OBJECTIVETo study the influence of heat stimulation on expression of coxsackie-adenovirus receptor (CAR) in keratinocytes (KCs) of mouse skin and the effect of CAR on production of cell growth factors by dendritic epidermal T lymphocytes (DETCs).

METHODS(1) Twenty BALB/c mice were divided into heat stimulation group (HS) and control group (C) according to the random number table, with 10 mice in each group. Mice in group HS were inflicted with scald milder than superficial-thickness by dressing wet hot gauze, which had been soaked in 100°C hot water for 3 min, in the hair removed area on the back for 1 to 3 s, while mice in group C were sham injured by dressing a wet gauze which had been soaked in water of room temperature for 3 min in the hair removed area on the back for 1 to 3 s. Square full-thickness skin specimens measuring 2.0 cm × 2.0 cm in size were obtained from the center of the bare skin. The expression of CAR in skin tissue sections were detected by immunohistochemistry staining. The mRNA and protein expression levels of CAR in skin tissue sections were respectively determined by real-time fluorescent quantitation RT-PCR and Western blotting. (2) KCs were isolated and cultured from full-thickness skin obtained from the trunk of 2 fetal BALB/c mice, and they were divided into 2 groups according to the random number table, with 5 wells in each group. The cells in group HS and group C were respectively cultured in 42°C and 37°C, 5% CO2 incubator for 1 h, and then all the cells were cultured in 37 °, 5% CO2 incubator for 6 h. The apoptosis of the cells and their expression of CAR were detected by flow cytometer. (3) Five BALB/c mice were sacrificed, and full-thickness skin was obtained from the trunk. The DETCs were divided into 7 groups according to the random number table after being isolated and purified from the skin specimens. Cells in group C were cultured without any stimulation, and cells in the 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 mg/L CAR groups were respectively cultured with corresponding concentration of recombinant mice CAR nutrient solutions, with 5 wells in each group. The contents of insulin-like growth factor I (IGF-I) and keratinocyte growth factor (KGF) were determined with ELISA. Data were processed with independent samples t test and one-way analysis of variance.

RESULTS(1) The immunohistochemistry staining showed that there was mild positive staining in the skin tissue sections of mice in group C, while the positive staining was more obvious in group HS. The positive staining was mainly located in KCs, hair follicles, and sweat gland epithelial cells, while no positive staining was observed in fibroblasts. The mRNA expression levels of CAR in skin tissue sections in group C and group HS were respectively 0.157 ± 0.027 and 0.773 ± 0.029. There was statistically significant difference between them (t = 3.052, P < 0.01). The protein expression levels of CAR in skin tissue sections in group C and group HS were respectively 0.23 ± 0.09 and 0.89 ± 0.14. There was statistically significant difference between them (t = 2.556, P < 0.05). (2) The apoptosis rates of KCs in group C and group HS were respectively (5.7 ± 1.3)% and (7.4 ± 1.7)% (t = 0.464, P > 0.05). The expression rates of CAR in KCs in group C and group HS were respectively (48 ± 6)% and (80 ± 8)%. There was statistically significant difference between them (t = 2.585, P < 0.05). (3) The contents of IGF-Iin culture supernatants in group C and 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L CAR groups were respectively (23.1 ± 1.8), (22.5 ± 2.1), (31.2 ± 2.5), (39.7 ± 2.3), (61.8 ± 3.5), (45.1 ± 2.8), and (29.0 ± 2.0) µg/L. There was statistically significant difference among 7 groups (F = 3.414, P < 0.05). The contents of KGF in culture supernatants in group C and 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L CAR groups were respectively (131 ± 9), (217 ± 12), (355 ± 21), (563 ± 21), (535 ± 34), (292 ± 20), and (245 ± 10) ng/L. There was statistically significant difference among 7 groups (F = 5.063, P < 0.01).

CONCLUSIONSThe expression of CAR in KCs would rise after HS. The optimum CAR concentration to increase IGF-I and KGF production in DETCs is low.