Chemokine networks in inflammatory skin diseases

ARTICLE

Auteur(s) : Saveria PASTORE, Francesca MASCIA, Feliciana MARIOTTI, Cristina DATTILO, Giampiero GIROLOMONI

Istituto Dermopatico dell’Immacolata, IRCCS, Via Monti di Creta 104, 00167 Roma, Italy

Article accepted on 25/03/2004

The recruitment of T cells and other leukocytes at the site of skin inflammation is a critical step for an efficient response to potentially dangerous signals as well as in the pathogenesis of chronic inflammatory skin diseases. Leukocyte trafficking encompasses a series of highly coordinated events and molecules, such as endothelial cell selectins, which mediate tethering and rolling of leukocytes on the vessel wall; chemokine receptors on leukocytes, whose engagement induces integrin activation; and integrins, which are responsible for firm adhesion preceding leukocyte extravasation. Leukocytes are then attracted into the dermal and epidermal compartments by gradients of chemoattractants produced by resident and immigrated cells, and maintained in situ by adhesion molecules [1]. The static components of the skin immune system, represented by keratinocytes, mast cells, endothelial cells and fibroblasts, as well as dendritic cells (DCs) and T cells, are now recognized as a major source of numerous chemoattractants, including lipid metabolites and chemokines. Certain chemokines (e.g. TARC/CCL17) and selectins are constitutively expressed on microvascular endothelial cells to allow an effective skin immunosurveillance [2], but most chemokines are produced in response to environmental signals such as inflammatory cytokines, pathogens and physical-chemical stressors [3].
This review will provide an overview of our current understanding of the complex chemokine network active during inflammatory skin diseases, in particular allergic contact dermatitis (ACD), psoriasis and atopic dermatitis (AD). The pattern of chemokine expression shows overlapping features but also important differences in these disorders (Table I) due to distinct sources and types of pro-inflammatory signals involved in chemokine induction, and the inherent capacity of resident skin cells to produce chemokines.

Table I. Chemokine expression in inflammatory skin diseases

Chemokines and their receptors

Chemokines are a superfamily of structurally related, small (67 to 127 amino acids) proteins that regulate the traffic of all leukocyte subsets [3-9]. They are classified in four subfamilies according to the position of two highly conserved cysteine residues at the N-terminus of their molecule, which are either separated by a single amino acid (CXC) or are adjacent (CC). CX₃C and C chemokines are minor structural subfamilies, with two cysteines separated by three aminoacids and a single cysteine motif, respectively. Upon secretion, chemokines bind to extracellular matrix and cell membrane proteoglycans forming stable gradients from their site of release. Together with SR-PSOX/CXCL16, fractalkine/CX₃CL1 forms an exception to this rule, as these two chemokines are expressed as membrane integral proteins destined to be shed from the membrane [9, 10]. Although their main function is to regulate cell trafficking, chemokines also govern leukocyte activation and differentiation [11, 12]. The specific effects of chemokines on target cells are mediated by seven transmembrane spanning, G-protein coupled receptors [13, 14]. To date, more than 50 human chemokines and about 20 chemokine receptor-encoding genes have been identified [http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html]. Chemokines appear to act in a network, constituted by many overlapping interactions between ligands and their receptors. Indeed, the chemokine-chemokine receptor axis is often highly promiscuous, allowing a single chemokine to bind different receptors and a chemokine receptor to be activated by distinct chemokines. This complex organization possibly aims to guarantee a robust protection even in the case of impairment of part of its components. However, some chemokines show a very high receptor and tissue specificity, and thus contribute to tissue-restricted leukocyte trafficking. CTACK/CCL27 is predominantly expressed in the epidermis and specifically binds CCR10 [15]. Also the TARC/CCR4 system has been proposed to be determinant for the selective homing of CCR4-bearing T lymphocytes to unperturbed and inflamed skin [2, 16]. Moreover, the same chemokine may initiate different signals according to the bound receptor: for instance Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 are agonists to CXCR3, but they also bind CCR3, antagonizing CCR3 agonists [17]. Similarly, eotaxin/CCL11 is a natural agonist for CCR3 and CCR5, and an antagonist for CCR2 and CXCR3 [18]. These data support the concept of the opposing roles of CCR3 and CXCR3 in Th1/Th2 polarization.

Chemokines in allergic contact dermatitis

ACD represents the prototype of T cell-mediated skin immune responses. Valuable animal models reproducing ACD exist, and the disease can be easily induced in humans, allowing a detailed analysis of the cell types and molecules involved in its induction, expression and regulation. ACD occurs in sensitized individuals at the site of hapten challenge. In the sensitization phase, the hapten penetrated into the skin is picked up by skin DCs, which then migrate to the draining lymph nodes and present the hapten-peptide-MHC complexes to naive antigen specific T lymphocytes. This process leads to the clonal expansion of specific T cells which can then be recruited to the skin. The migration of skin DCs to the lymph nodes is regulated by a profound rearrangement in the pattern of chemokine receptors, which allow DCs to encounter naive T cells [19, 20]. In mice deficient in CCR7 or its ligand SLC/CCL21, skin DCs do not migrate to the lymph nodes and ACD does not develop [21, 22]. In sensitized subjects, re-exposure to the antigen induces the activation and expansion of specific memory T cells. The inflammatory infiltrate is mainly composed of CD4⁺ and CD8⁺ T cells, monocytes and DCs, with an early and transient recruitment of neutrophils. The expression of both murine and human ACD correlates with the activity of hapten-specific CD8⁺ T cells, which exert their effector function through direct cytotoxic activity as well as via cytokine release [23]. CD4⁺ T cells play a more complex role, with Th1 and Th2 subsets contributing to disease expression and T regulatory cells to its modulation and termination [24, 25].
During ACD, leukocyte recruitment is under the control of a sequential and coordinated release of numerous chemokines from resident and immigrating cells (Table I). In a recent work, Goebeler et al. analyzed the pattern of chemokine expression in the skin at different time points after hapten application [26]. By in situ hybridization, they showed that MCP-1/CCL2 is detectable at 6 h after hapten challenge, whereas RANTES/CCL5 and MDC/CCL22 appear at 12 h, concomitantly with the infiltration of mononuclear cells in the dermis and epidermis. The expression of IP-10, Mig, TARC and PARC/CCL18 begins at 12 h and peaks at 72 h, paralleling the strong infiltration of lymphocytes. The critical role of MCP-1 in ACD is confirmed by the observation that transgenic mice overexpressing MCP-1 in basal keratinocytes show enhanced ACD responses together with an increased number of infiltrating DCs [27]. Activated endothelial cells can conceivably contribute to early leukocyte arrival in the skin by expressing both adhesion molecules and chemokines [28]. During the elicitation of ACD, upregulated expression of epidermal CTACK is already observed after 6 hours, and is detectable in the papillary dermis and on dermal microvessels at 24 h. Accordingly, the number of CCR10⁺ T cells increases dramatically at 24-48 h, first in the perivascular and subepidermal areas and then in the epidermis [29]. Mast cells can be importantly involved in neutrophil recruitment through the release of TNF-α and MIP-2, the functional analogue of human IL-8/CXCL8 in murine ACD [30]. Infiltrating monocytes and DCs are themselves a source of chemokines for successive boosts of lymphocyte arrival. The activation of some hapten-specific T lymphocytes into the skin leads to the production of cytokines like IFN-γ, TNF-α and IL-4, which in turn stimulate keratinocytes and other cells to produce IP-10, Mig and I-TAC, the ligands of CXCR3 [31-33]. The higher expression of CXCR3 and CCR4 on skin-homing nickel-specific CD8⁺ and CD4⁺ T cells suggests that trafficking of these two populations at the site of skin inflammation differs, with CD4⁺ and CD8⁺ cell recruitment primarily directed by the TARC/CCR4 and IP-10/CXCR3 axis, respectively [34].
Resolution of ACD is likely due to multiple mechanisms, including induction of T cell anergy and active suppression, and may involve several cell types. In this process IL-10 producing T regulatory cells might play a central role [35]. I-309 is produced by both keratinocytes and activated T cells, and is expressed in ACD skin with an earlier kinetics compared to IL-4 and IL-10 [36]. These data indicate that I-309/CCR8 may contribute relevantly to the termination of ACD through the recruitment of regulatory lymphocytes. Also CCR6, the receptor of MIP-3α/CCL20, seems to be involved in the modulation of ACD [37].

Chemokines in psoriasis

Psoriasis is a genetically determined skin disease characterized by aberrant proliferation and differentiation of keratinocytes as well as cutaneous inflammation. T cell-mediated immune mechanisms have a primary role in the pathogenesis of psoriasis [38, 39]. In particular, activated Th1 cells releasing IFN-γ and TNF-α stimulate keratinocytes to produce cytokines, chemokines and adhesion molecules, which further amplify the inflammatory response at the local level [40]. Various studies have documented a strong chemokine expression in psoriatic skin lesions (table I). Specifically, IL-8 and the related Gro-β/CXCL2 are strongly upregulated in psoriatic skin, and are responsible for the typical intraepidermal collection of neutrophils [41, 42]. MCP-1, RANTES, IP-10 and other CXCR3 ligands attract predominantly monocytes and Th1 cells [43, 44], whereas MIP-3α recruits immature Langerhans cells, DCs and cutaneous lymphocyte-associated antigen (CLA)⁺ T cells [45, 46]. In line with these observations, T cells bearing CCR4, CXCR3 and CCR6 are well represented in psoriatic skin lesions [47, 48]. In particular, CXCR3⁺CD8⁺ T cells are increased by ten-fold in psoriatic epidermis compared with their frequency in peripheral blood of patients with psoriasis [47]. MCP-4/CCL13 is also expressed in the basal layers of the psoriatic epidermis and together with MIP-3α can direct the traffic of immature DCs [48]. Also CTACK is abundantly present in basal and suprabasal keratinocytes of psoriatic lesions as well as in the dermis, together with a high number of CCR10⁺ T cells [49]. Interesting enough, in a very recent comparison of the inflammatory gene expression pattern of AD versus psoriasis skin lesions performed with microarray technology, both MCP-4 and CTACK transcripts were found much more strongly expressed in AD than in psoriasis, whereas the opposite held true for MIP-3α [42]. Expression of MIP-3α on dermal endothelial cells may have an important role in the arrest of CCR6⁺ immature DCs and memory T cells [45], while expression of TARC is important for the arrest of CCR4⁺ T cells [50]. Also MIP-1β/CCL4, active towards CCR1- and CCR5-bearing Th1 cells, immature DCs and monocytes, is strongly expressed in psoriatic lesions [42]. Dermal endothelial cells of psoriasis lesions, but not of AD, are also strongly positive for fractalkine, whose receptor (CX₃CR1) is preferentially expressed on Th1 cells and NK cells [51].
The genetic predisposition to psoriasis may include an altered control of inflammatory gene expression in the skin. In particular, psoriatic keratinocytes may have intrinsic defects leading to exaggerated synthesis of certain chemokines such as IL-8, MCP-1 and IP-10 [41, 52, 53], and also display enhanced expression of CXCR2, which can mediate a strong proliferative response of keratinocytes to IL-8 [40, 54]. Increasing evidence indicates that nitric oxide (NO) is involved in the maintenance of skin homeostasis as well as in the modulation of inflammatory reactions. Of note, high levels of NO have been measured in the skin affected with psoriasis, AD or ACD [55, 56]. In vivo, psoriatic skin treated with a NO releaser showed a reduction of IP-10, RANTES and MCP-1, but not of IL-8 expression by keratinocytes. Moreover, the number of CD14⁺ and CD3⁺ cells infiltrating the epidermis and papillary dermis diminished significantly. NO donors also suppressed ICAM-1 expression [53]. These results define NO donors as negative regulators of chemokine production by keratinocytes. The clinical efficacy of novel targeted immunomodulatory therapies of psoriasis, such as IL-10 and dimethylfumarate, is associated with a down-regulation of chemokine production and signaling pathway. In particular, administration of IL-10 to patients with chronic plaque psoriasis inhibited the expression of IL-8, its receptor CXCR2, and its inducer IL-17 [57, 58]. Consistently, dimethylfumarate suppressed IFN-γ-induced production of Gro-α/CXCL1, IL-8, Mig, IP-10 and I-TAC in keratinocytes and peripheral blood mononuclear cells [59].

Chemokines in atopic dermatitis

AD is a chronic inflammatory disease which results from complex interactions between genetic and environmental factors [60]. An altered lipid composition of the stratum corneum is responsible for the xerotic aspect of the skin, and may determine a higher permeability to allergens and irritants. Indeed, specific immune responses against a variety of environmental allergens are implicated in AD pathogenesis with a bias towards Th2 immune responses. Keratinocytes of AD patients exhibit a propensity to an exaggerated production of cytokines and chemokines, a phenomenon that can be relevant in promoting and maintaining inflammation, and may have a major role in localizing the atopic diathesis to the skin [61]. Eventually, a complex network of cytokines and chemokines contributes to establish a local milieu that favors the permanence of inflammation in AD skin (Table I).
The proportion of CD4⁺ T lymphocytes expressing the CCR4 receptor in the peripheral blood of patients with AD is higher when compared to healthy controls. In contrast, AD patients bear a lower percentage of circulating CXCR3⁺CD4⁺ T cells [62-64]. Moreover, the percentage of blood CCR4⁺CD4⁺ cells correlates with disease severity and IL-4 and IL-13 secretion by CD4⁺ T cells [64, 65]. The ligands for CCR4 are TARC and MDC, whose levels in the blood of AD patients correlate with disease activity [66, 67]. Both chemokines are abundantly produced by immature DCs and even more by mature DCs in vitro. TARC is expressed by microvascular endothelial cells in AD lesions, thus reasonably playing some role in the recruitment of CCR4⁺ T cells from the circulation [50], but also by epidermal keratinocytes [68]. In situ studies also showed that MDC immunoreactivity in AD skin is mostly confined to CD1a⁺CD83⁺ mature DCs, and identified this cell type as the major source of MDC in vivo [50, 69]. These data point to a relevant role for CCR4⁺ T cells in AD, and suggest that DCs may guide not only their activation but also their preferential accumulation in AD skin. Other chemokines that participate in the accumulation of T cells in AD include RANTES, MCP-1, eotaxin, MIP-3α, CTACK and IL-16. RANTES and MCP-1, which attract both Th1 and Th2 cells, are expressed by infiltrating leukocytes but especially by keratinocytes in diseased skin [52], and RANTES was found elevated in the serum of patients [70]. Together with its receptor CCR3, eotaxin is expressed in the dermis of AD lesions in particular by mononuclear cells and fibroblasts [71]. Also MIP-3α mRNA is found expressed in AD skin, although less abundantly than in psoriasis [42, 72]. Acute and chronic AD lesions exhibit strong CTACK expression, as previously commented [42], and numerous CCR10⁺ T cells [49]. Compared to psoriasis, AD lesions also present higher levels of PARC transcript expression [42]. This chemokine, whose receptor is not known yet, attracts both CD4⁺ and CD8⁺ naive T cells. Recently, an elevation of circulating IL-16 has been associated to active AD in children [73]. Increased expression of IL-16, active on CD4⁺ T cells, monocytes and eosinophils, was found in the epider mis of AD lesions, and Langerhans cells (LCs) were recognized as the most relevant source of this chemokine [74]. Lesional skin of AD patients exhibits an increased number of cells belonging to the DC lineage, which displays an upregulated expression of both high affinity (Fc RI) as well as the low affinity (Fc RII/CD23) IgE receptors. Of note, Fc RI engagement upregulates IL-16 production in LCs derived frℴm atopic donors [60]. Eventually, the selective uptake of allergens and the subsequent induction of specific T-cell responses further perpetuate skin inflammation. A possible association between a promoter polymorphism in the gene encoding IL-16 and polysensitization to contact allergens, but not AD, has been recently reported [75]. In the context of acute and chronic AD lesions, eosinophils are attracted by resident skin populations also through increased expression of CCR3 binding molecules, including RANTES, MCP-4 and eotaxin [42, 60]. A significant elevation of serum levels of eotaxin-3/CCL26, an exclusive CCR3 ligand, was found in patients with AD, and a correlation could be established with the disease activity [76]. In vitro studies showed that keratinocytes from AD patients produced increased amounts of RANTES, but reduced levels of IP-10, in response to IFN-γ or TNF-α when compared to keratinocytes from normal controls or psoriasis patients [52]. Keratinocytes cultured from AD patients also displayed abnormal production of spontaneous as well as IL-1α- and IFN-γ-induced GM-CSF release, when compared to healthy control keratinocytes [77, 78]. Numerous functional polymorphisms in the regulatory/coding regions of clusters of cytokine/chemokine genes, including RANTES, have been found in AD patients [79, 80], which could be implicated in an overproduction by keratinocytes. However, apart from genes coding for Th2 cytokines, polymorphisms for other inflammatory genes were not confirmed in other studies [81]. The distinct propensity of keratinocytes to produce higher than normal levels of growth factors (GM-CSF), chemokines (RANTES), and cytokines (TNF-α, thymic stromal lymphopoietin/TSLP) [82], may greatly stimulate DC differentiation from precursors, and recruit as well as activate DCs in AD skin. Possible triggers of keratinocyte activation in the very early phases may reasonably include altered epidermal permeability barrier functions [83], while environmental allergens do not seem effective [84].

Perspective

Since the discovery of the superfamily of chemokines and their receptors, there has been a considerable effort to define their peculiar role in the orchestration of leukocyte trafficking. Using a variety of experimental approaches, recent studies have provided irrefutable evidence that chemokines are essential mediators in the pathophysiology of inflammatory diseases, and thus good candidates for therapeutic strategies. Chemokine receptors rather than chemokines appear the appropriate therapeutic targets as they are more chemically tractable and play less redundant functions. Intense investigation is currently focused on the development of selective small-molecule chemokine receptor antagonists to possibly attenuate a variety of inflammatory disorders, including autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, and allergic life-threatening diseases such as asthma [85, 86]. The therapeutic validity of specifically targeting one or a few of the links of this complex molecular web represents a crucial issue. Concomitantly, the potential negative consequences of this type of therapy on effective defenses against microbial infections and tumor growth has to be seriously analyzed [87]. In spite of the challenge underlying this type of therapeutic approach, it can be reasonably expected that pharmacological impairment of chemokine/chemokine receptor axis will attract active investigation on dermatological diseases in the near future [1]. n

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