in IL-1α and TNF-α mrna levels two hours after hapten application to a mouse ear and levels remained high for the following twelve hours 5. In organotypic cultures using ex vivo skin and in HSEs, topical exposure to SDS results in increased IL-1α mrna levels for at least 8 hours 16. However, IL-1α has also been detected as a depot in the stratum corneum 30 and therefore this can be considered as a depot ready to be released upon exposure to environmental danger. In the HSE model, IL-1α protein release into culture supernatants peaks sharply within 2 hours after chemical exposure and then rapidly declines to basal levels within 4 hours after exposure, thus preceding increases in chemokine secretion. Taking into account that the chemical first has to penetrate the stratum corneum, stimulate I L-1 α transcription and/or release, the IL-1α has to diffuse throughout the culture including the collagen gel and into the culture medium, our findings strongly suggest that chemical exposure results in the immediate release of stored IL-1α from the stratum corneum and ProefschriftMT.indd :12:44

61 61 that this is the initial alarm signal to stimulate chemokine secretion. Although TNF-α could not be detected in our kinetics experiment until 14 hours after allergen or irritant exposure, anti-tnf-α could effectively block chemical mediated increases in CCL20 and CXCL8 secretion and recombinant TNF-α could significantly increase CCL20 and CXCL8 secretion. This indicates that TNF-α, as well as IL-1α, is involved in the regulation of CCL20 and CXCL8 and that TNF-α levels at earlier time-points may be below the detection limit of the ELISA and/ or the cytokine may be taken up to a large extent by the cells or matrix in the HSE. Unlike IL-1α, it has not been described that TNF-α can be stored in depots in the stratum corneum. Therefore transcription of TNF-α may be induced after allergen or irritant exposure rather than the immediate release of stored protein being involved. Interestingly, blocking allergen or irritant induced CCL20 and CXCL8 secretion with neutralizing antibodies to either anti- IL-1α or anti-tnf-α reduced chemokine secretion only to basal levels indicating that basal chemokine secretion is regulated in an IL-1α and TNF-α independent manner. In conclusion, IL-1α and TNF-α are released as primary alarm signals, which trigger the release of secondary CCL20 and CXCL8 chemokine signals. CCL27 is regulated in an IL-1α and TNF-α independent manner. Increased levels of CCL20, CXCL8 and CCL27 have the potential to initiate infiltration of immune responsive cells into an area of the skin which is exposed to either an allergen or an irritant. For a contact allergen this can result in a sensitised individual developing ACD whereas for a contact irritant this can lead to ICD. Acknowledgements The excellent technical assistance of P. Prins was greatly appreciated. ProefschriftMT.indd :12:45

62 62 References 1. Levin CY, Maibach HI. Irritant contact dermatitis: is there an immunologic component? Int Immunopharmacol 2002;2(2-3): Chapter 2 Induction of cytokine (IL-1α, TNF-α) and chemokine (CCL20, CCL27, CXCL8) alarm signals after allergen and irritant exposure 2. Smith HR, Basketter DA, McFadden JP. Irritant dermatitis, irritancy and its role in allergic contact dermatitis. Clin Exp Dermatol 2002;27(2): Smith CK, Hotchkiss SAM. Allergic contact dermatitis; chemical and metabolic mechanisms. 1 ed. London: Taylor and Francis; McFadden JP, Basketter DA. Contact allergy, irritancy and danger. Contact Dermatitis 2000;42(3): Enk AH, Katz SI. Early molecular events in the induction phase of contact sensitivity. Proc Natl Acad Sci 1992;89(4): Sauder DN, Mounessa NL, Katz SI et al. Chemotactic cytokines: the role of leukocytic pyrogen and epidermal cell thymocyte-activating factor in neutrophil chemotaxis. J Immunol 1984;132(2): Camp RD, Fincham NJ, Cunningham FM et al. Psoriatic skin lesions contain biologically active amounts of an interleukin 1-like compound. J Immunol 1986;137(11): Wood LC, Elias PM, Calhoun C et al. Barrier disruption stimulates interleukin-1 alpha expression and release from a pre-formed pool in murine epidermis. J Invest Dermatol 1996;106(3): Nakayama T, Fujisawa R, Yamada H et al. Inducible expression of a CC chemokine liver- and activation-regulated chemokine (LARC)/macrophage inflammatory protein (MIP)-3alpha/CCL20 by epidermal keratinocytes and its role in atopic dermatitis. Int Immunol 2001;13(1): Maruyama K, Zhang JZ, Nihei Y et al. Regulatory effects of gamma-interferon on IL-6 and IL-8 secretion by cultured human keratinocytes and dermal fibroblasts. J Dermatol 1995;22(12): Steude J, Kulke R, Christophers E. Interleukin-1-stimulated secretion of interleukin-8 and growthrelated oncogene-alpha demonstrates greatly enhanced keratinocyte growth in human raft cultured epidermis. J Invest Dermatol 2002;119(6): Boorsma DM, de Haan P, Willemze R, Stoof TJ. Human growth factor (HuGRO), interleukin-8 (IL-8) and interferon-gamma-inducible protein (Gamma-IP-10) gene expression in cultured normal human keratinocytes. Arch Dermatol Res 1994;286(8): Griffiths CE, Barker JN, Kunkel S, Nickoloff BJ. Modulation of leucocyte adhesion molecules, a T-cell chemotaxin (IL-8) and a regulatory cytokine (TNF-alpha) in allergic contact dermatitis (Rhus Dermatitis). Br J Dermatol. 1991;124(6): Homey B, Alenius H, Muller A et al. CCL27-CCR10 interactions regulate T-cell-mediated skin inflammation. Nat Med 2002;8(2): Schmuth M, Neyer S, Rainer C et al. Expression of the C-C chemokine MIP-3 alpha/ccl20 in human epidermis with impaired permeability barrier function. Exp Dermatol 2002;11(2): Gibbs S, Vietsch H, Meier U, Ponec M. Effect of skin barrier competence on SLS and waterinduced IL-1alpha expression. Exp Dermatol 2002;11(3): El Ghalbzouri A, Gibbs S, Lamme E et al. Effect of fibroblasts on epidermal regeneration. Br J Dermatol 2002;147(2): ProefschriftMT.indd :12:45

63 Ponec M, Gibbs S, Pilgram G et al. Barrier function in reconstructed epidermis and its resemblance to native human skin. Skin Pharmacol Appl Skin Physiol 2001;14 Suppl 1: Ponec M, Hasper I, Vianden GD, Bachra BN. Effects of glucocorticosteroids on primary human skin fibroblasts. II. Effects on total protein and collagen biosynthesis by confluent cell cultures. Arch Dermatol Res 1977;259(2): Smola H, Thiekotter G, Fusenig NE. Mutual induction of growth factor gene expression by epidermal-dermal cell interaction. J Cell Biol 1993;122(2): Ponec M, Kempenaar JA, De Kloet ER. Corticoids and cultured human epidermal keratinocytes: specific intracellular binding and clinical efficacy. J Invest Dermatol 1981;76(3): Tuschl H, Kovac R. Langerhans cells and immature dendritic cells as model systems for screening of skin sensitizers. Toxicol In Vitro 2001;15(4-5): Rustemeyer T, Preuss M, von Blomberg BME et al. Comparison of two in vitro DC maturation models for screening contact sensitizers using a panel of methacrylates. Exp Dermatol 2003;12(5): Schroder JM, Christophers E. Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells. J Immunol 1989;142(1): Homey B, Wang W, Soto H et al. Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2 (GPR-2, CCR10) binds the skin-associated chemokine CCL27 (CTACK/ALP/ILC). J Immunol 2000;164(7): Dieu-Nosjean MC, Vicari A, Lebecque, S, Caux C. Regulation of dendritic cell trafficking: a process that involves the participation of selective chemokines. J Leukoc Biol 1999;66(2): Liao F, Rabin RL, Smith CS et al. CC-Chemokine receptor 6 is expressed on diverse memory subsets of T-cells and determines responsiveness to macrophage inflammatory protein 3 alpha. J Immunol 1999;162(1): Newby CS, Barr RM, Greaves MW, Mallet AI. Cytokine release and cytotoxicity in human keratinocytes and fibroblasts induced by phenols and sodium dodecyl sulfate. J Invest Dermatol 2000;115(2): Terunuma A, Aiba S, Tagami H. Cytokine mrna profiles in cultured human skin component cells exposed to various chemicals: a simulation model of epicutaneous stimuli induced by skin barrier perturbation in comparison with that due to exposure to haptens or irritant. J Dermatol Sci 2001;26(2): Gahring LC, Buckley A, Daynes RA. Presence of epidermal-derived thymocyte activating factor/ interleukin 1 in normal human stratum corneum. J Clin Invest 1985;76(4): ProefschriftMT.indd :12:45

64 64 Chapter 3 CXCL8 secretion by dendritic cells predicts contact allergens from irritants ProefschriftMT.indd :12:45

65 65 Chapter 3 CXCL8 secretion by dendritic cells predicts contact allergens from irritants ProefschriftMT.indd :12:46

66 66 Chapter 3 CXCL8 secretion by dendritic cells predicts contact allergens from irritants ProefschriftMT.indd :12:46

67 67 CXCL8 secretion by dendritic cells predicts contact allergens from irritants Abstract Monocyte-derived dendritic cell functions have been explored for identification of contact allergens in vitro. Current methods, including measurement of changes in cell surface marker expression (e.g. CD83, CD86) do not provide a sensitive method for detecting the sensitising potential of a chemical. In this study, we investigated whether chemokine production by monocyte-derived dendritic cells is increased upon maturation and whether chemokine production can provide methodology for the detection of allergens. Monocyte-derived dendritic cells were exposed to allergens (nickel sulphate, cobalt chloride, palladium chloride, copper sulphate, chrome-(iii)-chloride, potassium dichromate, p-phenylenediamine and dinitrochlorobenzene) and irritants (sodium dodecyl sulphate, dimethylsulfoxide, benzalkoniumchloride and propane-1-ol). CD83 and CD86 expression was analysed by flow cytometry and chemokine production (CXCL8, CCL5, CCL17, CCL18, CCL19, CCL20, CCL22) was determined by ELISA. Significant up regulation of CD83 and CD86 expression could only be induced by three out of seven and five out of seven allergens, respectively. In contrast, CXCL8 production was significantly increased after stimulation with all allergens tested, whereas irritant exposure led to decreased CXCL8 production. All other chemokines tested, failed in identifying contact allergens. In conclusion, CXCL8 production, next to CD83 and CD86 up regulation, by monocyte-derived dendritic cells provides a promising in vitro tool for discrimination between allergens and irritants. ProefschriftMT.indd :12:46

68 68 Introduction Chapter 3 CXCL8 secretion by dendritic cells predicts contact allergens from irritants Both contact allergens and irritants have irritant properties. However, only the first can trigger specific T-cell responses in allergic individuals. Upon contact of the skin with an allergen, the first critical step in the development of contact sensitisation is capture of the antigen by Langerhans cells (LCs). Subsequently, LCs begins to mature, migrate from the epidermal layers via the lymphatic vessel to the T-cell zone of the draining lymph node and can present antigen to naïve T-cells. While the migration of LCs to the lymph node is orchestrated by chemokines 1, dendritic cells (DCs) themselves have also been shown to secrete chemokines 2. Hashimoto et al. (2000) 3 showed by serial analysis of gene expression that exposure of DC to lipopolysaccharide (LPS) resulted in DC maturation and a simultaneous increase in the transcription of a large variety of genes. Amongst the transcripts showing the greatest increase in RNA levels were a number of chemokines CCL5 (RANTES), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3β), CCL20 (MIP-3α), CCL22 (MDC) and CXCL8 (IL-8). The increase in transcription of most chemokines was more pronounced than the increase in CD83 and CD86 transcription. In vivo, an increased rate of chemokine secretion would be of major importance for triggering the influx of leukocytes to the site of inflammation and also for bringing leukocytes within close proximity of the maturing DC. Whether or not allergen induced DC maturation also results in altered chemokine secretion profiles is not yet known. If this were the case, allergen induced chemokine secretion would provide a powerful means of predicting potential contact allergens in vitro. Cultured monocyte-derived dendritic cells (modcs) have been shown to provide a promising tool for identifying potential allergens in vitro Similar to LCs, allergen induced modc maturation results in elevated expression of the cell surface markers CD83 and CD86. In this study, we investigated whether chemokine production by modcs is also increased upon allergen induced maturation and ultimately, whether chemokine production can be used as a sensitive method to identify allergens. MoDCs were exposed to a panel of seven allergens and four irritants. Next to chemokine production (CXCL8, CCL5, CCL17, CCL18, CCL19, CCL20 and CCL22), the expression of CD83 and CD86 was determined. The present study is the first of its kind to investigate chemokine secretion by dendritic cells after allergen or irritant exposure and indicates that chemokine secretion, in particular CXCL8 secretion, may provide a novel tool to identify potential contact allergens in vitro. Materials and methods Culture of monocyte-derived DCs DCs were generated as previously described 16. Briefly, monocytes were isolated from heparinized leukocyte-enriched buffy coats from different donors using Ficoll (Amersham Pharmacia Biotech, Uppsala, Sweden) and Percoll (Pharmacia Bioscience, Uppsala, Sweden) ProefschriftMT.indd :12:46

69 69 density gradient centrifugation. After several washes with Iscove s modified Dulbecco s medium (IMDM, BioWhittaker, Verviers, Belgium) containing 1% heat-inactivated fetal calf serum (FCS, Hyclone, Logan, USA), 7 x 10 6 monocytes (at least 85 percent CD14 + ) were plated in 80 cm 2 culture flasks (Nalge Nunc international, Roskilde, Denmark) and cultured in IMDM containing 10% FCS, 1% penicillin-streptomycin (Gibco, Paisley, United Kingdom), 50 μm dithiothreitol (Merck, Darmstadt, Germany), 1100 U/ml granulocyte-macrophage colony stimulating factor (GM-CSF, Novartis, The Netherlands) and 1000 U/ml interleukin-4 (IL-4, Strathmann Biotec, Hannover, Germany). Flasks were incubated at 37 C in 5% humidified CO 2. Every second day, 1100 U/ml GM-CSF and 1000 U/ml IL-4 were refreshed. Following 6 days of culture, immature DCs (idcs) were harvested and plated in either 12 well tissue culture plates (Corning incorporated, NY, USA) at approximately 0.8 x 10 6 cells per well or 24 well tissue culture plates at approximately 0.3 x 10 6 cells. DCs expressed an immature phenotype, characterized by CD1a +, CD83 -, CD86 low and HLA-DR + expression (less than 5 percent impurity of CD3 + cells). Chemical exposure The cultured idc were exposed for 48 hours to LPS (obtained from Escherichia coli 055:B5, Sigma, St Louis, MO, USA), aqueous nickel sulphate (NiSO 4, Merck), palladium chloride (PdCl 2, Sigma), cobalt chloride (CoCl 2, Sigma), copper sulphate (CuSO 4, Sigma), chrome- (III)-chloride (CrCl 3, Sigma), potassium dichromate (K 2 Cr 2 O 7, Sigma), p-phenylenediamine (PPDA, Sigma), sodium dodecyl sulphate (SDS, Sigma), DMSO (Riedel-de Haën, Seelze, Germany), benzalkoniumchloride (BCl 2, Sigma) or propane-1-ol (Riedel de Haën). Dinitrochlorobenzene (DNCB, Sigma) was dissolved in DMSO and applied at less than 0.1 % (v/v). In order to determine the maximum non-toxic dose of allergens and irritants to be studied a dose response study was performed in which modcs were exposed to seven allergens, NiSO 4, CoCl 2, PdCl 2, CuSO 4, Cr 3+ (CrCl 3 ) and Cr6+ (K 2 Cr 2 O 7 ), PPDA, DNCB and four irritants, SDS, DMSO, BCl 2, propan-1-ol for 48 hours. The concentration of chemicals was considered non toxic when at least 75% of cells were viable determined by annexin V and propidium iodide expression (Bender Medsystems, Vienna, Austria). In this way, the maximum nontoxic dose was identified as 600 μm NiSO 4 ; 300 μm CoCl 2 ; 50 μm CuSO 4 ; 300 μm PdCl 2 ; 1000 μm CrCl 3 ; 100 nm K 2 Cr 2 O 7 ; 200 μm PPDA; 12 μm DNCB; 300 μm SDS; 30 μm BCl 2 ; 140 mm Propan-1-ol; 280 mm DMSO. In some experiments, 100 ng/ml LPS or 15 ng/ml tumor necrosis factor (TNF-)α was added to the cells 6 hours after addition of the chemicals, total chemical exposure time remained 48 hours. Flow cytometry Expression of CD83 and CD86 was analysed by flow cytometry. Cell staining was performed using PE-labelled monoclonal antibodies: mouse anti-human-cd86-pe (IgG 1, Pharmingen, B&D systems) and mouse anti-human-cd83-pe (IgG 2b, Immunotech, Marseille, France). Isotype controls to assess non-specific binding were monoclonal mouse IgG 1 -PE (Pharmingen, B&D systems) or monoclonal mouse IgG 2b -PE (Immunotech). Cells were incubated with antibodies for 30 minutes, washed in PBS plus 0.1% BSA and 0.1% Sodium Azide and ProefschriftMT.indd :12:47

70 70 resuspended in the same buffer for FACS analysis. Flow cytometry was performed with a Coulter FACS-STAR. Histograms or dot plots were analysed using Cell Quest software. The relative mean fluorescence index (fold increase over control) was calculated by the following formula: mean fluorescence intensity of DC treated with chemicals/mean fluorescence intensity of non-exposed DC. Chapter 3 CXCL8 secretion by dendritic cells predicts contact allergens from irritants Detection of chemokine secretion For CXCL8 quantification, a Pelipair reagent set (Sanquin, Amsterdam, The Netherlands) according to the manufactures recommendation was used. The production of CCL5, CCL17, CCL18, CCL19, CCL20 and CCL22 was measured by ELISA pair antibodies obtained from R&D systems (Minneapolis, USA). Briefly, maxisorp nunc immunoplates (Nalge Nunc international) were coated overnight at room temperature with 2 μg/ml mouse anti-human CCL5 (clone ), 1 μg/ml mouse anti-human CCL17 (clone ), 2 μg/ml mouse anti-human CCL18 (clone 64507), 0.8 μg/ml mouse anti-human CCL19 (clone BAU01), 2 μg/ ml mouse anti-human CCL20 (clone ) or 2 μg/ml mouse anti-human CCL22 (clone ). Subsequently, plates were blocked with PBS containing 0.5 % BSA for 1 hour. After several washes, serial dilutions of the collected supernatants and provided standards were added. After 1 hour incubation and washing, biotinylated anti-human antibody was added (CCL5, CCL17, CCL18, CCL19, CCL20 or CCL22). After incubation 1:10000 diluted streptavadin-horse rabbit peroxidase (Sanquin) was added. The enzyme reaction was initiated by 0.2 mg/ml orthophenyle diamine (ICN pharmaceuticals, NY, USA) and stopped by adding H 2 SO 4. Absorbance was determined at 490 nm. The amount of chemokine present in the culture supernatants was calculated using a standard curve (the lower detection limit is shown in brackets) rhccl5 (15.6 pg/ml), rhcxcl8 (15.6 pg/ml), rhccl17 (15.6 pg/ml), rhc- CL18 (15.6 pg/ml), rhccl19 (15.6 pg/ml), rhccl20 (2.7 pg/ml) and rhccl22 (15.6 pg/ml). Data are presented as chemokine production by exposed cells minus chemokine production by control cells in picogram s or nanogram s per million cells. Data analysis All data are presented as mean ± SEM. Differences between two groups were evaluated by a paired two tailed Wilcoxon test, with the computer program MedCalc (Mariakerke, Belgium). P 0.05 (*), p (**) and p (***) was considered statically significant. Results CD83 and CD86 expression after allergen and irritant exposure MoDCs were exposed to non-toxic concentrations of a panel of seven allergens and four irritants and changes in CD83 and CD86 expression were determined by flow cytometry (Fig. 3.1 and 3.2). Chromium was studied both as hexavalent chromium (K 2 Cr 2 O 7 ) and trivalent chromium (CrCl 3 ). Exposure to irritants did not result in augmented CD83 or CD86 ProefschriftMT.indd :12:47

71 71 expression. Exposure to the allergen NiSO 4 resulted in the greatest increase in CD83 and CD86 expression when compared to other allergens (4.5 ± 0.5 fold increase in CD83 and 4.7 ± 0.7 fold increase in CD86 expression). For CD83, of the seven allergens tested only exposure to NiSO 4, CoCl 2 and CuSO 4 resulted in a significantly increased expression of this cell surface marker (Fig. 3.1). Regarding CD86, five out of the seven allergens tested resulted in a significantly increased expression (Fig. 3.2). Exposure to chromium III (CrCl 3 ) led to a significant increase in CD86 expression in all concentrations tested, whereas chromium VI (K 2 Cr 2 O 7 ) only increased CD86 expression when DCs were exposed to the maximum nontoxic dose. In conclusion, these results show that CD86 is a more sensitive marker than CD83 when discriminating allergens from irritants but neither of these markers provides robust methodology for this purpose. CD *** * *** * * NiSO 4 (µm) CoCl 2 (µm) CuSO 4 (µm) PdCl 2 (µm) RFICD CrCl 3 (µm) K 2 Cr 2 O 7 (nm) PPDA (µm) DNCB (µm) *** SDS (µm) BCl 2 (µm) Propan-1-ol (mm) DMSO (mm) Fig. 3.1: CD83 expression on modcs after exposure to contact allergens and irritants for 48 hours. Relative fluorescence index is shown (fold increase compared to unexposed control cultures) derived from at least four different donors. Asterisks indicate a significant increase or decrease in expression compared to respective controls, p 0.05 (*) and p (***). ProefschriftMT.indd :12:48

72 72 CD * *** *** 2 2 * * NiSO 4 (µm) CoCl 2 (µm) CuSO 4 (µm) PdCl 2 (µm) Chapter 3 RFICD * * * * CrCl3 (µm) K 2Cr 2O 7 (nm) * * * PPDA (µm) * * DNCB (µm) *** *** CXCL8 secretion by dendritic cells predicts contact allergens from irritants SDS (µm) BCl 2 (µm) Fig. 3.2: CD86 expression on modcs after exposure to contact allergens and irritants for 48 hours. Relative fluorescence index is shown (fold increase compared to unexposed control cultures) derived from at least four different donors. Asterisks indicate a significant increase or decrease in expression compared to respective controls, p 0.05 (*) and p (***). Effects of TNF-α and LPS on allergen and irritant mediated surface marker expression As maturing factors such as TNF-α and LPS have been shown to increase CD83 and CD86 expression, the question arose as to whether low concentrations of these maturing factors could further enhance the allergen mediated increases in CD83 and CD86 expression. Hence, sub-optimal concentrations of TNF-α (15 ng/ml) or LPS (100 ng/ml), corresponding to percent of the maturing dose, were added to the culture medium 6 hours after allergen or irritant exposure and cultures were harvested 42 hours later. The tested substances, NiSO 4 (300 μm), DNCB (6 μm), PPDA (100 μm) and SDS (150 μm) were added in sub-optimal concentrations corresponding to half the maximum non-toxic dose in order to avoid cytotoxicity. Addition of the maturing factors TNF-α and LPS increased CD83 and CD86 expression slightly in control cultures (for TNF-α 1.7 ± 0.1 and 1.4 ± 0.1 fold increase respectively and for LPS 1.4 ± 0.2 and 1.2 ± 0.0 fold increase respectively). Only the response to NiSO 4 -exposed modcs, which was already significantly increased without the addition of maturing factors, could be further increased by the addition of either TNF-α or LPS and then only with regards CD83 expression (TNF-α: 1.8 ± 0.3 and LPS: 1.9 ± 0.3 fold increase above NiSO 4 alone). Therefore, in distinguishing the allergens NiSO 4, DNCB and PPDA from the irritant SDS no increase in the sensitivity of the assay was obtained. Similar results were 0 Propan-1-ol (mm) DMSO (mm) ProefschriftMT.indd :12:49

73 73 obtained when maturing factors and test substances were added simultaneously to the modc cultures (data not shown). Chemokine secretion after allergen and irritant exposure In order to find a more sensitive marker than CD83 and CD86 in predicting a contact allergen from an irritant, chemokine secretion was analysed in the culture supernatants after exposure of modcs to allergens or irritants (Fig. 3.3, table I). CXCL8 was the only chemokine whose secretion was found to significantly increase after exposure to all seven allergens tested. In contrast to allergens, exposure to irritants showed no increase of CXCL8 production, rather a slight decrease of CXCL8 secretion was seen. Similar to CD83 and CD86, exposure to NiSO 4 resulted in a greater increase in CXCL8 secretion than was observed after exposure to other allergens. The increase in CXCL8 secretion stimulated by NiSO 4 was in the same order of magnitude to that observed after exposure to LPS. Notably, trivalent chromium (CrCl 3 ) significantly increased CXCL8 secretion, whereas hexavalent chromium (K 2 Cr 2 O 7 ) did not. CXCL * 50 * 50 * * * * NiSO 4 (µm) CoCl 2 (µm) CuSO 4 (µm) PdCl 2 (µm) Delta CXCL8 production (ng/10 6 cells) CrCl 3 (µm) * * K 2Cr 2O 7 (nm) * PPDA (µm) * ** DNCB (µm) SDS (µm) BCl 2 (µm) Propan-1-ol (mm) DMSO (mm) Fig. 3.3: CXCL8 secretion after exposure of modcs to contact allergens and irritants. Immature DCs were exposed to three non-toxic doses of allergens and irritants for 48 hours (see Materials and Methods). Culture supernatants were analysed by ELISA. The increase in chemokine secretion above that of unexposed cultures is shown. Values are mean ± SEM of at least three independent experiments. Asterisks indicate a statistically significant increase or decrease in secretion compared to respective controls, p 0.05 (*), p (**) and p (***). ProefschriftMT.indd :12:49

74 74 Although CCL5 secretion increased after exposure to all seven allergens, the increase was only significant after exposure to NiSO 4, PdCl 2 and CrCl 3. Furthermore, exposure to one of the four irritants tested (SDS) also resulted in an increased secretion of this chemokine. Again, NiSO 4 resulted in a greater increase in CCL5 secretion than other allergens and this was again in the same order of magnitude to that observed after LPS exposure. CCL20 was only detectable in culture supernatants after exposure of modcs to nickel sulphate or LPS. CCL19 was not detectable before or after exposure to any contact allergen or irritant tested (data not shown). CCL17, CCL18 and CCL22 secretion did not distinguish allergens from irritants (Table 3.1). Chapter 3 Table 3.1. Chemokine secretion after exposure of modcs to allergens, irritants or LPS a CXCL8 CCL18 CCL20 CCL22 CXCL8 secretion by dendritic cells predicts contact allergens from irritants a Immature DCs were exposed to the highest non-toxic dose of allergens and irritants for 48 hours. The concentration of chemokines in culture supernatants were analysed by means of ELISA. The increase in chemokine secretion above that of unexposed cultures is shown. Values are mean ± SEM of at least three independent experiments. b Basal secretion of unstimulated cells: 17.6 ± 4.5 ng CXCL8/10 6 cells, ± 60.0 pg CCL5/10 6 cells, 48.5 ± 11.7 ng CCL17/10 6 cells, 55.1 ± 18.4 ng CCL18/ 10 6 cells, 0.0 ± 0.0 pg CCL20/10 6 cells, ± ng CCL22/10 6 cells. Bold indicates a significant increase or decrease in secretion compared to the respective control (p at least 0.05). Discussion In this study, next to conventional phenotypic changes, chemokine secretion was investigated after exposure of modcs to contact allergens and irritants. We show that exposure of modcs to contact allergens results in an increased secretion of CXCL8, whereas exposure to irritants fails to induce CXCL8 secretion. The idea that chemokine secretion may provide ProefschriftMT.indd :12:51

75 75 an alternative method for detecting contact allergens from irritants was triggered by the findings of Hashimoto et al. (2000) 3 using serial analysis of gene expression. This study showed that LPS induced DC maturation resulted in marked increases in gene transcription of a number of chemokines, notably CXCL8, CCL5, CCL17, CCL18, CCL19, CCL20 and CCL22. This finding was confirmed on the protein level in studies which showed that increased (CXCL8, CCL5, CCL20, CCL22) secretion occurred during DC maturation induced by either LPS, TNF-α or CD40ligand 2;17. During the last decade, several investigators proposed test systems in which contact allergens could be distinguished from contact irritants Monocytes derived from peripheral blood or CD34 + progenitors, were differentiated to idcs and exposed to non-toxic concentrations of allergens or irritants. Aiba et al. (1997, 1999, 2000) and Tuschl et al. (2000, 2001) demonstrated that exposure of modcs to contact allergens (e.g. nickel chloride, nickel sulphate, cobalt chloride, oxazolone, 2,4,6-trinitrobenzene sulfonic acid, 2,4-dinitrochlororbenzene, α-hexylcinnamaldehyde, eugenol) in contrast to irritants (e.g. SDS, DMSO, benzalkonium chloride) resulted in up regulation of CD54, CD83, CD80, CD86 and HLA-DR 4;5;6;13;14. In agreement with these reports, we found that exposure of modcs to allergens resulted in increased CD83 and CD86 expression. However, the sensitivity of the markers was low and the assay did not identify all allergens tested and therefore can not be used to predict the sensitising capacity of a chemical. Additionally, it was found that exposure of modcs to allergens or irritants together with low concentrations of maturing factors (TNF-α and LPS) did not significantly improve the sensitivity of the assay. Our finding that CXCL8 secretion only increased upon allergen exposure and rather decreases upon irritant exposure makes this a very promising chemokine for predicting contact allergens from non allergens. In contrast, CCL5, CCL17, CCL18 and CCL22 secretion were remarkably unpredictable, possibly related to their high basal secretion. As CCL19 secretion was detectable neither after LPS exposure nor after allergen or irritant exposure, it is possible that this chemokine is only upregulated at the mrna level, but is not secreted by DC into culture supernatants. CCL20 was only secreted after exposure to LPS or NiSO 4. The fact that CXCL8 secretion is only increased upon exposure to contact allergens and not contact irritants suggests a critical role of CXCL8 in allergic responses. CXCL8 attracts cells expressing CXCR1 and CXCR2, like neutrophils and idcs, but also specific subsets of T-cells, including natural killer cells, natural killer T-cells 19 and CD8+ T-cells 20, which are all potent interferon-γ producing cells. Taking into account that CXCL8 is produced within 3 hours after stimulation with TNF-α or LPS in vitro 2 and within 8 hours in vivo 21, it is most likely that CXCL8 secretion plays a pivotal role in the skin during the onset of LC migration from the epidermal layers. Additionally, CXCL8 may also indirectly cause T-cell accumulation via neutrophil attraction. In response to CXCL8, neutrophils can release chemotactic mediators, notably defensins and cathepsin G, which attract T-cells 22;23. Exposure of DCs to nickel sulphate clearly resulted in a stronger up regulation of CD83 and CD86 expression and also a greater CXCL8, CCL5, CCL17 and CCL20 secretion than any other allergen tested. This unique potential of nickel sulphate to mature DC may be explained by the concomitant triggering of several signal transduction pathways by this ProefschriftMT.indd :12:51

76 76 Chapter 3 CXCL8 secretion by dendritic cells predicts contact allergens from irritants allergen, notably p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK) and nuclear factor-kb (NF-kB), whereas DNCB only activates p38 MAPK but not ERK or NF-kB Interestingly, trivalent chromium induced more CD86 expression in modc than hexavalent chromium. Moreover, CXCL8 secretion was significantly induced only by trivalent chromium and not by hexavalent chromium. It is thought that hexavalent chrome penetrates the skin and is then reduced enzymatically to trivalent chromium, which combines with proteins as the hapten 27. Our results support this view and indicate that LC present in the skin are triggered by the reduced form of the allergen chromium. Thus, although the pro-hapten PPDA induced clear up regulation of CD86 and production of CXCL8 by modcs, distinct prohaptens such as hexavalent chromium cannot be handled by modcs. In conclusion, we have shown that exposure of modcs to allergens and irritants leads to differential chemokine secretion and maturation marker expression. Exposure to contact allergens but not contact irritants results in increased secretion of CXCL8 by modcs. Interestingly, CXCL8 is a potent attractant for, next to neutrophils, different subtypes of T cells. These results suggest that CXCL8 has a pivotal role in the induction and possibly also in the elicitation of allergic contact dermatitis. Our results warrant further large scale analysis of panels of potential contact allergens and irritants. ProefschriftMT.indd :12:51

77 77 References 1. Caux C, Ait-Yahia S, Chemin K et al. Dendritic cell biology and regulation of dendritic cell trafficking by chemokines. Springer Semin Immunopathol 2000;22(4): Sallusto F, Palermo B, Lenig D et al. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur J Immunol 1999;29(5): Hashimoto SI, Suzuki T, Nagai S et al. Identification of genes specifically expressed in human activated and mature dendritic cells through serial analysis of gene expression. Blood 2000;96(6): Aiba S, Terunuma A, Manome H, Tagami H. Dendritic cells differently respond to haptens and irritants by their production of cytokines and expression of co-stimulatory molecules. Eur J Immunol 1997;27(11): Aiba S, Tagami H. Dendritic cells play a crucial role in innate immunity to simple chemicals. J Investig Dermatol Symp Proc 1999;4(2): Aiba S, Manome H, Yoshino Y, Tagami H. In vitro treatment of human transforming growth factor-beta1-treated monocyte-derived dendritic cells with haptens can induce the phenotypic and functional changes similar to epidermal Langerhans cells in the initiation phase of allergic contact sensitivity reaction. Immunology 2000;101(1): Hulette BA, Ryan CA, Gerberick GF. Elucidating changes in surface marker expression of dendritic cells following chemical allergen treatment. Toxicol Appl Pharmacol 2002;182(3): De Smedt AC, Van Den Heuvel RL, Zwi BN, Schoeters GE. Modulation of phenotype, cytokine production and stimulatory function of CD34+-derived DC by NiCl2 and SDS. Toxicol In Vitro 2001;15(4-5): De Smedt AC, Van Den Heuvel RL, Van Tendeloo VF et al. Phenotypic alterations and IL-1beta production in CD34+ progenitor- and monocyte-derived dendritic cells after exposure to allergens: a comparative analysis. Arch Dermatol Res 2002;294(3): Manome H, Aiba S, Tagami H. Simple chemicals can induce maturation and apoptosis of dendritic cells. Immunology 1999; 98(4): Rustemeyer T, Preuss M, von Blomberg BM et al. Comparison of two in vitro dendritic cell maturation models for screening contact sensitizers using a panel of methacrylates. Exp Dermatol 2003;12(5): Staquet MJ, Sportouch M, Jacquet C et al. Moderate skin sensitizers can induce phenotypic changes on in vitro generated dendritic cells. Toxicol In Vitro 2004;18(4): Tuschl H, Kovac R, Weber E. The expression of surface markers on dendritic cells as indicators for the sensitizing potential of chemicals. Toxicol In Vitro 2000;14(6): Tuschl H, Kovac R. Langerhans cells and immature dendritic cells as model systems for screening of skin sensitizers. Toxicol In Vitro 2001;15(4-5): Yoshida Y, Sakaguchi H, Ito Y et al. Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line. Toxicol In Vitro 2003;17(2): Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994;179(4): ProefschriftMT.indd :12:52

78 Nagorsen D, Marincola FM, Panelli MC. Cytokine and chemokine expression profiles of maturing dendritic cells using multiprotein platform arrays. Cytokine 2004;25(1): Yoshida Y, Sakaguchi H, Ito Y et al. Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line. Toxicol In Vitro 2003;17(2): Chuntharapai A, Lee J, Hebert CA, Kim KJ. Monoclonal antibodies detect different distribution patterns of IL-8 receptor A and IL-8 receptor B on human peripheral blood leukocytes. J Immunol 1994;153(12): Qin S, LaRosa G, Campbell JJ et al. Expression of monocyte chemoattractant protein-1 and interleukin-8 receptors on subsets of T cells: correlation with transendothelial chemotactic potential. Eur J Immunol 1996;26(3): Chapter 3 CXCL8 secretion by dendritic cells predicts contact allergens from irritants 21. Griffiths CE, Barker JN, Kunkel S, Nickoloff BJ. Modulation of leucocyte adhesion molecules, a T-cell chemotaxin (IL-8) and a regulatory cytokine (TNF-alpha) in allergic contact dermatitis (rhus dermatitis). Br J Dermatol 1991;124(6): Chertov O, Michiel DF, Xu L et al. Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem 1996;271(6): Tani K, Su SB, Utsunomiya I, Oppenheim JJ, Wang JM. Interferon-gamma maintains the binding and functional capacity of receptors for IL-8 on cultured human T cells. Eur J Immunol 1998; 28(2): Arrighi JF, Rebsamen M, Rousset F et al. A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-alpha, and contact sensitizers. J Immunol 2001;166(6): Aiba S, Manome H, Nakagawa S et al. p38 Mitogen-activated protein kinase and extracellular signal-regulated kinases play distinct roles in the activation of dendritic cells by two representative haptens, NiCl2 and 2,4-dinitrochlorobenzene. J Invest Dermatol 2003;120(3): Boisleve F, Kerdine-Romer S, Rougier-Larzat N, Pallardy M. Nickel and DNCB induce CCR7 expression on human dendritic cells through different signalling pathways: role of TNF-alpha and MAPK. J Invest Dermatol 2004;123(3): Burrows D. The dichromate problem. Int J Dermatol 1984; 23(4): ProefschriftMT.indd :12:52

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80 80 Chapter 4 Kinomic profiling of MUTZ-3 myeloid cells discriminates between allergens and irritant ProefschriftMT.indd :12:52

81 81 Chapter 4 Kinomic profiling of MUTZ-3 myeloid cells discriminates between allergens and irritant ProefschriftMT.indd :12:53

82 82 Chapter 4 Kinomic profiling of MUTZ-3 myeloid cells discriminates between allergens and irritant ProefschriftMT.indd :12:53

83 83 Kinomic profiling of MUTZ-3 myeloid cells discriminates between allergens and irritant Abstract As exposure to sensitisers (allergens) most commonly results in dendritic cell maturation, sensitisers are likely to initiate signal transduction pathways different to those triggered by non-sensitisers (irritants). In this study, kinomic profiling was performed in order to identify new parameters for the detection of potential sensitisers. Myeloid cell line MUTZ-3 cells were exposed to allergens: DNCB, NiSO 4, or irritant: SDS. After 30 or 90 minutes exposure, kinase activities were determined by phosphorylation of peptide substrates corresponding to distinct kinase activities. The PepChip microarray data showed that NiSO 4 and DNCB resulted in altered phosphorylation of 196 and 341 peptides, respectively, out of the 1024 peptides tested. In contrast, only 51 peptides showed altered phosphorylation by the irritant SDS. Overall, phosphorylation of 74 of the 1024 peptides was specific for allergen exposure. These peptides are reported substrates for several kinases which are components of membrane receptor, phospholipase, MAPK and cell cycle pathways. In conclusion, this study provides a preliminary comprehensive survey of kinase activities which are distinctive for allergen exposure in a dendritic cell-like cell line. Kinomic profiling may allow us to determine distinct allergen-specific dendritic cell maturation pathways and may provide a novel tool to distinguish potential sensitisers from non-sensitisers. Introduction Allergic contact dermatitis (ACD) is a T cell-mediated, delayed-type hypersensitivity immune response induced by allergens. The dendritic cells (DCs) of the epidermis, known ProefschriftMT.indd :12:53

84 84 Chapter 4 Kinomic profiling of MUTZ-3 myeloid cells discriminates between allergens and irritant as Langerhans cells (LCs), play a central role in the initiation of allergic skin responses. Following encounter with an allergen, LCs become activated, undergo maturation and differentiate into potent immunostimulatory DCs which are capable of presenting antigens to T-cells. Currently, extensive research is aimed at understanding the initial phase of ACD as this understanding may lead to the development of much needed in vitro assays to distinguish potential sensitisers (allergens) from non-sensitisers (irritants). The need for non-animal alternatives for sensitization testing is becoming all the more important because of existing and pending EU regulations. Research on the initial phase (induction) of ACD was greatly facilitated when a method to generate large numbers of dendritic cells in vitro from peripheral blood-derived monocytes (modcs) was introduced 1. This breakthrough enabled studies to be performed on the effects of allergens on cultured dendritic cells. It was first shown in 1997 that allergens, but not irritants, can induce maturation of modcs as analysed by differential expression of cell surface markers such as CD86 and HLA-DR 2. Later, we showed that modcs exposed to allergens secrete more CXCL8 than modcs exposed to irritants indicating that this chemokine may predict a contact allergen from an irritant 3. Next to modcs, various DC cell lines have been used in an attempt to predict whether or not a chemical is a sensitiser 4-5. Earlier, we reported on the promising qualities of the human myeloid leukaemia-derived MUTZ-3 cell line for this purpose 6. This proliferating DC progenitor cell line has a novel characteristic in that it can differentiate in a cytokine dependent manner into immature dendritic cells closely resembling either LCs or dermal DCs and can mature upon exposure to cytokine cocktails, Toll-like ligands or chemicals 7. It was also described that the MUTZ-3 line might be suitable to discriminate allergens from irritants 8. Therefore, we decided to use MUTZ-3 cells to further explore options for the development of robust in vitro assays to predict the potency of a sensitizing compound. As exposure to allergens typically results in DC maturation, it is most likely that different allergens trigger common signal transduction pathways which are different to those triggered by irritants. Indeed, several distinct signalling pathways such as mitogen-activated protein kinases (MAPKs) and nuclear factor-kb (NF-κB) have been found to be associated with allergen-induced DC maturation 9. In vitro exposure of DCs to the allergens dinitrochlorobenzene (DNCB) and nickel sulphate (NiSO 4 ), was reported to lead to activation and translocation of p38 MAPK and Jun N-terminal Kinase (JNK) and ultimately to the up-regulation of CD80, CD83, CD86 and CCR Next to p38 MAPK and JNK, NiSO 4 may also trigger extracellular signal-regulated kinases (ERK) and NF-κB Until now, the role of other signalling pathways in allergen or irritant exposed DCs has not yet been investigated. With the development of array technology, kinase signalling pathways can now be readily unravelled A peptide array has been used to determine the kinase phoshorylation events induced in peripheral blood mononuclear cells (PBMCs) upon stimulation with lipopolysaccharide (LPS) 13. The peptide array contains spatially addressed mammalian kinase substrates, allowing for a broad detection of signal transduction pathways. This peptide array can be used to make a comprehensive description of the kinome in a cellular context. Here, we applied this novel technology to gain insight into signal transduction pathways involved in allergen-induced MUTZ-3 maturation and also to identify potential markers for application in in vitro assays aimed at distinguishing sensitisers from non-sensitisers. ProefschriftMT.indd :12:53

85 85 Materials and methods Culture and exposure of MUTZ-3 cell line The human-derived cell line MUTZ-3 was obtained from the German collection of micro organisms and cell cultures (DSMZ, Braunschweig, Germany) This MUTZ-3 cell line was maintained in the proliferating DC precursor-like phenotype by culturing in MEM-alpha with ribonucleosides and deoxyribonucleosides (Gibco, Paisley, United Kingdom), 20% v/v heatinactivated fetal calf serum (FCS) (Hyclone, Logan, UT, USA), 1% penicillin-streptomycin (Gibco), 2 mm L-glutamine (Invitrogen, Breda, The Netherlands), 50 µm 2-mercaptoethanol (Merck, Darmstadt, Germany), supplemented with 10% conditioned medium from the human renal carcinoma cell line 56376; 16. Cells were harvested and plated in 6 wells plates (Corning Incorporated Life Sciences, Acton, MA, USA) at 5 x 10 6 cells per well in 5 ml 1% heat-inactivated FCS in IMDM (BioWhittaker, Verviers, Belgium) overnight. Thereafter, MUTZ-3 progenitor cells were maintained in 1% FCS/IMDM and exposed to either medium (negative control), maturation cytokine mix (positive control: 50 ng/ml tumor necrosis factor alpha (TNF-α) (Strathmann Biotec, Hannover, Germany), 100 ng/ml IL-6 (Strathmann Biotec), 25 ng/ml IL-1β (Strathmann Biotec), 1 μg/ml PGE 2 (Sigma-Aldrich, St Louis, MO, USA)); or non-toxic doses of the following allergens: NiSO 4 (300 μm, Merck); allergen: DNCB (12 μm, Sigma-Aldrich) or irritant: sodium dodecyl sulphate (SDS; 37.5 μm, Sigma-Aldrich). Nontoxic concentrations corresponded to half the chemical concentration which resulted in 75% relative viability compared to unexposed cultures after 24 hours exposure. After only 30 or 90 minutes exposure, MUTZ-3 cells were harvested and lysed for kinomic profiling by means of pepchip analysis. After 24 hours exposure, parallel MUTZ-3 cultures were harvested and stained for analysis of cell surface marker expression by means of flow cytometry analysis (see also flow cytometry section). Viability analysis was performed using an annexin V and propidium iodide expression analysis kit in accordance to the manufacturer s specifications (Bender Medsystems, Vienna, Austria). Flow cytometry Expression of CD1a, CD14, CD34, Langerin, DC-SIGN, CD86 or HLA-DR was analysed by flow cytometry. Cell staining was performed using mouse anti-human-cd1a-pe (IgG 1 ), mouse anti-human-cd14-fitc (IgG 2a ), mouse anti-human-cd34-pe (IgG 1 ), mouse anti-human-dc- SIGN-FITC (IgG 2a ), mouse anti-human-cd86-pe monoclonal antibody (IgG 1 ), mouse antihuman HLA-DR-FITC (IgG 2a, all from BD Biosystems, mountain view, CA, USA) or mouse anti-human-langerin-pe (IgG 1, Beckman Coulter, Oakley Court, UK). Monoclonal mouse IgG 1 -PE, IgG 2b -FITC or IgG 2a -FITC (BD Biosystems) was used as an isotype control to assess non-specific binding. Cells were incubated with antibodies for 30 minutes, washed in PBS plus 0.1% BSA (Sigma-Aldrich) and 0.1% sodium azide (Merck) and resuspended in the same buffer for FACS analysis. Flow cytometry was performed with a FACScan (BD Biosystems) and results were analysed using Cell Quest software (BD Biosystems). ProefschriftMT.indd :12:54

86 86 Cell lysates of MUTZ-3 cells Chemical or cytokine stimulation of 5 x 10 6 MUTZ-3 progenitor cells was terminated by washing in ice-cold PBS (unexposed control cultures were treated similarly). MUTZ-3 lysates following stimulation with control, MCM mimic, DNCB, NiSO 4 or SDS of two independent experiments were harvested and lysed with 100 μl lysis buffer (Cell Signaling Technology, Beverly, MA, USA) containing a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Thereafter, cell lysates were sonicated for 10 min and subsequently centrifuged for 10 min. Supernatants, containing the kinases, were used immediately for PepChip experiments. Chapter 4 Kinomic profiling of MUTZ-3 myeloid cells discriminates between allergens and irritant PepChip experiments The cell lysates (60 μl) of two independent experiments were added to 10 µl of filter-cleared activation mix, containing 50% glycerol (Merck), 50 μm ATP (Boehringer Mannheim), 60 mm MgCL 2 (Sigma-Aldrich), 0.05% Brij-35 (Sigma-Aldrich), 0.25 mg/ml BSA (Roche Applied Science), 2000 μci/ml 33 P-ATP (Amersham Biosciences, Little Chalfont, UK). The third generation peptide arrays (Pepscan Presto, Lelystad, The Netherlands), containing 1024 different pseudosubstrates spotted in triplicate on a glass slide were incubated with lysates containing activation mix at 37 C in a humidified stove for 120 min. Next, the peptide arrays were washed twice in PBS/Tween20, twice in 2 M NaCl/PBS, and twice in demineralized H 2 O and then air-dried. Slides were exposed to a phosphorimager plate (Molecular Dynamics, Sunnyvale, CA, USA) for 72 hours, and the density of the spots was measured with a phosphoimager Storm (Molecular Dynamics). Data acquisition and statistical analysis of peptide arrays Data acquisition of the peptide arrays was performed with a phosphoimager and array software (Bio-Rad Laboratories, Hercules, CA, USA). After data acquisition and quantification using median spot densities, the data were exported to a spreadsheet program (Microsoft Excel 2002, Microsoft Redmond, WA, USA). To correct for inter-array variation, normalisation of the spot densities was performed by subtracting the local background. In addition, the variation between arrays and the triplicates within the two individual experiments was reduced by normalisation to the 90 th percentile of the overall intensity of each array. Peptides corresponding to inconsistent data (SD between data points > 1.96 of the mean value) were excluded from further analysis. Consistent data for a peptide were averaged and included for dissimilarity measurement to identify kinases of which activity was either significantly increased or decreased. Differential kinase activities in lysates from allergen or irritant exposed MUTZ-3 cells were determined by significant fold change ratios of the combined values of phosphorylated peptides resembling a substrate for kinase activity. Significance analyses were performed using a minimal modification for the algorithm originally developed for microarray analysis (www-stat.stanford.edu/~tibs/sam). The full list of peptides spotted on the peptide array can be found online ( ProefschriftMT.indd :12:54

87 87 Results Effect of allergen or irritant exposure on maturation marker expression In order to define the phenotypic status of MUTZ-3 cells in our system, we measured the expression levels of a series of markers before stimulation. MUTZ-3 cells have the characteristics of undifferentiated myelo-monocytic cells. Only 14.7 ± 5.3 % of the cells showed CD86 expression. Almost all cells expressed HLA-DR (88.9 ± 2.4 %), however the mean expression of this molecule was low. Together, these features reflect the immature state of the MUTZ-3 cells. The cells do not express CD1a or Langerin, indicating that they do not have a Langerhans cell-like phenotype. The same holds true for the dermal DC marker, DC-SIGN, which was also negative. The phenotype of the undifferentiated MUTZ-3 immature progenitor cells was in agreement with the observations made by ourselves and others 7;16;18. Fig. 4.1: CD86 and HLA-DR expression on MUTZ-3 cells after exposure to contact allergens and irritant for 24 hours. MUTZ-3 progenitor cells were cultured in the presence or absence of contact allergens (NiSO 4, DNCB), irritant (SDS) and the positive control MCM. After 24 hours incubation, surface expression of markers on exposed MUTZ-3 cells was determined. In histogram plots, effects of allergens, irritant and positive control MCM mimic on CD86 and HLA-DR expression are shown (MCM: 100 ng/ml IL-6, 50 ng/ml TNF-α, 25 ng/ml IL-1β, 1 μg/ml PGE 2 ; NiSO 4 : 300 μm; DNCB: 12 μm; SDS: 37.5 μm). Bold lines represent expression of CD86 or HLA-DR. Dotted line represent isotype controls. In the upper right corner of the histogram plots, the mean fluorescence of the marker is shown. Results are representative of three independent experiments. It was shown that MUTZ-3 progenitor cells may be used to discriminate allergens from irritants 8. To evaluate the potential of the chemicals compared to the well-established maturing positive control MCM mimic to initiate maturation of MUTZ-3 cells in this study, CD86 and HLA-DR expression was measured with and without stimulation. As depicted in Fig. 4.1, MCM mimic upregulated the expression of both CD86 and HLA-DR. Allergen exposure ProefschriftMT.indd :12:55

88 88 (NiSO 4 and DNCB) also resulted in upregulation of these markers, whereas irritant exposure (SDS) resulted in downregulation of CD86 and HLA-DR. This is in line with our previous observations using monocyte-derived DCs in which these allergens induced maturation, in contrast to the irritant SDS which rather suppressed 3. Chapter 4 Kinomic profiling of MCM mimic treated or untreated MUTZ-3 cells Having confirmed that MUTZ-3 cells were capable of increasing cell surface expression of markers characteristic of DC maturation upon exposure to maturing factors, we performed kinome profiling by means of pepchip analyses. In silico array analysis allows to identify consensus amino acid phosphorylation sequences of most prominent kinases present in the mammalian genome. As reported earlier, the peptide sequences are derived from known kinase substrates. It should be noted that the tables show potential kinases which may or may not be present within MUTZ-3 lysate but which are known to phosphorylate the peptide motives. Some kinases are shown next to more than one peptide since the peptide can in principle be phosphorylated by more than one kinase. Arrays were constructed by spotting chemically synthesized soluble peptides, which were covalently coupled to polymer-coated glass substrates. Each array consisted of 1024 peptides each spotted in triplicate areas on a single slide 13. Kinomic profiling of MUTZ-3 myeloid cells discriminates between allergens and irritant Fig. 4.2: Phosphorylation of peptide arrays by cellular lysates of control or MCM mimic stimulated MUTZ-3 cells. MUTZ-3 cells were either stimulated with MCM mimic (B) or left untreated (A), followed by lysis of the cells and incubation of the peptide array in the presence of [γ- 33 P]-ATP. The array consisted of 1024 peptides spotted in triplicate peptide regions. Results shown are representative of such a triplicate peptide region on an array for one of the two independent experiments. Each spot represents phosphorylation of a specific substrate through kinase activity in the different lysates. Square = increased phosphorylation, circle = decreased phosphorylation. ProefschriftMT.indd :12:56