Effects Of Topical Pimecrolimus On High-dose Ultraviolet B-irradiated Epidermal Langerhans Cells And Relevant Mechanism Study

Background Topical calcineurin inhibitors(TCI),pimecrolimus cream and tacrolimus ointment,can block T-cell proliferation and have been mainly used for the treatment of atopic dermatitis.Epidermal Langerhans cells(LC)-mediated immunosurveillance is one important pathway against tumorigenesis.Epidermal LC are key mediators of skin immunity.They process antigen and migrate to local draining lymphnodes from epidermis,expressing CD1 a and Langerin(CD 207).Epidermal immature LC can discern and ingest abnormal antigen,and LC mature gradually in the process of migrating to the draining lymphnodes and then stimulate T lymphocytes,the morphology of LC becomes rounder,and the dendrites become shorter in the process of migration under the role of various cytokines and molecules.As LC gain functional maturity,they express HLA-DR and co-stimulatory molecules CD83 and CD86.Martires,et al.reported that low-dose short-term UV irradiation-induced changes were minimally affected by pimecrolimus,which showed that there were more epidermal HLA-DR+ LC and CD83+ LC in pimecrolimus+UV group compared to UV-only group.In our previous small sample experiments,we observed that a single high-dose UVB irradiation could induce epidermal CD1a+ LC reduction,however,the CD1a+ LC number of UVB+pimecrolimus increased.K?lgen,et al.reported that the possible mechanisms for significant epidermal LC depletion caused by single high-dose UVB irradiation in human skin are apoptosis and migration,especially the increase of LC migration.Many cytokines,adhesion molecules and chemokines play roles in the process of epidermal LC migration.The increase in the level of TNF-? and IL-1?,and the decrease in the level of E-cadherin could promote LC migration from epidemis to dermis.Herein,we propose a hypothesis that topical pimecrolimus could reverse the epidermal LC reduction induced by high-dose UVB irradiation by inhibiting the epidermal LC migration.To verify this hypothesis,we will use immunohistochemistry,flow cytometry,reverse transcription-PCR(RT-PCR),Western blot and other means to investigate the effects of topical pimecrolimus on high-dose UVB-irradiated epidermal LC and possible molecular mechanism.Methods 1.ImmunohistochemistryForty fresh human foreskin tissues were randomly divided into 4 groups of 10 tissues each as follows: Group A,control;Group B,tissues topically applied once with pimecrolimus 1% on epidermal side;Group C,tissues irradiated once with 180 m J/cm2 UVB on epidermal side;Group D,tissues topically applied pimecrolimus after UVB irradiation.All the tissues were cultured at 37�C.After that,four time points were set as follows:(a)at 0 h;(b)at 18 h;(c)at 24 h;(d)at 48 h.Each tissue was cut into 4 pieces corresponding to 4 time points,for each group.The tissues were collected at every time point respectively.Part of each skin specimen was fixed with formalin until further processing for immunohistochemical staining of CD1 a,Langerin and HLA-DR.The numbers of epidermal CD1a+,Langerin+ and HLA-DR+ LC were all counted for five successive fields in high magnification(HM,X 400)with light microscopy.2.Flow cytometry The culture medium was collected at every time point respectively.The percentage of CD1a+ cells in the culture medium was detected by means of flow cytometry.3.RT-PCR and Western blot The above collected tissues were detected for the m RNA levels of cytokines and molecules(TNF-?,IL-1? and E-cadherin)related to LC migration by means of RT-PCR.Verify the cytokines and molecules with significant changes at the protein level by using Western blot.Results 1.Effects of topical pimecrolimus on high-dose UVB-irradiated epidermal LC numbersThe epidermal CD1a+ LC numbers had no significant differences at different time points,for control and pimecrolimus-only group.For UVB-only group,the epidermal CD1a+ LC numbers at 18 h,24h and 48 h all had obvious reduction compared to CD1a+ LC number at 0h.At 18 h,24h and 48 h,the numbers of epidermal CD1a+ LC of UVB+pimecrolimus group were all had significant increase compared to UVB-only group.Similar results were obtained with the immunohistochemical staining for Langerin.The epidermal HLA-DR+ LC numbers had no significant differences at different time points,for control,pimecrolimus-only and UVB+pimecrolimus group.For UVB-only group,the epidermal HLA-DR+ LC numbers at 18 h,24h and 48 h all had a slight but not significant decrease in comparison to HLA-DR+ LC number at 0h.2.Effects of topical pimecrolimus on high-dose UVB-irradiated epidermal LC migrationFrom 18 h to 48 h,the percentages of CD1a+ cells in the culture medium all showed a significantly increasing trend for each group.For pimecrolimus-only group,the percentages of CD1a+ cells in the culture medium had no significant differences compared to control group at 18 h,24h and 48 h.At 18 h,24h and 48 h,the percentages of CD1a+ cells of control group were all significantly less than UVB-only group and UVB+pimecrolimus group,and the percentages of CD1a+ cells of UVB-only group were significantly more than UVB+pimecrolimus group.3.Effects of topical pimecrolimus on cytokines and molecules related to LC migration after high-dose UVB irradiationRT-PCR For both UVB-only group and UVB+pimecrolimus group,the TNF-? m RNA relative expression had significantly increasing trends from 0h to 48 h.The TNF-? m RNA relative expression of UVB-only group was obviously higher than control group at 18 h,24h and 48 h.For UVB+pimecrolimus group,the TNF-? m RNA relative expression was significantly lower compared to UVB-only group.For both UVB-only group and UVB+pimecrolimus group,the IL-1? m RNA relative expression had significantly increasing trends from 0h to 48 h.The IL-1? m RNA relative expression of UVB-only group was obviously higher than control group at 18 h,24h and 48 h.For UVB+pimecrolimus group,the IL-1? m RNA relative expression had no significant difference compared to UVB-only group.For both UVB-only group and UVB+pimecrolimus group,the E-cadherin m RNA relative expression had significantly declining trends from 0h to 48 h.The E-cadherin m RNA relative expression of UVB-only group was obviously lower than control group at 18 h,24h and 48 h.For UVB+pimecrolimus group,the E-cadherin m RNA relative expression was significantly higher at 24 h and 48 h,but had no significant difference at 18 h,compared to UVB-only group.Western blotFor both UVB-only group and UVB+pimecrolimus group,the TNF-? protein relative expression had significantly increasing trends from 0h to 48 h.The TNF-? protein relative expression of UVB-only group was obviously higher than control group at 18 h,24h and 48 h.For UVB+pimecrolimus group,the TNF-? protein relative expression was significantly lower compared to UVB-only group.For both UVB-only group and UVB+pimecrolimus group,the E-cadherin protein relative expression had significantly declining trends from 0h to 48 h.The E-cadherin protein relative expression of UVB-only group was obviously lower than control group at 18 h,24h and 48 h.For UVB+pimecrolimus group,the E-cadherin protein relative expression was significantly higher compared to UVB-only group.Conclusion This study found that high-dose UVB irradiation could induce significant human epidermal LC reduction,however,topical pimecrolimus cream could reverse the LC reduction induced by high-dose UVB irradiation,where the possible mechanism might be the inhibition of epidermal LC migration via inhibition of the increase in the level of TNF-? expression and the decrease in the level of E-cadherin expression induced by high-dose UVB irradiation.