| Background and ObjectiveAt present, the mechanisms of some immune related skin dermatoses like psoriasis, vitiligo and atopic dermatitis are still unclear, but it is known to all that they seem highly relevant to immuno-inflammation, especially in and around the skin lesions. So the involvement of such inflammatory cytokines and cellular surface antigens in these kinds of dermatoses comprises a large immune net, once it loses balance, skin diseases occur, and it also directly affects illness period and/or condition, and the choice of clinical treatment.We know that ultraviolet is quite relevant to our human skin. Keratinocytes and melanoytes are the two main cells in the skin as the targets for UV irradiation, and narrow-band UVB is now extensively applied to treat some immune-related skin dermatoses like psoriasis, vitiligo and atopic dermatitis, and it seemed to have got a significant and satisfactory treatment effectiency, but the mechanism is still not completely elucidated while immunosuppression takes the effect to some degree. Reasons may include to activate inhibitory T lymphocytes, then intervent the proliferation of antigen specific CD8~+ effect T lymphocytes, ultimately inhibit the cellmediated immunoreaction; Keratinocytes can excrete many kinds of immune inhibitory cytokines and neuropeptides, andα-melanocyte stimulating hormone(α-MSH) is especially important because it can inhibit the production and viability of lots of inflammatory cytokines; it can down-regulate the expression of co-stimulating molecules like B7(CD80,CD86) on APCs; it can also down-regulate TNF-αinduced CD54 expression and IL-1βinduced NF-κB expression, etc.CD40 is a member of the TNF receptor family of cell surface proteins expressed on many kinds of cells. It is a kind of co-stimulating molecule mainly expressed on APCs, which was discovered in 1985, and was found to express on keratinocytes and melanocytes in 1996 and 2002, IFN-γcould up-regulate CD40 expression on these cells. Its ligation with CD40L(gp39) is known to be an important pathway leading to skin inflammatory disorders such as psoriasis and AD through regulating the release and expression of some cytokines and molecules, playing an important role in inflammation, now known as CD40/gp39 pathway.As Interferon-γis often used to stimulate the expression of leukocyte differentiation antigens in vitro, and it also takes effects in vivo, thus we choose IFN-γas a stimulator/regulator of cell differentiation antigens on karatinocytes and melanocytes in our experiments.We hold the whole experiments in order:①To investigate and compare response abilities of normal human melanocytes and keratinocytes to UVB irradiation under different dosages and/or different time intervals by light microscopy, cell count and MTT assay.②To investigate whether UVB irradiation affects IFN-γinduced overexpression of surface antigens on normal human keratinocytes and melanocytes in vitro. Methods include a pre-culture of cells(KC, MC) with recombinant human IFN-γ, then were irradiated with UVB. FACS analysis, RIA or ELISA were used to detect the expressions of surface antigens, concentrations ofα-MSH or levels of cytokines respectively.③To investigate whether UVB irradiated keratinocyte medium could affect IFN-γinduced surface antigen expressions on melanocytes. Methods were similar to②. Taken together, some mechanisms for UVB irradiation to treat psoriasis, vitiligo, etc. immune related dermatoses will be disscussed.Methods1. Cell culture: Healthy foreskin was obtained from urinology department. Cells were cultured with KC-SFM and remained cells with MCDB153 medium. After supplying the new medium every 3 days, the pure cultured keratinocytes and melanocytes could be harvested in 1-2 weeks. Cells could be applied for experiment after 2-3 passages.2. UVB irradiation: The cultured cells were plated in 96-well plate or in 10cm dish. The former was used for detecting cellular viability, the latter for observing cell morphology. Cells were divided into irradiation and sham-irradiation group, irradiation group would be irradiated by designed UVB in PBS(15cm away from light source).3. Cell morphology observation: After 24h,48h and 72h of UVB irradiation, microscope(Olympus, CH type) was used to observe cell morphology change.4. Cell counting and cellular viability detection: 0h,24h,48h,72h after irradiation, MTT assay was used to detect cellular viability and cell counting to record proliferation curves.5. Cell surface antigen detection: To harvest sham or irradiated conditioned cells with 0.25% protease into lml EP pipe. Treatment with FITC-labeled CD40 monoclone antibody, and then mixed for flow cytomestry detection.6.α-MSH detection: RIA was adopted, samples does not cross with ACTH,β-MSH andγ-MSH. Samples were divided into contols,300IU/ml IFN-γgroup and 30mJ/cm~2 UVB group, and the supernatants were harvested by Oh, 24h, 48h, 72h and 96h after irradiation。7. Cytokine determination: To harvest the supernatant from each group, manipulate IL-6, IL-8 and TNF-αdetection according to ELISA kit instructions.8. Statistical analysis: To analyze experimental data with SPSSll.0 Statistical software including t test and ANOVA.Results1. Compared with controls, UVB-irradiated keratinocytes were damaged morphologically, cell growth was inhibited and cellular activity decreased dose- and time-dependently. On the contrary, no obvious damage happened to melanocytes when low-dosage UVB was administrated. However, their cellular activity got even higher. But with the increase of UVB dosage, they also showed similar change to that of keratinocytes. In addition, the cellular activity ratios calculated from various intervals after low-dosage UVB irradiation(72h/48h and 72h/24h ) were reversed between the two kinds of cells. The MTT ratio showed a decrease in keratinocytes(1.81 and 1.23), while an increase in melanocytes (0.94 and 1.01).2. Normal human keratinocytes expressed low but detectable CD40, CD54 and MHC-II, which could be significantly, enhanced by IFN-γstimulation (P＜0.01). The expression oft he antigens on the IFN-γ-treated cells was markedly down-regulated after UVB irradiation (P＜0.01) Meanwhile, the concentration ofα-MSH increased with to the maximum (P＜0.01) by 72h after irradiation. In addition, the expression of CD54 and the release of IL-8 of keratinocytes were specially related to the CD40 expression and could be magnified through ligation with sCD40L.3. Melanocytes could express low but detectable CD40 molecule, IFN-γcould upregulate CD40 expression, UVB could not affect CD40 expression orα-MSH excretion. UVB irradiated keratinocyte medium could inhibit IFN-γinduced CD40 expression on melanoytes.Conclusion1. The photodamage of UVB irradiation on keratinocytes and melanocytes shows quite different. Melanocytes show a stronger resistant ability to UVB irradiation compared with keratinocytes.2. UVB irradiation down-regulates IFN-γinduced expressions of surface antigens like CD40 on keratinocytes, this may be highly relevant withα-MSH, which is a strong autocrine immunosuppressive factor. UVB could not affect CD40 expression on melanocytes directly, but could inhibit IFN-γinduced CD40 expression on melanocytes indirectly through keratinocytes to secreteα-MSH. CD40 overexpression is significant for the development of immune dermatoses, and UVB is a potent intervention by reducing CD40 overexpression or by disrupting the CD40/gp39 pathway. |
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