Investigating The Influence Of Langerhans Cells On Keratinocyte Proliferative Response To Chemical And Ultraviolet B (uvb) Exposure

Initially, the Girardi lab demonstrated that LC facilitate chemical carcinogenesis by showing lower levels of proliferation in LC-deficient (Langerin-diptheria toxin A transgenic) mice relative to LC-intact littermate controls. Thus, we sought to explore the breadth of potential influences of LC on keratinocyte responses (hypertrophy, apoptosis, and clonal expansion) in two classic models of carcinogenesis (chemical and UVB), by using immunohistochemical (IHC) and immunofluorescent (IF) methods of cell identification. At baseline, LC-deficient compared to LC-intact mouse skin showed comparable proliferation levels as measured by Ki-67 IHC (13.6±2.6 vs 12.7±2.8 Ki-67+ cells, NS) and epidermal thickness (7.9±1.8 vs 6.9±2.8 micrometers, NS). After a single application of mutagen DMBA, followed by repeated applications of tumor promoter TPA (x 5.5 wks), LC-depleted skin showed smaller increase in epidermal proliferation (31.5 ± 7.7 vs 25.7 ± 7.4 # Ki-67+ cells, P=0.007) and minimal epidermal thickness (59.2 ± 28.4 vs 25.8 ± 8.9 micrometers, P\u3c0.0001), as compared to LC-intact skin. There was a similar trend of decreased epidermal proliferation and decreased minimal epidermal hypertrophy in LC-depleted (Lang-DTA) skin after chronic TPA-alone (x 4 wks, P=0.0002 and P\u3c0.0001, respectively), or DMBA-alone application (P=0.0001 and P\u3c0.0292, respectively). To address the potential influence of LC on acute UVB-induced keratinocyte damage, mice were irradiated with a single dose of 3,360J/m2. There was a trend toward increased epidermal apoptosis (cleaved caspase-3, P\u3c 0.0001 and sunburn cells). Chronic low-dose UVB (500-1500 J/m2, 5d/wk x 5-9wks) induced mutant keratinocyte islands over-expressing p53 (CM5) in both LC-intact and LC-deficient skin, with a statistical trend towards greater levels within LC-intact skin. After 5 and 9 weeks of chronic UVB exposure Lang-DTA mouse skin, compared to NLC, had a smaller number of mutated clones per mm2 (P=0.0489 and P=0.004, respectively). After 5 weeks of chronic UVB followed by 3 weeks of no UVB (and DT injection at weeks 6, 7 and 8), there was a smaller number of mutant clones per mm2 in Lang-DTR mouse skin versus NLC (P\u3c 0.0001). For this group, statistical significance was also reached for mean number of mutated clones/mm2 per group in Lang-DTR vs NLC mice (P=0.0106). After 5 weeks of chronic UVB, `replacement\u27 LC are associated with mutant clones in Lang-DTA mice, as compared to areas without clones (P\u3c 0.0001). Statistical significance was also reached for mean density per mouse (P=0.0106). A similar association was seen in NLC mice after 5 weeks of chronic UVB, where LC were associated with mutant clones in NLC mouse skin as compared to areas without clones (P= 0.0047). After chronic UVB, GL3+ dendritic cell numbers were decreased, but still present, in Lang-DTA and NLC mice, many located within or near hair follicles and some seen interacting with LC. After 5 weeks of chronic UVB, Lang-DTA mice have approximately 12 times less langerin+ DC compared to NLC mouse skin. In summary, LC exert diverse, measurable influences on chemically induced keratinocyte proliferation and acute and chronic UVB-induced keratinocyte apoptosis