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Psoriasis is a common chronic inflammatory disease with characteristic skin alterations and functions as a model of immune-mediated disorders. Cytokines have a key role in psoriasis pathogenesis. Here, we demonstrated that out of 30 individually quantified cytokines, IL-19 showed the strongest differential expression between psoriatic lesions and healthy skin. Cutaneous IL-19 overproduction was reflected by elevated IL-19 blood levels that correlated with psoriasis severity. Accordingly, anti-psoriatic therapies substantially reduced both cutaneous and systemic IL-19 levels. IL-19 production was induced in keratinocytes by IL-17A and was further amplified by tumor necrosis factor-α and IL-22. Among skin cells, keratinocytes were found to be important targets of IL-19. IL-19 alone, however, regulated only a few keratinocyte functions. While increasing the production of S100A7/8/9 and, to a moderate extent, also IL-1β, IL-20, chemokine C-X-C motif ligand 8, and matrix metalloproteinase 1, IL-19 had no clear influence on the differentiation, proliferation, or migration of these cells. Importantly, IL-19 amplified many IL-17A effects on keratinocytes, including the induction of β-defensins, IL-19, IL-23p19, and T helper type 17-cell- and neutrophil-attracting chemokines. In summary, IL-19 as a component of the IL-23/IL-17 axis strengthens the IL-17A action and might be a biomarker for the activity of this axis in chronic inflammatory disorders.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a downstream molecule of p38, involved in the production of TNF-alpha, a key cytokine, and an established drug target for many inflammatory diseases. We investigated the role of MK2 in skin inflammation to determine its drug target potential. MK2 deficiency significantly decreased plasma TNF-alpha levels after systemic endotoxin application. Deficient mice showed decreased skin edema formation in chronic 2-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritative dermatitis and in subacute 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity. Surprisingly, MK2 deficiency did not inhibit edema formation in subacute 2,4-dinitrochlorobenzene (DNCB)-induced contact allergy and even increased TNF-alpha and IL-1beta levels as well as granulocyte infiltration in diseased ears. Ear inflammation in this model, however, was inhibited by TNF-alpha neutralization as it was in the subacute DNFB model. MK2 deficiency also did not show anti-inflammatory effects in acute DNFB-induced contact hypersensitivity, whereas the p38 inhibitor, SB203580, ameliorated skin inflammation supporting a pathophysiological role of p38. When evaluating possible mechanisms, we found that TNF-alpha production in MK2-deficient spleen cells was strongly diminished after TLR stimulation but less affected after T-cell receptor stimulation. Our data suggest that MK2, in contrast to its downstream effector molecule, TNF-alpha, has a rather elusive role in T-cell-dependent cutaneous inflammation.

Histone deacetylase inhibitors are under investigation in the clinic as a new class of anti-cancer therapeutics. While recent studies have also suggested their potential as inhibitors of a wide spectrum of inflammatory reactions, the anti-inflammatory mechanism of action of these compounds is not fully defined. We show here that the histone deacetylase inhibitors MS-275 and SAHA induce the generation of regulatory T cells (Tregs) from anti-CD3/anti-CD28-stimulated human CD4(+)CD25(-) T cells. These Tregs express the regulatory T cell-associated transcription factor Foxp3 and display suppressive activity against CD4(+)CD25(-) T cell proliferation. Topical treatment with histone deacetylase inhibitors also induces Foxp3 expression in the draining lymph nodes and the skin in the context of a murine contact hypersensitivity model. These findings suggest that Treg generation may serve as a novel mechanism by which histone deacetylase inhibitors regulate the immune response, and provide an additional rationale for the use of histone deacetylase inhibitors in the treatment of inflammation.

A mixture of different fumaric acid esters (FAE) is established for systemic therapy of psoriasis, a frequent inflammatory skin disease. The main active compound of FAE, however, has not been identified so far, and the mechanisms of activity are only partially understood. We analyzed the impact of FAE on in vitro immune function and aimed to gain knowledge about the mode of action. Dimethylfumarate (DMF) and diethylfumarate (DEF), but not fumaric acid, methylhydrogenfumarate and ethylhydrogenfumarate, exhibited potent depression of inflammatory cytokine secretion (e.g., tumor necrosis factoralpha, IL-12, and IFNgamma) in activated human peripheral blood mononuclear cells. Moreover, solely DMF and DEF inhibited alloreactive T-cell proliferation in mixed leukocyte reaction. Interestingly, these immunosuppressive effects were accompanied by the strong induction of the anti-inflammatory stress protein heme oxygenase 1 (HO-1). Supplementation with exogenous glutathione (GSH), which is known to bind DMF, prevented both HO-1 induction as well as the anti-inflammatory effects of DMF. Moreover, inhibition of HO-1 activity restored the diminished IL-12 and IFNgamma production after FAE treatment. These results suggest that DMF acts as active compound within the FAE mixture and at least partially mediates its immunomodulatory activity by the induction of the anti-inflammatory stress protein HO-1 ascribed to the functional depletion of reduced GSH.

Ultraviolet irradiation represents a well-established treatment modality for inflammatory skin diseases. The aim of this study was to investigate the mechanisms of ultraviolet B radiation-induced keratinocyte insensitivity towards interferon-gamma. Flow cytometric analyses indicated that ultraviolet B radiation temporarily inhibits the interferon-gamma-induced activation of primary keratinocyte and HaCaT cells as measured by reduced intercellular adhesion molecule-1 (CD54) and HLA-DR upregulation. Western blot experiments have suggested that this is mediated by the ultraviolet B radiation-induced inhibition of signal transduction and transcription factor-1 phosphorylation. Neither interleukin-10 neutralization nor interleukin-10 addition had any effect on the ultraviolet B radiation-induced inhibition of interferon-gamma induced intercellular adhesion molecule-1 expression. Furthermore, the supernatant from ultraviolet B-irradiated cells failed to inhibit the interferon-gamma-induced CD54 and HLA-DR upregulation in nonradiated HaCaT cells. Moreover, irradiated cells from whom the supernatant was withdrawn 4 h after irradiation still showed a diminished interferon-gamma-induced response after 24 h. Thus, not soluble but intracellular factors might be involved in the ultraviolet B radiation-induced inhibition of interferon-gamma-induced keratinocyte activation. Therefore, we analyzed the expression of members of suppressors of cytokine-signaling (SOCS) molecules using real-time polymerase chain reaction. We found a fast and strong upregulation of SOCS1 and SOCS3 but not of SOCS2 after ultraviolet B radiation. Similarly, ultraviolet B radiation induced the expression of these particular SOCS molecules in lesional psoriatic skin. As SOCS molecules are known inhibitors of signal transduction and transcription factor phosphorylation, which is essential for interferon-gamma-induced intercellular adhesion molecule-1 and HLA-DR upregulation, this may explain the interferon-gamma unresponsiveness after ultraviolet B radiation.

1Alpha,25-dihydroxyvitamin D3, the biologically active form of vitamin D3, is a potent immunomodulatory molecule; however, its clinical use as an immunosuppressant is limited due to its strong effects on calcium homeostasis and the risk of associated side-effects. Here, we present a representative of a novel class of vitamin D analogs that exhibits potent immunosuppressive activity in a murine model of contact hypersensitivity when applied systemically and is efficacious also at nonhypercalcemic dosages. In vitro analysis revealed a binding affinity of ZK 191784 to the vitamin D receptor comparable with 1,25-dihydroxyvitamin D3. This compound inhibits lymphocyte proliferation and secretion of tumor necrosis factor alpha and interleukin-12 in monocytes in a concentration-dependent manner, but with reduced potency and efficacy than 1,25-dihydroxy-vitamin D3. Treatment of human monocytes with this analog significantly reduces expression of major histocompatibility complex class II, B7.1, and intercellular adhesion molecule-1 equipotent to 1,25-dihydroxyvitamin D3. Interestingly, the compound failed to induce vitamin D-induced differentiation of human promyelocytic leukemia cell line HL-60 to monocytes and was capable of antagonizing the action of 1,25-dihydroxyvitamin D3. In vivo, as analyzed in mice the compound potently inhibits the contact hypersensitivity when applied systemically. ZK 191784 has a clear therapeutic advantage over 1,25-dihydroxyvitamin D3 by inducing immunosuppressive effects also at concentrations that do not cause hypercalcemia. ZK 191784 is the first representative of a novel class of vitamin D analogs that might have therapeutic potential in T cell-mediated immune disorders.

The ability of interleukin-10 therapy to reduce the severity of exacerbated psoriasis has been demonstrated recently. Considering the immunobiologic properties of this cytokine we investigated the effects of long-term interleukin-10 application on the immune system and duration of psoriasis remission. We performed a placebo-controlled, double-blind, phase II trial using interleukin-10 in patients with chronic plaque psoriasis in remission. Patients received subcutaneous injections with either interleukin-10 (10 microg per kg body weight; n = 7) or placebo (n = 10) three times per week until relapse or study termination after 4 months. The treatment was well tolerated. In the placebo group almost all patients (90%) showed a relapse during the observation period. In contrast to this, only two of seven patients (28.6%) relapsed in the interleukin-10-treated group. Kaplan-Meier analysis revealed a significantly lower relapse incidence in the interleukin-10 than in the placebo group (p = 0.02). The mean relapse-free interval time was 101.6 +/- 12.6 d in the interleukin-10 group in comparison with 66.4 +/- 10.4 d in the placebo group. Immunologic activity of interleukin-10 application was indicated by an increase in soluble interleukin-2 receptor plasma levels and higher ex vivo interleukin-4 secretion capacities. Remarkably, a significant negative correlation was demonstrated between the interleukin-4 secretion capacity and Psoriasis Area and Severity Index score (r = -0.36, p < 0.01). Our data suggest that interleukin-10 therapy is immunologic effective, decreases the incidence of relapse and prolongs the disease-free interval in psoriasis. Its value should be further determined in larger trials and for the prevention of re-exacerbation of other inflammatory disorders with a similar immunologic profile.

Beneficial effects of interleukin-10 therapy and lower endogenous interleukin-10 formation compared with atopic dermatitis and cutaneous T cell lymphomas indicated that interleukin-10 is a key cytokine in psoriasis. The interleukin-10 promoter is highly polymorphic, with two informative microsatellites, interleukin-10.G and interleukin-10.R. In order to understand whether interleukin-10 itself is a predisposing gene for the psoriasis susceptibility we analyzed interleukin-10 promotor polymorphism in patients. The distribution of interleukin-10.G and interleukin10.R microsatellite alleles did not vary between patients (n = 78) and healthy controls (n = 80). In addition, when the psoriasis patients were stratified according to age of onset (younger than 40 y of age, or age 40 and older), no difference in allele distribution was observed; however, a clear differential distribution was revealed at the interleukin10.G locus when patients were stratified according to whether they had a positive family history of psoriasis (p = 0.04). This difference was due to an over-representation of the interleukin10.G13 allele in those patients with familial disease (40.4% vs 19.6%, Chi-square = 7.292, p = 0.007). The positive association of allele interleukin10.G13 with familial psoriasis was especially true when patients with an early onset (< 40 y of age) of the disease were compared with those patients with early onset against a nonfamilial background (39.6% vs 14.5%, Chi-square = 8.959, p = 0.003). Patients with age-of-onset of less than 40 were 4-fold [odds ratio = 3.85 (1.55--9.62)] more likely to have a psoriatic family background if they carried this interleukin10.G13 allele. These data suggest that the interleukin-10 locus contributes to the heritability of psoriasis susceptibility.

Interleukin-10 is an important anti-inflammatory and immunosuppressive cytokine with major impact on several immune reactions, including regulatory mechanisms in the skin. Recently, we performed a phase II trial in psoriatic patients receiving subcutaneously interleukin-10 over 7 wk. The clinical response suggested that interleukin-10 might represent a novel anti-psoriatic drug. In order to understand better the mode of action and to elucidate the effects of systemic interleukin-10 treatment on the skin immune system, skin punch biopsies from sites different from interleukin-10 injection were analyzed. Biopsies were obtained from the patients before, at the end, and 3 wk after interleukin-10 therapy. The results are reported here. Histologic examination showed a decrease of several parameters reflecting the psoriatic disease activity as acanthosis and extension of the horny layer. Immunohistologic examination demonstrated decreasing numbers of infiltrating T cells, dermal CD1a+ cells, and a diminished proliferation of epidermal cells. Using a novel, quantitative reverse transcriptase-polymerase chain reaction approach a significant shift within the cytokine pattern was found. Interleukin-10 therapy led to a decrease of cutaneous interleukin-8 and interleukin-10 mRNA expression. Whereas no significant changes of interleukin-6, tumor necrosis factor-alpha, and interferon-gamma expression were found, interleukin-4 was strongly upregulated suggesting a shift from a type 1 towards a type 2 cytokine pattern. The changes within the local cytokine pattern seem to be disease-related, as an inverse course was found in a single interleukin-10 nonresponding patient. Our findings demonstrate considerable effects of systemic interleukin-10 application on the skin immune systems, which might contribute to the anti-psoriatic activity of interleukin-10.

Clonal T cells have been demonstrated in skin lesions of all stages of cutaneous T-cell lymphomas (CTCLs). However, there are conflicting data regarding the CTCL stage at which dissemination of clonal cells into peripheral blood occurs. Although the multifocal occurrence of cutaneous CTCL lesions and T-cell recirculation suggest an early appearance of neoplastic cells in the blood, circulating clonal T cells have only been detected in advanced stages. We investigated their occurrence by a highly sensitive polymerase chain reaction (PCR) assay amplifying T-cell receptor gamma rearrangements and subsequent heteroduplex temperature gradient gel electrophoresis (HD-TGGE) of the amplification products. Circulating clonal T cells were found in 26 of 45 patients with mycosis fungoides (MF), six of seven with Sezary's syndrome (SS), 10 of 13 pleomorphic CTCLs, and three of four unclassified CTCLs. Corresponding skin specimens carried clonal T cells in 29 of 40 MF, three of four SS, 12 of 12 pleomorphic, and two of two unclassified CTCL patients. Except for the blood specimen of a psoriatic patient, all samples of 60 controls (psoriasis vulgaris, atopic dermatitis, and healthy volunteers) revealed polyclonal amplification products. In 30 of 32 CTCL patients carrying a clonal rearrangement in blood and skin, identity of both clones was indicated by HD-TGGE and confirmed by sequencing six of these cases. We found an unexpected high frequency of identical clonal T cells in peripheral blood and skin of CTCL patients, including early stages of MF. This supports the concept of an early systemic disease in CTCL and raises new questions concerning the pathogenesis.

Indolent, primary cutaneous T-cell lymphomas (CTCL) are characterized by hyper-proliferation of malignant T-helper cells in the skin with a favorable prognosis in the early stages. Cytotoxic T cells (CTLs) are believed to be of major importance for tumor surveillance, but there is not yet sufficient evidence for a systemic anti-tumor response in mycosis fungoides (MF). On the contrary, there are hints of systemic immunodepression. We wondered whether signs of a systemic anti-tumor response were demonstrable in peripheral blood of patients with MF and CD30+ pleomorphic T cell lymphoma. Using multiparameter flow cytometry, we investigated blood samples from 39 CTCL patients at different stages and compared them with those from patients with psoriasis, atopic dermatitis, and healthy volunteers. In CTCL patients, an elevated number of lymphocytes expressing natural killer cell markers were found, as well as considerable T-cell activation, indicated by increased percentages of T cells expressing HLA-DR, IL-2 receptor alpha-chain, and transferrin receptor. The CD8+ T cells, which were the most strongly activated T-cell subset, were of polyclonal origin, as shown by their usage of different T-cell receptor families. The enhanced expression of activation antigens was associated with an increased proportion of CD8+ T cells with high expression of the adhesion molecule LFA-1, demonstrating the capacity for migration of these cells. These CD8+ effector cells are suspected to be CTLs and may be responsible for the favorable prognosis of indolent, primary CTCL. Interestingly, a stage-dependent decrease in T-cell activation antigen expression was observed, suggesting the development of a lack in tumor surveillance in advanced MF stages. Further investigations are necessary to verify whether any of the parameters determined are of predictive value for prognosis and response to therapy in CTCL.

Cytokines are believed to play an important role in the pathogenesis of cutaneous T cell lymphoma. Data regarding the local cytokine pattern in mycosis fungoides (MF) are partly conflicting. Recent studies have suggested a shift from type 1 to type 2 cytokine pattern because IL-4 and IL-5 mRNA have been more frequently detected in lesions of advanced stages. Another study has described a type 1 cytokine pattern in MF lesions. None of the previous studies of cytokine mRNA expression in MF, however, used quantitative methods, and therefore only the presence of a cytokine, but not the level of expression, could be determined. To gain better insight into the development of cytokine pattern during tumor progression we used semiquantitative reverse transcriptase-polymerase chain reaction to analyze cytokine mRNA expression in MF skin lesions at different stages. Biopsies from patients with patch (n = 11), plaque (n = 6), and tumor (n = 3) stage MF were compared with biopsies from patients with pleomorphic T cell lymphoma (n = 5), psoriasis (n = 7), atopic dermatitis (n = 5), and nonlesional skin (n = 8). MF progression was associated with significantly higher IL-10 and lower interferon-gamma mRNA expression. Moreover, the stage-dependent increase in IL-10 mRNA expression was also found in paired samples from individual patients. Unlike in pleomorphic T cell lymphoma, however, typical T helper 2 cells did not seem to be the source of increasing IL-10 in advanced MF, because stage-independent IL-4 mRNA was rarely detected, suggesting contribution of nonlymphoid cells to local IL-10 production. The overexpression of IL-10 in MF may be of importance for tumor progression, because this immunosuppressive cytokine might be involved in downregulation of immunologic tumor surveillance.