Inflammatory skin disease in K14/p40 transgenic mice: evidence for interleukin-12-like activities of p40

The proinflammatory cytokine interleukin-12, a p35/p40 heterodimer, is produced by resident cells in skin and has been implicated as a pathogenetic factor in T-cell-mediated skin diseases. Secretion of heterodimeric interleukin-12 is always accompanied by production of p40 monomer and p40/p40 homodimer. To investigate the possible in vivo role of p40 per se, we generated mice that constitutively express monomeric and homodimeric p40 in basal keratinocytes. These mice spontaneously developed an eczematous skin disease that was characterized by hyperkeratosis, focal epidermal spongiosis, and a mixed inflammatory infiltrate composed of T cells (CD4+), macrophages, eosinophils, mast cells, and few neutrophils. Fluorescence-activated cell sorter analysis of transgenic epidermal cell suspensions revealed induction of major histocompatibility complex class II molecules on keratinocytes and a 2-3-fold increase in the content of Langerhans cells. Cytokines produced by these activated epidermal cells include interleukin-1alpha and tumor necrosis factor alpha. The skin disease in K14/p40 mice was similar to that of littermate mice that received injections of interleukin-12, suggesting overlapping in vivo functional properties. As induction of interferon-gamma is a major function of interleukin-12, we tested the in vitro ability of transgenic p40 to induce interferon-gamma. In contrast to interleukin-12, transgenic p40 did not stimulate interferon-gamma secretion by cultured splenocytes. We conclude that transgenic p40 and interleukin-12 are equally capable of initiating cutaneous inflammation. Despite these in vivo similarities, there is a clear functional difference between interleukin-12 and transgenic p40 in vitro, suggesting that interferon-gamma is not a major factor contributing to interleukin-12-like activities of transgenic p40.     

Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene

Interleukin-18 is a potent inducer of interferon-gamma by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-beta-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-gamma expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-gamma-dependent inflammatory processes through expression, activation, and secretion of interleukin-18.     

Differential expression and function of Toll-like receptors in Langerhans cells: comparison with splenic dendritic cells

Toll-like receptors are key elements in pathogen recognition by the host immune system. Although the expression pattern and functions of Toll-like receptors have been studied in a variety of cytokine-induced dendritic cells, it remains unknown whether Toll-like receptor stimulation influences maturation and cytokine production of authentic Langerhans cells. We purified murine epidermal Langerhans cells along with splenic dendritic cell using a panning method. Langerhans cells expressed Toll-like receptor 2, 4, and 9 but not 7, the pattern of which suggests Langerhans cells are the closest to one of the murine dendritic cell lineage, CD11c+11b+8 alpha-4-. Then we stimulated Toll-like receptor 2, 4, and 9 with the corresponding ligand, Staphylococcus aureus Cowan 1, lipopolysaccharide, and CpG, and found that all of these stimuli upregulated expression of B7-1 and B7-2 in splenic dendritic cells but not in Langerhans cells. As in human Langerhans cells, stimulation of murine Langerhans cells with Staphylococcus aureus Cowan 1, lipopolysaccharide, and CpG overall resulted in T helper 1-polarizing cytokine production (namely, induction of IL-12p40 and inhibition of TARC (thymus and activation-regulated chemokine)/CCL17). Exceptionally, lipopolysaccharide exhibited no effect on IL-12p40 production by Langerhans cells and inhibited IL-12p40 production by splenic dendritic cells. These results may represent the functional heterogeneity between Langerhans cells and splenic dendritic cells, and are important for better understanding of innate immunity to bacterial infections differentially regulated in the skin and spleen. MeSH terms: Toll-like receptors, Langerhans cells, dendritic cells.     

Interleukin-17 is produced by both Th1 and Th2 lymphocytes, and modulates interferon-gamma- and interleukin-4-induced activation of human keratinocytes

Interleukin-17 is a T-cell-derived cytokine, detected in skin affected by allergic contact dermatitis and psoriasis, which regulates keratinocyte expression of adhesion molecules and chemokines. In this study, we have analyzed whether interleukin-17 production segregates with a particular T helper (Th) cell subset, and have examined the capacity of interleukin-17 to modulate the activation of keratinocytes induced by Th1 and Th2 cytokines. A panel of 80 nickel-specific CD4+ T cell clones (36 Th0, 30 Th1, and 14 Th2) was isolated from peripheral blood or lesional skin of allergic contact dermatitis patients. Significant amounts (> 50 pg per ml) of interleukin-17 were released by about 50% of activated Th0, Th1, and Th2 cells. Interleukin-17 alone and in cooperation with interleukin-4, or to a lesser extent with interferon-gamma, decreased the interleukin-1 receptor antagonist to interleukin-1alpha ratio in the supernatants as well as in cell lysates from keratinocytes. In addition, interleukin-17 stimulated the release of growth-regulated oncogene-alpha, granulocyte-macrophage colony stimulating factor, and interleukin-6, with synergistic or additive effects when used together with interferon-gamma or interleukin-4. Interleukin-17 and interleukin-4 also increased stem cell factor release, a function that was inhibited by interferon-gamma. Moreover, interleukin-17 and interleukin-4 enhanced interferon-gamma-induced expression of intercellular adhesion molecule 1, but not CD40, on keratinocytes. The constitutive expression of interleukin-17 and interferon-gamma receptors on keratinocytes was not modulated by interleukin-17, interferon-gamma, or interleukin-4, whereas the interleukin-4 receptor was significantly downregulated by interferon-gamma. As a whole, the results indicate that interleukin-17 can participate relevantly in T-cell-mediated skin immune responses by amplifying both interferon-gamma- and interleukin-4-induced activation of keratinocytes.     

Inhibitory effect of 1α,25-dihydroxyvitamin D3 on allogeneic lymphocyte stimulation and Langerhans cell maturation

Abstract 1α,25-Dihydroxyvitamin D₃ (calcitriol) has been shown to inhibit the proliferation of peripheral blood mononuclear cells induced by allogeneic Langerhans cells in a human mixed epidermal cell lymphocyte reaction. The potent antigen-presenting function of Langerhans cells is gained during culture. We tried to dissect the effect of calcitriol on lymphocyte proliferation and Langerhans cell maturation in a murine mixed epidermal cell lymphocyte reaction using unfractioned epidermal cells as a source for Langerhans cells. First, calcitriol was added upon setup of the mixed epidermal cell lymphocyte reaction using cultured epidermal cells as antigen-presenting cells, and this was found to inhibit the proliferation of lymphocytes in a time- and concentration-dependent manner. When calcitriol was added only during preculture of freshly isolated epidermal cells, the subsequent mixed epidermal cell lymphocyte reaction was also inhibited. In addition, the growth of keratinocytes and the production of granulocyte/macrophage colony-stimulating factor during preculture of epidermal cells was completely inhibited. Supplementation with this growth factor only partially restored the proliferative response of lymphocytes. These results suggest that calcitriol inhibits both the alloantigen-driven stimulation of naive T cell and Langerhans cells maturation. Further experiments with purified Langerhans cells are required to elucidate the mechanism of action of calcitriol on Langerhans cell maturation. Received: 18 February 1998     

Gene transfer of secreted-type modified interleukin-18 gene to B16F10 melanoma cells suppresses in vivo tumor growth through inhibition of tumor vessel formation

Interleukin-18 is a novel cytokine identified as a strong inducer of interferon-gamma. Interleukin-18 has been shown to have similar bioactivities to interleukin-12 and to have antitumor efficacy in experimental models. In this study, we investigated whether the introduction of the interleukin-18 gene to B16F10 melanoma cells can induce antitumor response or not. Before the transfection, we modified the interleukin-18 gene to enable transfected tumor cells to secrete bioactive interleukin-18, because interleukin-18 does not have a signal sequence and requires processing by the interleukin-1 converting enzyme to attain the mature form. We found that B16 melanoma cells transduced with hybrid cDNA consisting of the interferon-beta signal sequence and mature interleukin-18 sequence, but not native interleukin-18, secreted a large amount of interleukin-18 and exhibited retarded tumor growth when injected in syngeneic mice. The antitumor effect was mostly abrogated by administration of anti-interferon-gamma antibody, but was not affected by in vivo depletion of CD8+ T cells or natural killer cells. Histologic analysis revealed that vascularization was markedly reduced and that necrosis was extensively induced in interleukin-18-secreting B16F10 melanoma (B16/IL18) tissues, whereas abundant tumor vessel formation was observed in B16/IL18 tissues of interferon-gamma-neutralized mice. We also found that chemokines, interferon-inducible protein-10 and monokine induced by interferon-gamma, were produced in B16/IL18 tissues and that the expression of both chemokines was dependent on that of interferon-gamma in the tumor tissues. Further, we showed that B16 melanoma cells secreted both chemokines in response to interferon-gamma. In addition, the expression of angiogenin, an angiogenic factor of melanoma, in B16 melanoma cells was reduced by interferon-gamma treatment. These results indicate that gene transfer of secreted-type interleukin-18 to B16F10 melanoma cells is a useful method of triggering an antitumor response without any systemic adverse effects and that the antitumor efficacy is mainly mediated by antiangiogenic activity, which is possibly involved in at least two dynamic changes induced by interferon-gamma inside B16 melanoma cells: the upregulation of antiangiogenic chemokines, interferon-inducible protein-10 and monokine induced by interferon-gamma, and the downregulation of angiogenic factor, angiogenin.     

Alterations of immune functions in barrier disrupted skin by UVB irradiation

BACKGROUND: While immunologic events elicited by acute barrier disruption or UVB irradiation have been studied in detail, the biological sequel of multiple insults to the skin is not well understood. OBJECTIVE: Since the skin would receive a variety of simultaneous stimuli in daily life, we tested the effects of sequential treatments with barrier disruption and UVB exposure on skin immunity. METHODS: Earlobes of BALB/c mice received tape-stripping and subsequently low-dose UVB exposure. Control mice were treated with either tape-stripping or UVB. The expression of surface markers and cytokine production in Langerhans cells and keratinocytes and the elicitation response of contact hypersensitivity were compared. RESULTS: By flow cytometry, tape-stripping augmented the expression of MHC class II, CD54, CD80, CD86 and CD40 on Langerhans cells, whereas UVB decreased the expression of some of these molecules. Combination of tape-stripping and UVB induced largely intermediate levels between these two. Upon stimulation with L cells expressing CD40L, Langerhans cells from tape-stripped and UVB-irradiated earlobes strongly transcribed mRNA for interleukin-1beta compared to each treatment. In keratinocytes, tape-stripping or UVB slightly upregulated tumor necrosis factor-alpha and interleukin-1alpha production at both mRNA and protein levels, whereas these two treatments synergistically increased the production of these cytokines. The in vitro hapten-presenting ability of Langerhans cells to trinitrophenyl-immune lymph node T cells ranked first in tape-stripping, second in tape-stripping plus UVB and third in UVB, and so did the intensity of elicitation responses in contact hypersensitivity to a hapten, picryl chloride. CONCLUSION: It is suggested that barrier disruption and UVB antagonize with each other in contact hypersensitivity as a reflection of their effects on Langerhans cell antigen-presenting function, but they synergize in cytokine production by both Langerhans cells and keratinocytes.     

Identification and characterization of the low-affinity receptor for immunoglobulin E (FcepsilonRII/CD23) on murine Langerhans cells

CD23 is a low-affinity receptor for immunoglobulin E and expressed on various hemopoietic cells. Although human epidermal cultured Langerhans cells express CD23, the study to identify CD23 on murine Langerhans cells has so far failed. In this study, using highly enriched (> 95%) Langerhans cells from murine epidermis obtained by the panning method, we investigated whether murine Langerhans cells express CD23. As the result of a series of experiments using fluorescence activated cell sorter analysis and the polymerase chain reaction method, it was revealed that CD23 is expressed on cultured Langerhans cells, but not on freshly isolated Langerhans cells. Comparison of the DNA sequence of polymerase chain reaction products of CD23 from cultured Langerhans cells with that from spleen leukocytes demonstrated that there were the same sequences between the two polymerase chain reaction products. The expression of CD23 on cultured Langerhans cells was downregulated when Langerhans cells were cultured with keratinocyte-derived cytokines: interleukin-1alpha, interleukin-18, macrophage colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. Moreover, it was shown that murine IgE bound to cultured Langerhans cells and this binding was partially inhibited when Langerhans cells were cultured with monoclonal antibody against CD23 (B3B4). Thus this study revealed murine cultured Langerhans cells do express CD23 and the discrepancy from previous reports may be due to the influence of cytokines derived from keratinocytes. Furthermore, the finding that murine cultured Langerhans cells bind IgE through CD23 suggests that CD23 on murine Langerhans cells may be involved in IgE-mediated immune responses.     

Interleukin-18 and the costimulatory molecule B7-1 have a synergistic anti-tumor effect on murine melanoma; implication of combined immunotherapy for poorly immunogenic malignancy

Interleukin-18 has been described recently as a cytokine secreted primarily by Kupffer cells. Furthermore, it has been shown that it has significant anti-tumor effects, which are mediated by T cells and natural killer cells, in a manner similar to interleukin-12. Here, we report the evaluation of the effects of the systemic administration of interleukin-18 in combination with B7-1 (CD80) expressed on tumor cells [interleukin-18 + B7-1] on the growth of murine B16 melanoma in vivo. After the subcutaneous inoculation of B16 melanoma, B16 tumors grew progressively in immunocompetent syngeneic C57BL/6 mice. Mice treated with either interleukin-18 or immunized with B7-1-transduced B16 did not demonstrate significant anti-tumor effect. The combination of the two treatments, however, resulted in dramatic suppression of melanoma formation, tumor growth, and a significant improvement in survival. Inhibitory effects of [interleukin-18 + B7-1] on lung metastasis in mice were also detected. Additionally, mice treated with [interleukin-18 + B7-1] showed an increase of natural killer cytotoxicity and interferon-gamma production in vivo. Unlike [interleukin-18 + B7-1], [interleukin-12 + B7-1] did not have a strong anti-tumor effect against B16 melanoma. Histologic characterization after the [interleukin-18 + B7-1] treatment confirmed the infiltration of natural killer cells into the tumor, suggesting that natural killer cells may be involved in the [interleukin-18 + B7-1]-induced anti-tumor effect. This finding was confirmed by showing that depletion of NK1.1+ cells before immunization inhibits the [interleukin-18 + B7-1]-induced anti-tumor effect. Depletion of CD3+ cells in vivo also decreased the anti-tumor effect of [interleukin-18 + B7-1], suggesting the importance of CD3+ T cells. Collectively, combination with interleukin-18 and B7-1 expression has synergistic anti-tumor effects against B16 murine melanoma.     

Counterregulation of interleukin-18 mRNA and protein expression during cutaneous wound repair in mice.

Recent work has suggested interleukin-18 to represent a proinflammatory cytokine that contributes to systemic and local inflammation. As the process of cutaneous wound healing crucially involves an inflammatory phase of repair, we investigated the regulation of interleukin-18 during the repair process. In non-wounded skin we observed high levels of interleukin-18 mRNA, whereas corresponding interleukin-18 protein was expressed only at low basal levels. Upon injury, we found a rapid and large induction of interleukin-18 protein expression, which is directly correlated with decreasing mRNA levels within the wound. Immunohistochemical analysis revealed different sites of expression in the wounded area, with keratinocytes as one major source of interleukin-18 production. The counterregulation of interleukin-18 mRNA and protein expression during wound repair in vivo might represent a general mechanism for interleukin-18 expressional regulation, as cytokine-stimulated keratinocytes exhibit a similar downregulation of interleukin-18 mRNA that is directly associated with increasing interleukin-18 protein levels in vitro. The rapid induction of interleukin-18 during wound healing suggests a role for interleukin-18 within the early phase of repair rather than a role in costimulation of interferon-gamma release from T cells, which are present in high numbers within the wounded area only during the late inflammatory phase of repair.     

Differentiated keratinocytes are responsible for TNF-alpha regulated production of macrophage inflammatory protein 3alpha/CCL20, a potent chemokine for Langerhans cells

The recruitment of immature dendritic cells into the epidermis is a key step in the development of cutaneous immunity, although the mechanism remains to be clarified. Recently, it was reported that both macrophage inflammatory protein 3alpha (MIP-3alpha)/CCL20 produced by keratinocytes and TNF-alpha are important in recruiting Langerhans cells (LC) to the epidermis. In this study, we examined the production of MIP-3alpha by human keratinocytes stimulated with TNF-alpha. Cultured keratinocytes showed enhanced expression of MIP-3alpha mRNA and protein when stimulated with TNF-alpha. In addition, conditioned medium from TNF-alpha-stimulated keratinocyte cultures induced the migration of L1.2 cells expressing CCR6. We next examined the production of MIP-3alpha in stratified keratinocytes and found that, in contrast to non-stratified keratinocytes, stimulation with TNF-alpha increased the expression of MIP-3alpha mRNA and protein. Moreover, skin samples grown in organ culture and treated with TNF-alpha showed MIP-3alpha in the keratinocytes of the spinous layer, but not in the basal layer, by immunofluorescence staining. Based on these results, we postulate that MIP-3alpha produced by keratinocytes in the spinous layer in response to TNF-alpha stimulation is a key chemokine responsible for the epidermal recruitment of Langerhans cells.     

Evaluation of apoptotic cells induced by ultraviolet light B radiation in epidermal sheets stained by the TUNEL technique

Two major components of epidermal cells, keratinocytes and Langerhans cells, are injured by ultraviolet light B radiation, resulting in sunburn cell (apoptotic cell) formation, impaired function, and a reduced number of Langerhans cells. Quantitative analysis of Langerhans cell damage is usually performed using epidermal sheets, whereas that of keratinocytes has been performed by counting the number of sunburn cells in vertical tissue sections. In this study we assessed the influences of ultraviolet light B radiation on epidermal cells by apoptotic cell formation, using murine epidermal sheets stained by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique. Ten to 75 mJ per cm2 of ultraviolet light B radiation induced apoptotic cells in abdominal skin of C3H mice. The cells were induced in 6 h after 50 mJ per cm2 of ultraviolet light B irradiation with the peak in number in 24 h, 18.8 +/- 5.0 per mm2 and 97.7 +/- 7.4 per mm2, respectively. One week later, the apoptotic cells were not visualized. As C3H/He, BALB/C, and C57BL/6 mice showed almost the same frequency of apoptosis in epidermal sheets from 50 mJ per cm2 ultraviolet light B-irradiated skin, the induction of the cells by ultraviolet light B radiation did not depend on the genetic trait of the mouse. Xeroderma pigmentosum type A gene-deficient mice, however, showed a greater induction of apoptotic cells (216.9 +/- 25.2 per mm2) by ultraviolet light B radiation than xeroderma pigmentosum type A wild-type mice (89.5 +/- 13.6 per mm2) and conventional mice. Pretreatment with a SPF 60 sunscreen agent was quite effective in reducing the induction of apoptotic cells. Using confocal laser scanning microscopy and double staining, 1.5 +/- 2.7% of apoptotic cells were Ia-positive cells in 24 h after 50 mJ per cm2 of ultraviolet light B radiation. Apoptotic Ia-positive cells were not observed 48 h after the radiation. On the other hand, no apoptotic dendritic epidermal T cells were observed in up to 75 mJ per cm2 of ultraviolet light B radiated skin. Thus, nearly all apoptotic cells were keratinocytes, and Langerhans cells and dendritic epidermal T cells appeared resistant to ultraviolet light B-induced apoptosis. Compared with the assessment in vertical tissue sections, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique with epidermal sheets appeared to be a more physiologically relevant method for quantitative evaluation of apoptotic epidermal cells induced by ultraviolet light B radiation.     

Interleukin-16 supports the migration of Langerhans cells, partly in a CD4-independent way

Migration of cutaneous dendritic cells is essential for the induction of primary immune responses. Chemotaxis plays an important part in guiding migrating cells through the skin. Therefore, we investigated the influence of interleukin-16, a potent chemoattractant, on the migratory properties of cutaneous dendritic cells. Interleukin-16 added to murine and human skin explant cultures, enhanced emigration of Langerhans cells as well as dermal dendritic cells out of the skin. In contrast to tumor necrosis factor-alpha, intradermally injected interleukin-16 did not reduce the density of Langerhans cells suggesting a chemotactic rather than a mechanistic migration-inducing effect of interleukin-16. In support of these findings, the known migration-promoting effect of tumor necrosis factor-alpha in skin explant cultures could be neutralized by anti-interleukin-16 antibody and vice versa, indicating different but cooperative ways of action for both cytokines. In whole skin explant cultures blocking of the interleukin-16 effect was also achieved with a monoclonal antibody against CD4, the receptor for interleukin-16. In contrast, in cultures of murine epidermis alone no blocking by anti-CD4 became obvious and in CD4-deficient mice Langerhans cell migration in response to interleukin-16 was maintained. This suggests that another receptor for interleukin-16 might be operative for Langerhans cells in the mouse epidermis. Finally, we detected interleukin-16-positive cells in the dermis of skin explants, tumor necrosis factor-alpha-treated and contact allergen-treated skin. Taken together, it seems likely that locally secreted interleukin-16 might serve to enhance the migration of cutaneous dendritic cells and optimize the response to foreign antigen encountering the skin.     

Evidence for involvement of the epidermal platelet-activating factor receptor in ultraviolet-B-radiation-induced interleukin-8 production

Ultraviolet B radiation has been shown to generate cutaneous inflammation in part through inducing oxidative stress and cytokine production in human keratinocytes. Amongst the proinflammatory cytokines synthesized in response to ultraviolet B radiation is the potent chemoattractant interleukin-8. Though the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and keratinocytes express PAF receptors linked to cytokine biosynthesis, it is not known whether PAF is involved in ultraviolet-B-induced epidermal cell cytokine production. These studies examined the role of the PAF system in ultraviolet-B-induced epidermal cell interleukin-8 biosynthesis using a novel model system created by retroviral-mediated transduction of the PAF-receptor-negative human epidermal cell line KB with the human PAF receptor. Treatment of PAF-receptor-expressing KB cells with the metabolically stable PAF receptor agonist carbamoyl-PAF resulted in increased interleukin-8 mRNA and protein, indicating that activation of the epidermal PAF receptor was linked to interleukin-8 production. Ultraviolet B irradiation of PAF-receptor-expressing KB cells resulted in significant increases in both interleukin-8 mRNA and protein in comparison to ultraviolet-B-treated control KB cells. Pretreatment with PAF receptor antagonists inhibited both carbamoyl-PAF-induced and ultraviolet-B-induced interleukin-8 production in the PAF-receptor-positive cells, but not in control KB cells. Similarly, treatment of the PAF-receptor-expressing primary cultures of human keratinocytes or the human epidermal cell line A-431 with carbamoyl-PAF or ultraviolet B radiation resulted in interleukin-8 production that was partially inhibited by PAF receptor antagonists. These studies suggest that the epidermal PAF receptor may be a pharmacologic target for ultraviolet B radiation in skin and thus may act to augment ultraviolet-B-mediated production of cytokines such as interleukin-8.     

Cyclosporin increases granulocyte/macrophage colony-stimulating factor (GM-CSF) activity and gene expression in murine keratinocytes.

Keratinocytes produce multiple cytokines in response to a variety of stimuli. The release of interleukin 1 (IL-1) from keratinocytes may be significant in initiation of cutaneous inflammation, and the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) is thought to be important in the regulation of antigen-presenting function by epidermal Langerhans cells. Because cyclosporin inhibits interleukin 2 release from T cells, it has been suggested that cyclosporin may function as an anti-inflammatory agent within the epidermis through inhibition of keratinocyte cytokine release. This investigation examined the direct effect of cyclosporin on the production of GM-CSF by murine keratinocytes and the keratinocyte cell line PAM 212. GM-CSF bioactivity increased in cell supernatants from keratinocytes exposed in vitro to 1 microgram/ml cyclosporin for up to 24 h. GM-CSF and IL-1 mRNA levels in keratinocytes cultured under similar conditions or in the presence of lipopolysaccharide also increased. The lack of inhibition of GM-CSF expression following cyclosporin treatment is consistent with recent observations in T cells and is opposite to the effect of cyclosporin on interleukin 2.     

Epidermal CD8+ T cells in chronic plaque psoriasis are Tc1 cells producing heterogeneous levels of interferon-gamma

The majority of epidermal CD8+ T cells in chronic plaque psoriasis are activated Tc1 cells producing interferon-gamma and no interleukin-4, a small proportion of which express NK-T receptors. To quantitate their level of cytokine production and characterize them further, CD8+ T cells were isolated from epidermal cell suspensions of lesional biopsies from 24 patients with chronic plaque psoriasis. T-cell lines (TCL) were established by culture of CD8+ T cells with feeders and IL-2 for 11 days and expansion with PHA. Ten TCL were stained for surface markers; 6 were cloned with PHA by limiting dilution. Interferon-gamma, interleukin-4 and interleukin-10 production was measured by ELISA after PMA/anti-CD3 activation of 15 TCL and 39 CD8+ T-cell clones. The 10 TCL stained were CD8alphabeta+ (93.3%), T-cell receptor-alphabeta+ (99.5%), costimulatory molecule CD28+ (90.1%), with a small CD8alphaalpha+ population (2.3%). No NK-T-cell receptor CD158a or CD158b expression was detected, whilst CD94 was expressed on 6.2% of cells in 6/9 TCL. All the TCL and 37/39 CD8+ T-cell clones produced interferon-gamma but no or minimal interleukin-4 or interleukin-10. The TCL produced a wide range of interferon-gamma levels (138 to 15,020 pg/ml). Clones from 3 patients showed low levels (60 to 1,410 pg/ml), from 2 patients high levels (6,105 to 43,040 pg/ml) and from 1 patient a wide range (405 to 36,010 pg/ml) of interferon-gamma production. Thus epidermal CD8+ Tc1 cells in chronic plaque psoriasis produce highly heterogeneous levels of interferon-gamma, which may reflect clinical diversity.     

Upregulation of interleukin-18 expression in mouse primary keratinocytes induced to differentiate by calcium

Interleukin-18 (IL-18) is an immunomodulatory cytokine that stimulates interferon-gamma production by T helper cells. Recently, basal keratinocytes have been shown to constitutively express IL-18, and IL-18 expression increases in the suprabasal keratinocytes in psoriatic lesions. In the study reported here we showed that in mouse epidermis, IL-18 immunoreactivity was markedly increased in the granular and cornified layers. To further investigate whether differentiated keratinocytes synthesize more IL-18, we examined the expression of mouse IL-18 in primary mouse keratinocytes induced to differentiate by calcium, an in vitro cell culture system mimicking keratinocyte differentiation in the epidermis. We demonstrated that IL-18 mRNA and protein in cultured keratinocytes were increased by calcium treatment in a time- and dose-dependent manner. The upregulation of IL-18 was associated with an increase in keratinocyte differentiation markers, and was dependent on the synthesis of new RNAs and proteins. However, the IL-18 protein in the cytoplasm was predominantly in the precursor form, and no increase in IL-18 activity was detected in the culture medium treated with calcium. Furthermore, blocking the calcium-induced keratinocyte differentiation with protein kinase C inhibitor inhibited the upregulation of IL-18 expression. These findings suggest that IL-18 is synthesized in keratinocytes mainly in the inactive precursor form, and its expression is upregulated as basal keratinocytes differentiate in the epidermis.     

结合珠蛋白在正常人表皮细胞及HaCaT细胞中的表达

目的 检测正常人表皮细胞及HaCaT细胞中结合珠蛋白(Hp)mRNA及蛋白的表达。方法 用原位杂交及RT-PCR法检测正常人表皮细胞及HacaT细胞中Hp mRNA的表达，用免疫组化法检测正常人表皮中Hp的表达；用免疫组化法及蛋白质免疫印迹法检测HaCaT细胞中Hp的表达。结果 在正常人表皮角质形成细胞(KC)及HaCaT细胞中Hp mRNA表达阳性；正常人表皮朗格汉斯细胞(LC)中Hp mRNA表达阴性；正常人表皮内可见Hp阳性的树突状细胞。用免疫组化法在每个HaCaT细胞胞质中未见明显Hp染色，但用蛋白质免疫印迹法在HacaT细胞中检测到Hp蛋白。结论 正常人KC及HaCaT细胞有合成Hp能力，正常人表皮LC无合成Hp的能力，在HaCaT细胞中有少量Hp表达。