DOI 10.1111/j.1365-2133.2007.08264.

Immune response proles in human skin

T. Meyer, * E. Stocketh and E. Christophers
*Institute for Immunology, Clinical Pathology and Molecular Medicine, Lademannbogen 61, 22339 Hamburg, Germany Department of Dermatology, University of Berlin, HTCC, Chariteplatz 1, 10117 Berlin, Germany Department of Dermatology, University of Kiel, Schittenhelmstrae 7, 24105 Kiel, Germany

Summary
Correspondence
Thomas Meyer. E-mail: meyer@labor-lademannbogen.de

Accepted for publication

4 September 2007

Key words adaptive immunity, epithelial defense, innate immunity, TH17 cell, toll-like receptor Conicts of interest
ES acts as a consultant to Meda Pharma. TM and EC declare no conicts of interest.

In addition to the function as a physical barrier human skin has been shown to be an important immune organ displaying various defense mechanisms, which can be divided into three major functional compartiments: (i) Epithelial defense, which is characterized by antimicrobial peptides and proteins (AP) and which can be induced in inammatory lesions but also in the absence of inammation. (ii) Innate-inammatory immunity, which involves recognition of microbial compounds by particular receptors like Toll-like receptors (TLR) and subsequent activation of signalling pathways resulting in expression of pro-inammatory cytokines and interferons, as well as genes of adaptive immunity. Interferon a (IFNa) produced by plasmacytoid dendritic cells (DC) may stimulate myeloid DC to produce IL-12 resulting in classical T-cell activation or to produce IL-23 activating IL-17 producing T-cells (IL-23 IL-17 pathway). (iii) Adaptive immunity, which is based on antigen presenting cells, T-cells and B-cells and which is characterized by specicity and memory. In contrast to epithelial defense and innateinammatory immunity, adaptive immune functions provide slowly reacting protection. Recent improvements of our knowledge of dysregulated immune pathways associated with inammatory skin diseases represent an important basis of novel immunomodulatory treatment modalities.
Epithelial defense has only recently been discovered in human skin3 whereas the leukocyte driven inammatory response has been known since a long time as an innate (previously called nonspecic) arm.4 The third arm (adaptive immunity) has by far been given greatest attention in medicine. With specicity and memory as outstanding features this system provides powerful, however, slowly reacting protection of unlimited diversity. In addition, adaptive immunity is of principal importance in immune surveillance against viruses and transformed cells. On the other hand in humans this (adaptive) skin immune system5 appears responsible for a greater number of diseases than any of the other two defense systems. In the following some aspects of this skin immune system will be briey discussed.

With a surface area of approximately 17.000 cm2 human skin is one of the largest organs of the body. Located at the interface between environment and living organ systems skin serves a variety of functions. These include 1 maintaining an effective barrier against loss of body uids and protection against chemical and physical injury, 2 protection against UV injury together with adequate radiation damage repair and 3 the capacity for rapid wound healing along with a powerful defense armentarium against invading microorganisms. Human skin is covered by the epidermis, which partly consists of 1720 layers of coherent, attened cells, the Stratum corneum.1,2 As skin is constantly exposed to the outside world physical injury is among the most common threats eventually leading toward lethal outcomes. Thus, during phylogeny a variety of defense systems has emerged which actively serve to maintain cutaneous integrity and immunity. Players participating in this scenario consist of protective antimicrobial peptides (APs) as well as inammatory cells and signal substances in conjunction with sessile skin cells forming a skin immune armentarium. Functionally this consists of three major compartments: 1 Epithelial defense 2 Innate-inammatory immunity and 3 Antigen-elicited, adaptive immunity.
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Epithelial defense
Chemical protection by peptides and proteins has rst been observed in insects (Drosophila) and plants.6,7 The rst antimicrobial peptide (AP) found in human skin was Lysozyme.8 At present about ten APs have been detected in human skin (Table 1) and more of these compounds will be discovered. RNAse 7 and psoriasin (S100A7) represent constitutively expressed APs, whereas human beta defensin (hBD) 24 as

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2 Immune response proles in human skin, T. Meyer et al. Table 1 Antimicrobial peptides in human skin Lysozyme Human beta defensin 1 (hBD1) hBD 24 RNAse 7, 8 Psoriasin CAP 18 LL-37 Dermcidin

dendritic cells (DC) and memory T cells (seen with hBD2 and hBD3) as well as neutrophils, T cells and monocytes (seen with LL-37).13,14

Innate (inammatory) immune response

In order to elicit inammatory responses innate immunity employs germline-encoded pattern recognition receptors to recognize microbial compounds. TLRs have been shown to act as primary sensors by binding of particular structures present on bacteria, viruses, fungi as well as protozoa [also called pathogen associated molecular patterns (PAMPs)15]. There are 10 members of the TLR family in humans (Table 2). Organ systems exposed to environmental inuences e.g. skin, lung, and intestines show different colonisation with microbial symbionts. In consequence, gut epithelium as compared to skin or lung shows different defense strategies in host-bacterial interaction.16 For these nonsterile organs with permanent contact to micro-organisms it is important to prevent constant activation of innate immune functions, which would potentially compromise organ function. To maintain homeostasis several regulatory mechanisms have evolved that control innate immunity. Due to the heterogeneity of microbial colonization homeostasis is achieved through different, organ-specic mechanisms involving TLRs. This includes differences in TLR activation thresholds, tolerance, and varying receptor proles in distinct organs, as well as different expression of negative regulators of TLR signalling and environmental molecules regulating TLR function (e.g. IFNa, IL-10 or TGFb [Transforming growth factor beta]).16 Binding of PAMPs to TLRs reects early infection with activation of cellular signalling pathways including transcription factor NFkB (Nuclear factor kappa B) and activation of MAP (mitogen-activated protein) kinases p38 and JNK (jun amino terminal kinase). These factors regulate expression of many genes involved in inammation and immunity. Thus, proinammatory cytokines and interferons, but also adaptive immune functions such as MHC (major histocompatibility
Table 2 Toll-like receptors and their ligands

well as LL-37 are inducible. As skin is practically soaked with (mostly keratinocyte derived) defense molecules (within microgram range per g tissue)4 their immediate availability provides a powerful chemical shield for instance in wounding. In fact most of the peptides and proteins are discovered in inamed skin, notably in psoriatic scales.9 Interestingly, only few of the APs exhibit broad antimicrobial activities (e.g. hBD3), instead the majority is directed against either gram-negative (e.g. hBD1, hBD2) or gram-positive bacteria or fungi (Lysozyme, RNAse7, CAP 18 LL-37). A degree of specicity is also seen with psoriasin which primarily acts against E. coli.10 Principal producers of APs are keratinocytes. They respond to a variety of stimuli including IL-1, TNFa (Tumour necrosis factor alpha), IGF-1 (Insulin-like growth factor 1) as well as bacterial membrane substances.3 Recently, IL-22 has been detected as potent cytokine stimulating keratinocytes to produce defense molecules, proteases and growth factors (see below). While regulatory pathways have become better understood,3 so far receptors for AP induction have been hiding from detection. One may assume that receptors different from known innate receptors e.g. Toll-like receptors (TLRs)11 are active. So far studies have shown induction of hBD2 via TLR4.12 In certain situations APs may participate in the adaptive immune response by serving as chemoattractants for immature

Receptor Ligands TLR 1 TLR 2 TLR 3 TLR 4 TLR 5 TLR 6 TLR 7 TLR 8 TLR 9 TLR10 Triacylated lipopeptidesa Peptidoglycan, bacterial lipoprotein, zymosan, lipoteichoic acid, LPS (Porphyromonas gingivalis, Leptospira interrogans), GPI-anchor proteins (Trypanosoma cruzi.) ds RNA LPS (gram-negative bacteria), F-Protein (RSV), Hsp60, Fibronectin domain A Flagellin MALP-2 (Mycoplsma)b, phenol-soluble modulin (Staphylococcus epidermidis)b ssRNA, Loxoribine, Bropirimine, Guanosine analogs, Imiquimod, Resiquimod ss RNA, Resiquimod, Loxoribine, Bropirimine Unmethylated CpG-DNA (bacteria and viruses) No ligands found yet

LPS, Lipopolysaccharid; GPI, glycosylphosphatidylinositol; MALP, macrophage activating lipopeptide. a Ligands recognized by TLR1 + TLR2 b Ligands recognized by TLR2 + TLR6

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Immune response proles in human skin, T. Meyer et al. 3

LPS (P. gingivalis) PG (S. aureus)

LPS (E. coli) dsRNA TLR4

ssRNA, CpG-DNA Imiquimod, Resiquimod

TLR2

MD2/ CD14

TIR

TRAM

TLR3
MAL MyD88 TIR TRIF

TIR MyD88 TRIF MAL TIR MyD88

TLR7,8,9

Fig 1. Differential gene expression of TLRsignalling. LPS, Lipopolysaccharid; PG, Proteoglycan; TIR, Toll-IL1-receptor; MAL, MyD88 adaptor-like; TRIF, TIR-related adaptor protein inducing interferon; TRAM, TRIFrelated adaptor molecule; IKK, Inhibitor of NFkB kinase; TBK, TANK-binding kinase; IRAK, Interleukin-1 receptor associated kinase; IRF, Interferon regulatory factor.

IKK complex

TBK-1

IKK complex

IRAK-1

NFkB

IRF3

NFkB

IRF7

TNF, IL1, IL6, IL12 IL8, MIP1a RANTES CD40, CD80, CD86

IFN

TNF, IL1, IL6, IL12 IL8, MIP1a RANTES CD40, CD80, CD86

IFN

complex)-proteins, co-stimulatory signals and adhesion molecules become activated.17,18 Furthermore, signal transduction induced upon ligand binding to TLR favours a complex interplay of a growing number of adaptor proteins.19 MyD88 was the rst adaptor molecule identied. TLR and MyD88 associate via common Toll-IL1receptor domain, which results in recruitment of kinases (IRAK1 and IRAK4). Phosphorylated IRAK1 then leads to activation of protein kinase TAK1 (TGF-activated kinase) which nally liberates NFkB from the Inhibitor of NFkB kinase complex. This pathway of NFkB activation has been seen with IL1R, but also represents the main pathway of TLR signalling, except for TLR3. Next to MyD88 several other adaptor proteins like MyD88 adaptor-like, TIR-related adaptor protein inducing interferon (TRIF) and TRIF-related adaptor molecule have been identied to be involved in TLR signalling. Importantly, different TLRs signal through different combinations of adaptors, resulting in activation of different transcription factors and diverse gene induction (see Fig. 1). Within the context of this report it is of relevance that ligand binding to intracellular receptors TLR7 and TLR 9 mainly results in activation of IRF7 and induction of IFNa, an important factor for antiviral defense and as shown below in a certain group of diseases.20

Toll-like receptor expression in different cells and tissues

Expression of TLRs has been detected in various human tissues with varying expression levels.21 In general, organs involved in immune response or exposed to environment, e.g. spleen, skin and lungs were found to have signicantly higher TLR expression, than for instance the brain, liver and skeletal muscles.21
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TLR expression is also increased in peripheral blood mononuclear cells with large differences of TLR expression levels between different cell types. For example, TLR 2, 4 and 8 are expressed predominantly in monocytes and macrophages22,23 with the highest level shown for TLR2 in monocytes. Bloodderived myeloid dendritic cells (mDC) express TLR 2, 3, 4, 5 and 8,2325 while TLR 7, 9 and 10 were found mainly on plasmacytoid dendritic cells (pDC) and B-cells.22,26 Of importance pDCs are the primary class I IFN producing cells.27 Interestingly, Imiquimod and Resiquimod act as TLR7 agonists28 and were shown to induce IFNa and IFNx in pDCs isolated from blood.29 In human skin TLRs are expressed in both DC and keratinocytes. Human keratinocytes constitutively express mRNA of TLR 1, 2, 3, 4, 5, 6, 9 and 10 not, however, of TLR 7 and 8.3032 Functionality was also shown for TLR 2, 3, 4, 5 and 9.30,31,33 Langerhans cells (LC) were also shown to express TLR 1, 2, 3, 5, 6, and 10.34 In another study LCs also respond to TLR 4, TLR 7 and TLR 9 ligands.35 Among DCs, however, TLR 8 is usually expressed in mDCs, while TLR 7 was found predominantly in pDCs.23 Recently, the accumulation of pDC-like cells has been described in the dermis of mice topically treated with TLR 7 agonist Imiquimod.36 pDCs were also identied in psoriatic skin lesion.37 Importantly, topical application of TLR7 agonist Imiquimod exacerbates the psoriatic lesion,38 indicating that pDCs are major effector cells of the immune response induced by Imiquimod.

Th17-induced inammation
Recently, further differentiation of immune defense has been elaborated.39,40 This arm involves cytokines as well a distinct set of lymphocyte subpopulations. Central role in this pathway

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4 Immune response proles in human skin, T. Meyer et al.

pDC

mDC IL12 IFN

Th1

IFN

IL23, TGF, IL6 EC

G-CSF, IL8, NO G-CSF

Th17 IL17 IL22 E MP KC KC

Fig 2. The IL-23 Th17 pathway in inammation. pDC, plasmacytoid dendritic cell; mDC, myeloid dendritic cell; Th17, IL-17 producing T cell; EC, endothelial cell; PMN, polymorphonuclear cell; MP, macrophage; F, broblast; E, epithelial cell; KC, keratinocyte; APs, antimicrobial peptides.

PMN

TNF, IL1, IL6

APs Proteases

is played by pDC and a new T cell subtype called Th17.40 As shown by Nestle et al.37 pDC can be activated by a variety of ligands including virus RNA, HSP (Heat shock protein) or bacterial products to produce IFNa with production being 1000 times higher compared to any other cell. The responder cell for this excess IFNa is noted as mDC. Following stimulation via IFNa these latter cells produce IL-12, whereby classical T cell activation, marked by secretion of IFNc, granzyme and preforin and others, proceeds.41 mDC are also seen to produce IL-23 which is structurally related to IL-12, sharing a common subunit (p40). IL-12 is known as the key promoter of naive T cells to multiply and differentiate into Th1 cells and cytotoxic T cells, while IL-23 causes activation of a IL-17 producing T cell, which is hence called Th17.39,40 Recent evidence suggests that under the co-stimulatory effect of TGFb and IL6 Th17 cells develop from nave T cells. Th17 cells once activated by IL-23 become important actors in the ensuing inammatory program in that IL-17 stimulates various cells to produce pro-inammatory cytokines: under the stimulus of IL-17 stromal cells, endothelial cells and others produce IL-1, TNFa, IL-6, IL-8 and GM-CSF [Granulocytemacrophage colony stimulating factor].42 These mediators are chemokines themselves (e.g. IL-8) or powerful stimulators of leukocyte activation and migration.43 The IL-23 IL-17 pathway represents an important proinammatory arm of the innate defense with leukocyte inltration and phagocytosis as ultimate goals. It bears relevance to psoriasis and it has been suggested that psoriasis rather than being a Th1 disease actually is a Th17 disease.40 Th17 cells have recently been shown also to produce IL-22 and further, a receptor for IL-22 is expressed by keratinocytes and other epithelial cell e.g. in the gut.4446 This ligand-receptor binding in keratinocytes causes up-regulation of defense molecules (e.g. defensins), but also proteases, S100 proteins and chemokines.4749

Apparently by these two arms of the innate defense armentarium (IL-17 and IL-22) not only inammation is initiated but also keratinocytes become active players (see Fig. 2). In this context keratinocytes not only respond to environmental pathogens by producing antimicrobial defense molecules and also, via the IL-22 receptor, interact with endogenous dendritic cell derived signals. One of the key TLRs capable for sensing viral nucleotides on pDCs is TLR7, which also binds Imiquimod. Several reports have shown that Imiquimod applied to skin can exacerbate psoriasis.50,51 As IL-23 and IL-17 are found to be up-regulated in psoriasis52,53 this IL-17 regulated pathway could play a signicant role in initiating psoriatic lesions. Attraction of pDCs, which are almost absent in normal skin, to inamed skin areas is mediated by chemotactic peptides like IL8 and complement split products, like C5a and its desarginated form C5a-des-Arg. These act as powerful chemoattractants and are present in increased amounts in psoriatic skin lesions.54,55 The complement cascade consists of a set of proteolytic enzymes and represents a component of the humoral innate immune system. Complement can be activated by three separate pathways (classical antibody-mediated, alternate properdine-induced, and lectin MASP (Mannose binding lectinassociated serine protease)-mediated).56 Activation nally leads to formation of membrane attack complex, which kills bacteria by cell lysis. During complement activation the split products C3a and C5a are generated, which have anaphylactic and chemotactic properties. In serum both are des-arginated by carboxypeptidase N to less potent fragments C3a-des-Arg and C5a-des-Arg. C5a-des-Arg, like C5a binds to C5a receptor (C5aR).57 Recently, expression of C5aR and C3aR was shown in immature pDCs of skin lesions from patients with LE and contact dermatitis.58 Considering signicant amounts of C5a and C5a-des-Arg in psoriatic skin lesions complement mediated attraction might be relevant for inltration of pDCs in psoriatic skin.
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Immune response proles in human skin, T. Meyer et al. 5

Induction of adaptive immune functions

A crucial step of the induction of adaptive immune reactions is reected by activation of DC, which are professional antigen processing and presenting cells. In skin epidermal LC and dermal dendritic cells (DDC) represent the most important types of DC. Resident epidermal LCs are characterized by expression of CD1a, CD1c, Langerin, E-cadherin and membrane ATPase and by the presence of Birbeck granules. These rod-shaped membrane structures are absent in resident DDC. Also, in contrast to LC, DDC do not express Langerin, E-cadherin, and membrane ATPase, but do express clotting factor FXIIIa, as well as CD1b and CD1c.59 Dendritic cells take up and process peripheral antigens, and after migration to lymphoid organs, present them to nave T cells. In addition binding of PAMPs to TLRs results in activation of DC by NFkB-induced expression of chemokines, cytokines, MHC class I and II antigens, co-stimulatory signals, and adhesion molecules.19 Migration of skin DCs to draining lymph nodes through afferent lymphatics is associated with functional and phenotypic changes. While migrating centrally LCs loose their Birbeck granules and downregulate CD1, langerin and Ecadherin. In contrast, due to TLR activation, surface proteins important for T-cell activation like MHC-proteins, CD40, CD58, ICAM-1, CD80, and CD86, are induced.59 During interaction with nave CD4 T-cells, which have entered the lymph nodes through high endothelial venules, DCs can induce differentiation of these cells. Usually, differentiation into two subsets is distinguished: Th1, induced by IL12 and promoting cellular immunity and Th2, induced by IL4 and promoting humoral immunity.41 Two pathways of antigen presentation in DCs have been described: an endocytic pathway, which results in binding of exogenous antigens into MHC II molecules for presentation to CD4+ T cells, and an endogenous pathway which results in association of endogenous antigens to MHC I molecules for presentation to CD8+ T cells.60 Moreover, a third pathway of antigen uptake and presentation exists, which depends on cross presentation and represents an important mechanism for presentation of exogenous antigens to CD8 T cells through a process called cross priming.61 In addition, nonprotein antigens (mycobacterial glycolipids) can be internalized after binding to langerin. After loading to CD1 molecules in Birbeck granules glycolipids are recycled to cell membrane and presented to CD4 and CD8 T cells and NK (Natural Killer) cells.62,63 In addition to immunostimulatory functions of LCs and DDCs in response to potential pathogens skin DCs exert regulatory functions to maintain homeostasis of the skin and to prevent excessive inammatory reactions.64,65 In the absence of danger signals under steady state conditions, LCs take up epidermal antigens and transport them to local lymph nodes. Here they act as promoters of peripheral tolerance to skin antigens by inducing anergy or apoptosis of nave T cells or by stimulation of regulatory T cells (Treg, CD4+, CD25+).66
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Effectors of adaptive immunity

T cells become activated by interaction with DC in local drain ing lymph nodes. Only nave T cells with TCRs, specic for the antigens presented, are stimulated. In addition to TCRMHC-antigen interaction (tri-molecular complex) co-stimulatory signals are necessary to induce clonal expansion of appropriate T cells (dened by antigen specicity). Co-stimulatory signals are represented by interactions of surface molecules, like CD40-CD40L, CD28-CD80 86 and CD2-CD58, as well as cytokines (IL-1, IL-6 TNFa). Absence of these secondary signals results in nonreactivity (anergy) to the respective antigens. Activated T cells leave the lymph node and enter the skin by interaction with specic homing receptors. Most important is the interaction of CLA (cutaneous lymphocyte antigen) with E-selectin expressed on endothelial cells of inamed skin. CLA is not expressed in nave T cells, but found in about 30% of circulating memory T cells and T cells in inamed skin are mainly CD45Ro- and CLA-positive.67,68 Next to CLA, several chemokine receptors (CCR4, CCR10) are also associated with skin homing of T cells.69,70 Among T cells, subpopulations with different functions are distinguished. CD4+ T cells (T helper cells) are mainly involved in immune responses to foreign antigens, while CD8+ T cells (cytotoxic T cells) provide antiviral and antitumor immune reactions. According to the cytokines produced, T helper cells were divided into Th1 and Th2 cells: Th1 cells produce IL-2, TNFa and IFNc and induce cell mediated immune responses. Th2 cells produce IL-4, IL-5, IL-10 and IL13 and promote humoral immune responses, as well as growth of eosinophils and IgE production. Th2 responses are frequently associated with allergic diseases, like atopic dermatitis. Th17 cells represent another subset of CD4 and CD8 T cells, which are generated different from Th1 and Th2 cells, as described above. As shown Th17 cells were suggested to be involved in the pathogenesis of psoriasis and other autoimmune diseases.46,71 Other populations of T cells with suppressive regulatory properties, important to control autoreactive immune responses, were recently identied. CD4+ CD25+ regulatory T cells (Tregs) have suppressive effects and induce anergy and tolerance by cell contacts probably via membrane-bound factors. The exact mechanism is still unknown, but does not require soluble suppressive cytokines. Th3 and type 1 T regulatory cells (Tr1) are also immunosuppressive, which, in contrast to Tregs, depends on production of immunosuppressive cytokines (IL-10, TGFb).72

Outlook
In human approximately 2000 separate disease entities are presently recognized and nearly half of these are inammatory. Thus the identication of inammatory pathways in skinincluding innate and adaptive, as well as stimulatory and regulatoryhas been revolutionized by the rapidly increasing knowledge especially of innate immunity. All of

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6 Immune response proles in human skin, T. Meyer et al.

this makes the skin an appropriate target for immunomodulatory therapy. As our knowledge of underlying immune dysregulations in skin diseases is improving, activation or suppression of distinct immune functions by topical application of immune response modiers (the number of which is steadily increasing) will provide extending treatment modalities in near time.

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