Keratinocytes Express CD80 And Mechanism Of Co-cultured Keratinocytes Induce Local Tolerance

It was previously thought that keratinocytes did not constitutively express MHC class II antigens and were unable to generate CD80 and CD86, the CD28 counterligands with costimulatory activity. Due to their less immunogenic, long-term survival and immunological tolerance of cultured keratinocytes allografts were initially claimed, but the later studies found that the cultured allografts did not survive permanently. The precise mechanisms of ultimate rejection of keratinocytes are not very clear. This study focus on the possible reason for cultured allogeneic keratinocytes transplantation rejection and the mechanisms of local tolerance induced by co-cultured murine keratinocytes.Keratinocytes were isolated from neonatal C57BL/6 (H-2b) and BALB/c (H-2d) mice and purified by defined cultured medium. They were cultured with or without 10% calf serum for 1, 4, and 7days respectively. Anti-IA/IE (H-2d/b) monoclonal antibodies were used to identify the remaining cells without Langerhans cells. Theresults of FACS and confocal microscope showed that CD80 could be detected onkeratinocytes only till 4d and 7d, but not 1d, while CD86 could not be detected all the time. Bone marrow-derived dendritic cells (DCs), used as a control, strongly expressedboth CD80 and CD86. RT-PCR analysis confirmed this result. Expression of the mRNA for CD80 (729 bp) amplified significantly from the 1st day to 7th day, as expression of the control /3-actin (253 bp), but CD86 (544 bp) was not detected. DCs cultured for 10 days significantly expressed both CD80 and CD86. Considering Langerhans cells expressed CD86 more widely and prominently than CD80, these results indicated that the cultured cells did not contain Langerhans cells and that murine keratinocytes expressed CD80. Moreover, mixed culture of lymphocytes with syngeneic/allogeneic keratinocytes (MLR) was performed. The results showed that Keratinocytes cultured in 10% calf serum for 7d stimulated effectively allogeneic rather than syngeneic T cell proliferation (3H-TdR cpm 13651?415 vs 4063?26). Interestingly, keratinocytes cultured in serum-free medium for 7d stimulated effectively not only allogeneic but also syngeneic T cell proliferation (3H-TdR cpm 7617?18vs7591?35).Our findings demonstrated for the first time that costimulatory molecule CD80 can be expressed on keratinocytes in vitro. As expression of MHC class I antigens and CD80, cultured murine keratinocytes could activate allogeneic T-cell significantly. These data provided a possible explanation for ultimate rejection of the allogeneic keratinocytes. The novel results also call into question the generally accepted notion that keratinocytes are immunologically naive due to their inability to provide effective secondary signals.The typical acute rejection did not appear after transplantation of co-culture allogeneic/syngeneic keratinocytes in rabbit, mice and human models. The current explanation is that syngeneic kerainocytes induce local tolerance by activating IL-10-secreting T cells. For our study that newborn murine keratinocytes expressed CD80 but not CD86 upon culture. Thus, we hypothesized that the co-cultured donor and recipient keratinocytes could influence the expression of costimulatory moleculeeach other without the participation of immunocytes, and it might be related to local tolerance.BALB/c and C57BL/6 keratinocytes were co-cultured for 7days with the following ratios: 25% to 75%, 50% to 50%, and 75% to 25%, while homogeneous keratinocytes as the control groups. Keratinocytes proportions did not show any difference between chimeric and homogeneous groups during 2 weeks' culture with or without serum. This was consistent with other investigators' data that the cultured chimeric epithelial sheets were composed of two cell types in the same ratio as the initial cell plating. Mixed cultures of lymphocytes with chimeric/homogeneous keratinocytes were performed, and CD80 expression level on keratinocytes was detected by mean fluorscence intensity (MFI) of FACS analysis. A strong, dose-dependent suppression of T cel...