Flow cytometric analysis of cytokine receptors on human Langerhans' cells. Changes observed after short-term culture.

It is well established that granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 and tumour necrosis factor-alpha (TNF-alpha) are involved in Langerhans' cell (LC) development and dendritic cell traffic. However, little is known about the pattern of cytokine receptors on human LC and their modulation during different stages of maturation. The expression of cytokine receptors was studied by flow cytometry on both freshly isolated LC (fLC) and 72-hr cultured LC (cLC). Epidermal cell suspensions enriched in LC were obtained after skin trypsinization and Ficoll-Hypaque gradient. LC were identified by their CD1a positivity. Although the majority of fLC were positive for the alpha chain of GM-CSF receptor (GM-CSFR), the beta chain of GM-CSFR was detected only on 15% of CD1a+ cells. fLC were also positive for IL-1 receptor (IL-1R) type 1, IL-1R type 2, 75,000 molecular weight TNF receptor (TNFR) and interferon-gamma receptor (IFN-gamma R). IL-6R and its transducing signal gp130 were present in a subset of fLC. Granulocyte colony-stimulating factor receptor (G-CSFR), macrophage colony-stimulating factor receptor (M-CSFR), the alpha and beta chain of IL-2R, IL-4R, IL-7R, IL-8R and 55,000 molecular weight TNFR were not detected on fLC. After culture, LC up-regulated the expression of both the alpha and beta chains of GM-CSFR, IL-1R type 2, alpha and beta chains of IL-2R, IL-6R and gp130. In contrast, IL-1R type 1 and 75,000 molecular weight TNFR were down-modulated and the expression of IFN-gamma R was not affected by culture. These results suggest that LC undergo changes in the cytokine receptor repertory during in vitro maturation.     

Increased expression of interleukin-4 receptors on psoriatic epidermal cells.

Abnormal keratinocyte proliferation and differentiation, the main characteristics of psoriasis, may be induced and maintained by cytokines produced by activated resident and recruited inflammatory cells in lesional skin. As the epidermal cytokine profile is clearly altered in psoriasis and because increased expression of interleukin-4 receptor (IL-4R) has been reported in some epithelial proliferative diseases, we investigated the expression of IL-4R on psoriatic epidermal cells (ECs). The expression of IL-4R on freshly isolated ECs from healthy skin and untreated psoriatic lesions was studied by immunostaining using an IL-4R-specific antibody and by examining their capacity to bind biotinylated recombinant human IL-4 using flow cytometry. The number of IL-4R+ ECs and the number of binding sites per cell were significantly increased on psoriatic ECs as compared with healthy control ECs. Immunostaining confirmed these results, whereby staining was mainly observed in the lower epidermal layers. In addition, an increased IL-4R mRNA expression was also observed in psoriatic epidermis using a digoxigenin-labeled IL-4R RNA probe. In short-term in vitro cultures, lipopolysaccharide/phorbol-myristate-acetate-stimulated and unstimulated psoriatic ECs did not produce any immunoreactive IL-4. The results of this study together with the reported increased expression of IL-4R in epithelial neoplasias suggest an association between overexpression of IL-4R and abnormal keratinocyte activation and proliferation.     

Nitric oxide donors suppress chemokine production by keratinocytes in vitro and in vivo

Nitric oxide (NO) is involved in the modulation of inflammatory responses. In psoriatic skin, NO is highly produced by epidermal keratinocytes in response to interferon-gamma and tumor necrosis factor-alpha. In this study, we investigated whether the NO donors, S-nitrosoglutathione (GS-NO) and NOR-1, could regulate chemokine production by human keratinocytes activated with interferon-gamma and tumor necrosis factor-alpha. In addition, we studied the effects of the topical application of a GS-NO ointment on chemokine expression in lesional psoriatic skin. NO donors diminished in a dose-dependent manner and at both mRNA and protein levels the IP-10, RANTES, and MCP-1 expression in keratinocytes cultured from healthy patients and psoriatic patients. In contrast, constitutive and induced interleukin-8 production was unchanged. GS-NO-treated psoriatic skin showed reduction of IP-10, RANTES, and MCP-1, but not interleukin-8 expression by keratinocytes. Moreover, the number of CD14(+) and CD3(+) cells infiltrating the epidermis and papillary dermis diminished significantly. NO donors also down-regulated ICAM-1 protein expression without affecting mRNA accumulation in vitro, and suppressed keratinocyte ICAM-1 in vivo. Finally, NO donors inhibited nuclear factor-kappa B and STAT-1, but not AP-1 activities in transiently transfected keratinocytes. These results define NO donors as negative regulators of chemokine production by keratinocytes.     

In vivo effects of cytokines on psoriatic skin grafted on nude mice: involvement of the tumour necrosis factor (TNF) receptor

Following engraftment of human involved psoriatic skin to nude mice there is a partial normalization of pathology associated with a loss of inflammatory leucocytes. However, the epidermis remains hyperproliferative, which may reflect a primary defect. The roles of TNF-α, IL-1 and IL-6 in epidermal hyperproliferation of grafted psoriatic lesions were investigated. Before and after treatment, grafts were analysed to determine epidermal thickness and labelling index (LI). HLA-DR, intercellular adhesion molecule-1 (ICAM-1), and TNF receptor (TNF-R; p75 and p55) expression were determined by immunoperoxidase staining. Psoriatic epidermis was found consistently to be negative for p55 TNF-R and p75 TNF-R before grafting. Following engraftment, TNF-R-positive cells (i.e. p55 by keratinocytes; p75 by epidermal dendritic cells) were identified throughout the epidermis. Higher numbers of p75 TNF-R epidermal dendritic cells were found in grafts following a course of TNF-α, IL-6 or IL-1 treatment. The p55 form of the TNF-R expressed by keratinocytes was significantly elevated after treatment with TNF-α or IL-6. HLA-DR and ICAM-1 were also expressed in these grafts. TNF-α, anti-IL-1, and anti-IL-6 treatment induced a marked decrease in the epidermal thickness and LI of psoriatic graft tissue, correcting the hyperproliferation associated with psoriatic epidermis. Supraphysiological levels of TNF-α may saturate and consequently down-regulate their own receptors, leading to a paradoxical inhibitory effect.     

Cellular localization of interleukin-8 and its inducer, tumor necrosis factor-alpha in psoriasis.

The importance of immunologic mechanisms in psoriasis has been deduced from the ability of immunosuppressive therapies to ameliorate this common and chronic skin disease. Certainly the histology of psoriatic lesions suggests a dialogue between the hyperplastic keratinocytes and infiltrating T lymphocytes and macrophages. To begin dissecting the cytokine network involved in the pathophysiology of psoriasis, the location, in both epidermal and dermal compartments, of tumor necrosis factor-alpha, interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha at the protein and/or mRNA levels were identified. Tumor necrosis factor-alpha was selected as a potentially key regulatory cytokine, first because it induces cultured keratinocyte interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha production, and second because intercellular adhesion molecule-1 expression by keratinocytes in psoriatic epidermis had been identified previously. Using immunohistochemical localization, tumor necrosis factor-alpha was identified in 12 psoriatic lesions as intense and diffuse expression by dermal dendrocytes (macrophages) in the papillary dermis (without significant staining of endothelial cells, mast cells, or dermal Langerhans cells), and focally by keratinocytes and intraepidermal Langerhans cells. Functional interaction between the dermal dendrocytes and keratinocytes was suggested by the presence of interleukin-8 expression of suprabasal keratinocytes immediately above the tumor necrosis factor-alpha-positive dermal dendrocytes. Interleukin-8 mRNA and transforming growth factor-alpha mRNA were detectable in the epidermal roof of psoriatic lesions, but neither was detectable at the protein or mRNA levels in any normal skin specimens. Treatment of cultured human keratinocytes with phorbol ester (which experimentally produces psoriasiform changes on mouse skin) or tumor necrosis factor-alpha also increased interleukin-8 and transforming growth factor-alpha mRNAs. Further elucidation of the cellular and molecular basis for the genesis and evolution of psoriasis will provide the framework for a better evaluation of the cause and treatment of this skin disease.     

Lesional psoriatic T cells contain the capacity to induce a T cell activation molecule CDw60 on normal keratinocytes.

In this report we demonstrate, that in psoriatic skin, basal and suprabasal keratinocytes express CDw60. The CDw60-specific monoclonal antibody, UM4D4, has recently been shown to recognize the 9-O-acetylated disialosyl group on ganglioside GD3. The CDw60 antigen on cultured keratinocytes also seems to be identical with the 9-O-acetylated disialosyl group, because the anti-UM4D4 binding was markedly reduced after neuraminidase treatment of keratinocytes. To examine whether factors from T cells in psoriatic lesions are responsible for the overexpression of CDw60 on keratinocytes, T cell lines obtained from lesional skin were initiated and cloned by limiting dilution. Factors released from 19 of 19 activated T cell clones up-regulated CDw60 expression on cultured normal keratinocytes. T-cell-secreted cytokines, including interleukin (IL)-2, IL-3, IL-4, IL-6, IL-13, transforming growth factor-beta, granulocyte/macrophage colony-stimulating factor, and interferon-gamma were tested for their capacity to modulate keratinocyte CDw60 expression. IL-4 and IL-13 strongly up-regulated the expression of CDw60; by contrast, interferon-gamma down-regulated keratinocyte CDw60 expression. Interestingly, IL-13 may in part be responsible for the T-cell-induced up-regulation of CDw60, because anti-IL-13 partly neutralized this effect of the T cell supernatant. In conclusion, CDw60 expression on psoriatic epidermal keratinocytes is likely induced by intralesionally activated T cells and may in part be due to IL-13. These findings would represent a novel mechanism by which T cells participate in the pathogenesis of psoriasis.     

In situ localization of interleukin-6 in normal skin and atrophic cutaneous disease.

Interleukin-6 (IL-6) is a multifunctional cytokine that participates in the inflammatory and immune responses. In human skin, keratinocytes produces IL-6, although the in vivo role of this cytokine is unknown. In the present study we investigated the in situ localization of IL-6 in normal epidermis (n = 10) and in a group of skin diseases characterized by epidermal atrophy. Formalin-fixed paraffin-embedded skin biopsies from patients with clinical and histopathological features consistent with localized scleroderma (n = 10), systemic scleroderma (n = 5), lichen sclerosus et atrophicus (n = 9) and balanitis xerotica obliterans (n = 7) were tested using polyclonal antibodies and avidin-biotin-peroxidase immunostaining. We demonstrated the presence of IL-6 in normal epidermis and in atrophic skin diseases. In normal skin there was moderate intercellular and intracellular reactivity detected using a high antibody concentration. In specimens with epidermal atrophy we detected intense cytoplasmic and intercellular immunostaining using a lower antibody concentration. The immunoreactivity was independent of the epidermal thickness. Plasma IL-6, measured by radioimmunoassay, was not elevated in plasma from patients with localized or systemic scleroderma. Increased IL-6 in the epidermis of selected skin diseases suggests that IL-6 may be related to the pathophysiology of dermatologic diseases characterized by epidermal atrophy.     

Expression of eotaxin, an eosinophil-selective chemokine, parallels eosinophil accumulation in the vesiculobullous stage of incontinentia pigmenti

Incontinentia pigmenti (IP) is an X-linked dominant genodermatosis primarily affecting female children. The initial vesiculobullous stage of IP is characterized clinically by inflammatory papules, blisters, and pustules, and histopathologically by acanthosis, keratinocyte necrosis, epidermal spongiosis and massive epidermal eosinophil infiltration. The cause of this multisystem disease is attributed to the mutations of an X-linked regulatory gene, termed nuclear factor-kappaB essential modulator (NEMO). The exact mechanism of epidermal eosinophil accumulation has not yet been determined. We explored the possible role of an eosinophil-selective, nuclear factor-kappaB-activated chemokine, eotaxin, in the accumulation of eosinophils in the initial stage of the disease. Monoclonal antibody (6H9) specific for human eotaxin strongly labelled the suprabasal epidermis of IP skin, paralleling the upper epidermal accumulation of eosinophils, but did not label the epidermis of normal skin or lesional skin from patients with other inflammatory skin diseases not characterized by prominent eosinophil accumulation, namely dermatitis herpetiformis and selected cases of atopic dermatitis lacking significant numbers of eosinophils. In addition, endothelial cells in lesional skin of IP also exhibited strong expression of eotaxin, which correlated with perivascular and intravascular eosinophil infiltration. We also examined the in vitro effects on epidermally derived eotaxin of several cytokines that were nuclear factor-kappaB-activated and/or known to induce eotaxin expression. In normal human keratinocytes, proinflammatory cytokines either independently (IL-1alpha) or synergistically (tumour necrosis factor-alpha (TNF-alpha)/ interferon-gamma (IFN-gamma) and TNF-alpha/IL-4) up-regulated eotaxin expression. These studies suggest that release of cytokines during the initial inflammatory stage of IP induces epidermal expression of eotaxin, which may play a role in the epidermal accumulation of eosinophils.     

Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures

Caspase-14 is a nonapoptotic caspase family member whose expression in the epidermis is confined to the suprabasal layers, which consist of differentiating keratinocytes. Proteolytic activation of this caspase is observed in the later stages of epidermal differentiation. In psoriatic skin, a dramatic decrease in caspase-14 expression in the parakeratotic plugs was observed. Topical treatment of psoriatic lesions with a vitamin D3 analogue resulted in a decrease of the psoriatic phenotype and an increase in caspase-14 expression in the parakeratotic plugs. To investigate whether vitamin D3 directly affects caspase-14 expression levels, we used keratinocyte cell cultures. 1alpha,25-Dihydroxycholecalciferol, the biologically active form of vitamin D3, increased caspase-14 expression, whereas retinoic acid inhibited it. Moreover, retinoic acid repressed the vitamin D3-induced caspase-14 expression level. In addition, the use of organotypic skin cultures demonstrated that 1alpha,25-dihydroxycholecalciferol enhanced epidermal differentiation and caspase-14 activation, whereas retinoic acid completely blocked caspase-14 processing. Our data indicate that caspase-14 plays an important role in terminal epidermal differentiation, and its absence may contribute to the psoriatic phenotype.     

Type 2 helper T-cell cytokines induce morphologic and molecular characteristics of atopic dermatitis in human skin equivalent

Both the immune system and the epidermis likely have an important role in the pathogenesis of atopic dermatitis (AD). The objective of the present study was to develop a human skin equivalent model exhibiting morphologic and molecular characteristics of AD in a controlled manner. Skin equivalents generated from normal adult human keratinocytes were stimulated with type 2 T-helper cell (Th2) cytokines IL-4 and IL-13, and morphologic features and gene expression of the epidermis were studied. Th2 cytokines induced intercellular edema similar to spongiotic changes observed in lesional AD as assessed at histopathologic analysis and electron microscopy. Furthermore, genes known to be specifically expressed in epidermis of patients with AD such as CAII and NELL2 were induced. In contrast, expression of psoriasis-associated genes such as elafin and hBD2 was not changed. Th2 cytokines caused DNA fragmentation in the keratinocytes, which could be inhibited by the caspase inhibitor Z-VAD, which suggests that apoptosis was induced. In addition, up-regulation of the death receptor Fas was observed in keratinocytes after Th2 cytokine stimulation. IL-4 and IL-13 induced phosphorylation of the signaling molecule STAT6. It was concluded that the skin equivalent model described herein may be useful in investigation of the epidermal aspects of AD and for study of drugs that act at the level of keratinocyte biology.     

MHC class II antigen expression is not induced on murine epidermal keratinocytes by interferon-gamma alone or in combination with tumour necrosis factor-alpha

Epidermal keratinocytes are induced to express MHC class II molecules in a variety of disease states associated with immune activity. To investigate the mechanism of this process we have exposed murine and rat keratinocytes to a variety of lymphokines and monitored changes in their MHC molecule expression. Murine cultured keratinocytes were treated with recombinant interferon-gamma (IFN-gamma), and MHC antigen expression quantified by flow cytometry. IFN pretreatment resulted in the up-regulation of class I molecule expression, but no class II expression was detected. In addition, cultured murine keratinocytes exposed to a combination of recombinant tumour necrosis factor (TNF)-alpha and IFN-gamma, or crude lymphocyte supernatants, failed to show positive membrane staining for class II molecules. However, rat keratinocytes cultured under conditions identical to murine cells were induced to express class II molecules after IFN-gamma pretreatment. The inability of IFN to induce class II expression on murine keratinocytes appears not to result from cell culture, as subcutaneous injection of IFN fails to induce epidermal class II antigen expression. However, class II expression can be induced on rat epidermis in vivo. Thus, the response of epidermal keratinocytes to IFN-gamma appears to show species variation.     

The keratinocyte-derived cytokine IL-7 increases adhesion of the epidermal T cell subset to the skin basement membrane protein laminin-5.

Human epidermis contains a subset of epidermal T cells that can mount an immune response by migrating through the skin and into the peripheral lymphnodes to proliferate before re-entering the epidermis. The cytokine IL-7 is shown to be localized to the basement membrane of normal human skin. Furthermore, culturing in the presence of IL-7 causes increased adhesion of epidermal T cells but not peripheral blood T cells to the major epidermal basement membrane protein, laminin-5. The mechanism for increased T cell adhesion to laminin-5 is due, at least in part, to an increase in the cell surface expression of the integrin alpha3beta1. Epidermal T cells cultured in IL-7 that are strongly adherent to laminin-5 are shown by flow cytometry to consist of a variety of subsets; therefore, the increase in cell adhesion is not due to an outgrowth of one T cell subset during culturing. We hypothesize that in vivo, exposure to IL-7 is required for epidermal T cell adhesion to laminin-5.     

Induction of acute phase response genes in keratinocytes following exposure to oligodeoxynucleotides

Keratinocytes have the ability to take up oligodeoxynucleotides (ODN) and plasmid DNA probably by receptor-mediated endocytosis. Despite the use of DNA for antisense and gene therapy little is known about the regulation of genes following exposure to nucleic acids. To systematically identify gene regulation in keratinocytes upon exposure to ODN we screened human cytokine DNA arrays containing 383 different genes and found interleukin (IL) 1alpha, IL-1beta, integrin-beta(1), alpha-tubulin, and follistatin highly induced, while most genes were unaffected. The time course and concentration dependence for IL-1alpha and follistatin expression were analyzed by standard northern blot technique. ODN of different length and sequence induced comparable amounts of IL-1alpha and follistatin. Their induction was independent of negative charge and of several proinflammatory compounds such as lipopolysaccharides, IL-1beta, and interferon-gamma but was partly inhibited by activin A. In summary, our study revealed several genes of the acute phase protein family that are induced in a non-sequence-specific manner following the exposure of normal human keratinocytes to ODN. Therefore it is tempting to speculate that upon internalization ODN bind to an intracellular receptor (e.g., Toll-like receptor 9) which mediates signaling.     

Interleukin (IL)-18 induces Langerhans cell migration by a tumour necrosis factor-alpha- and IL-1beta-dependent mechanism

Following skin sensitization a proportion of epidermal Langerhans cells (LC) are stimulated to leave the skin and to migrate, via afferent lymphatics, to draining lymph nodes where they accumulate as immunostimulatory dendritic cells (DC). It has been demonstrated previously that tumour necrosis factor-alpha (TNF-alpha), an inducible product of epidermal keratinocytes, and interleukin (IL)-1beta, produced exclusively by LC in murine epidermis, provide important signals for the initiation of this response. Recently, it has been demonstrated that IL-18, a cytokine produced by both LC and keratinocytes within the epidermis, may also participate in immune responses induced following skin sensitization. In the present investigations, the ability of IL-18 to contribute to the regulation of LC migration and the accumulation of DC in draining lymph nodes has been examined. It was found that, like IL-1beta, IL-18 administered intradermally to mice resulted in a significant reduction in epidermal major histocompatibility complex (MHC) class II+ LC densities and a marked increase in lymph node DC numbers. Using neutralizing anti-TNF-alpha and blocking anti-type I IL-1 receptor (IL-1RI) antibodies, it was shown also that the induction by IL-18 of both LC mobilization and DC accumulation in regional lymph nodes was dependent upon availability of TNF-alpha and the integrity of IL-1RI signalling. Furthermore, using IL-1beta converting enzyme (caspase-1) knockout mice, IL-18-induced LC migration was found to have a mandatory requirement for active IL-1beta. Importantly, not only was IL-18 able to contribute to the regulation of LC migration, it was found to be essential for the manifestation of these processes in response to topical sensitization with the contact allergen oxazolone.     

Development and validation of human psoriatic skin equivalents

Psoriasis is an inflammatory skin disease driven by aberrant interactions between the epithelium and the immune system. Anti-psoriatic drugs can therefore target either the keratinocytes or the immunocytes. Here we sought to develop an in vitro reconstructed skin model that would display the molecular characteristics of psoriatic epidermis in a controlled manner, allowing the screening of anti-psoriatic drugs and providing a model in which to study the biology of this disease. Human skin equivalents generated from normal human adult keratinocytes after air exposure and stimulation by keratinocyte growth factor and epidermal growth factor displayed the correct morphological and molecular characteristics of normal human epidermis whereas the psoriasis-associated proteins, hBD-2, SKALP/elafin, and CK16, were absent. Skin equivalents generated from foreskin keratinocytes were clearly abnormal both morphologically and with respect to gene expression. When normal skin equivalents derived from adult keratinocytes were stimulated with psoriasis-associated cytokines [tumor necrosis factor-α, interleukin (IL)-1α, IL-6, and IL-22] or combinations thereof, strong expression of hBD-2, SKALP/elafin, CK16, IL-8, and tumor necrosis factor-α was induced as shown by quantitative polymerase chain reaction and immunohistochemistry. Retinoic acid but not cyclosporin A was found to inhibit cytokine-induced gene expression at both the mRNA and protein levels. These results illustrate the potential of this disease model to study the molecular pathology and pharmacological intervention in vitro.     

Effects on rat T-helper cell proliferation by syngeneic epidermal cells exposed to IFN-gamma in vivo

In several conditions in the skin, characterized by T-cell infiltration, keratinocytes are induced to synthesize and express class II transplantation antigens. The biological significance of this induced expression is still not understood. In this study, class II antigens were induced on rat ear keratinocytes by local intradermal injections into the ear of rat recombinant interferon-gamma (IFN-gamma). Epidermal cell suspensions prepared from these ears contained more than 50% class II-expressing cells, as judged by immunocytochemistry, compared with less than 5% in untreated epidermis. When comparing the capacity of these to different epidermal cell populations to stimulate a syngeneic PPD-specific T-helper cell line, it was found that IFN-gamma-exposed epidermal cells induced a lower T-cell response to PPD than did normal epidermal cells. This discrepancy could not be explained by either an infiltration of inflammatory cells into the epidermis of IFN-gamma-treated ears or by a difference in interleukin-1 production as determined in culture supernatants. The addition of indomethacin to cultures with IFN-gamma-exposed epidermal cells restored the T-cell response to PPD to that of normal epidermal cells, suggesting an inhibitory effect of prostaglandins. Our data indicate that epidermal cells exposed to IFN-gamma in vivo can suppress an antigen-specific T-cell proliferation.