Abstract:

Liang Yinghong, Wei Ming, Tu Ling, Liu Jia, Gong Yanjie, Zhang Yihua Clinical Laboratory, Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Corresponding author: Wei Ming, Email: gushiweiming@126.com 【Abstract】 Objective To evaluate changes of Rho kinase （ROK） activity in peripheral blood T lymphocytes from patients with atopic dermatitis （AD）, and to analyze their clinical significance. Methods Eight milliliters of heparin-anticoagulated blood samples were collected from 60 patients with AD and 60 healthy human controls followed by separation of T lymphocytes and sera from these blood samples as well as culture of isolated T lymphocytes with 10% fetal bovine serum for 24 hours. Both patient- and control-derived T lymphocytes were classified into two groups to be cultured with patient- or control-derived sera. In addition, some patient-derived T lymphocytes were classified into 4 groups: Y27632 group treated with the Rho kinase-specific inhibitor Y2763, CD3/CD28 group treated with anti-CD3/anti-CD28 monoclonal antibodies, Y27632 + CD3/CD28 group treated with Y27632 and anti-CD3/anti-CD28 monoclonal antibodies, and control group treated with patient-derived sera. Subsequently, Western-blot analysis was performed to evaluate ROK activity in cells, methyl thiazolyl tetrazolium （MTT） assay to evaluate proliferative activity of T lymphocytes, and ELISA to measure interleukin 6 （IL-6） and IL-10 levels in supernatants of T lymphocytes. Results ROK activity was significantly lower in fresh T lymphocytes from patients than in those from healthy controls （2.47% ± 0.89% vs. 0.65% ± 0.35%, t = 2.729, P < 0.05）. After 24-hour culture with 10% fetal bovine serum in vitro, ROK activity was significantly decreased in patient-derived T lymphocytes compared with those before culture （0.70% ± 0.38% vs. 2.47% ± 0.89%, t = 2.658, P < 0.05）, but no significant difference was observed between patient- and control-derived T lymphocytes （0.70% ± 0.38% vs. 0.63% ± 0.32%, t = 1.010, P > 0.05）. Compared with T lymphocytes cultured with control-derived sera, those cultured with patient-derived sera showed significantly increased ROK activity （F = 8.22, P < 0.001）. Concretely speaking, ROK activity was significantly higher in patient-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera （2.41% ± 0.87% vs. 0.76% ± 0.41%, P < 0.05）, and higher in control-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera （2.17% ± 0.85% vs. 0.64% ± 0.33%, P < 0.05） at 24 hours. Y27632 could significantly inhibit the proliferation of as well as secretion of IL-6 （F = 18.68, 22.95, respectively, both P < 0.001） by patient-derived T lymphocytes, but had insignificant effects on secretion of IL-10. The cellular proliferative activity and IL-6 supernatant level were significantly lower in the Y27632 group than in the control group, and lower in the Y27632 + CD3/CD28 group than in the CD3/CD28 group （all P < 0.05）. Conclusion Aberrant activation of ROK exists in T lymphocytes from patients with AD, which may play a certain role in the pathogenesis of AD.