Effect of HLA-DR expression on IL-1 production by keratinocytes

There is increasing evidence that keratinocytes may participate in the inflammatory processes and immune response of skin. Keratinocytes can be induced to express functional immunocompetent molecules such as HLA-DR antigen. In addition, they secrete a wide range of inflammatory cytokines including IL-1. In order to better understand the immunologic role of keratinocyte, it is necessary to quantitate the amount of IL-1α produced by cultured human keratinocytes and evaluate the effect of HLA-DR expression on the production of IL-1α by keratinocytes. In this study, we employed the radioimmunoassay to measure the amount of IL-1α produced by cultured human keratinocyte. Human keratinocytes were isolated from neonatal foreskins and grown in MCDB 153 medium. HLA-DR expression was induced by 30 U/ml of r-IFN-γ. The supernatants were assayed for IL-1α. The results were as follows: 1. When culture medium was not replaced until 72 hours, cell numbers of keratinocytes without treatment of r-IFN-γ were 0.80 ± 0.26 x 10⁶, 1.16 ± 0.21 x 10⁶, 1.33 ± 0.21 x 10⁶ at 24, 48 and 72 hours, respectively. When culture medium was replaced every 48 hours, cell numbers of keratinocytes with treatment of r-IFN-γ were 0.72 ± 0.29 x 10⁶, 1.06 ± 0.26 x 10⁶ and 1.06 ± 0.25 x 10⁶ at 24, 48 and 72 hours, respectively. Antiproliferative effect of r-IFN-γ was significant at 48 and 72 hours (p = 0.046, p = 0.003). When culture medium was replaced every 48 hours, cell numbers of keratinocytes without treatment of r-IFN-γ were 1.16 ± 0.21 x 10⁶, 1.69 ± 0.27 x 10⁶, 2.01 ± 0.18 x 10⁶ at 48, 96 and 144 hours, respectively. Cell numbers of keratinocytes with treatment of r-IFN-γ were 1.06 ± 0.26 x 10⁶, 1.51 ± 0.20 x 10⁶ and 1.67 ± 0.17 x 10⁶ at 48, 96 and 144 hours, respectively. Antiproliferative effect of r-IFN-γ was significant at 48 and 144 hours (p = 0.046, p = 0.0001). 2. Relatively lower concentration of IFN-γ (30 U/ml) can induce HLA-DR expression on keratinocytes. The percent of HLA-DR positive keratinocytes were 42.15 ± 4.87% by 48 hours. Then it decreased to 14.26 ± 2.85% by 144 hours. 3. When culture medium was not replaced, the levels of IL-1α from supernatants of cultured keratinocytes were 1.513 ± 0.774, 0.778 ± 0.299 and 0.720 ± 0.274 fmol/10 cells at 24, 48 and 72 hours, respectively. The level is highest at 24 hours and then it decreased. 4. When the percentage of HLA-DR positive keratinocytes were relatively high, 42.15 ± 4.87% and 38.59 ± 3.74% at 48 an 72 hours, respectively, the levels of IL-1α were 1.009 ± 0.320 and 1.002 ± 0.301 fmol/10⁷ cells, respectively. They were significantly higher than r-IFN-γ untreated groups, of which IL-1α levels were 0.778 ± 0.299 and 0.720 ± 0.274 fmol/10⁷ cells, respectively (p = 0.059, p = 0.007). When the percentage of HLA-DR positive keratinocytes were decreased, the differences of IL-1α level between two groups were not significant. In summary, the results indicate that the amount of IL-1α produced by keratinocytes are relatively low and keratinocytes may participate in the immune process of the skin, and that the immunologic role of HLA-DR⁺ keratinocytes might be mediated via an increase production of IL-1α from HLA-DR⁺ keratinocytes.