Toll样受体4对HLA-B27相关前葡萄膜炎患者外周血单个核细胞分泌细胞因子的影响

Effects of Toll like receptor 4 on cytokines excreted by PBMC in HLA-B27 associated anterior uveitis

目的通过对比正常人与HLA-B27相关前葡萄膜炎恢复期患者外周血单个核细胞(peripheral blood mononuclear cells,PBMC)受脂多糖(lipopolysaccharide,LPS)刺激及抗Toll样受体4(Toll like receptor4,TLR4)抗体(HTA125)干预后分泌细胞因子的变化,探讨TLR4在HLA-B27相关前葡萄膜炎发病机制中的作用。方法选取HLA-B27阳性前葡萄膜炎恢复期患者10例,相同性别及年龄段的HLA-B27阴性正常人10例作为对照。抽取研究对象外周血分为以下5组体外培养PBMC:(1)正常人非LPS刺激组:加入终浓度为1mg·L-1的PBS进行培养;(2)正常人LPS刺激组:加入终浓度为1mg·L-1的LPS进行培养;(3)HLA-B27阳性患者非LPS刺激组:加入终浓度为1mg·L-1的PBS进行培养;(4)HLA-B27阳性患者LPS刺激组:加入终浓度为1mg·L-1的LPS进行培养;(5)HLA-B27阳性患者LPS+HTA125干预组:终浓度为5mg·L-1的HTA125预先干预PBMC 30 min后再加入1mg·L-1的LPS共同培养。分别于培养4h、8h、12h、24h后,利用ELISA法测定细胞培养上清液中的TNF-a、IL-10和(4)(5)组中IL-6的浓度。结果 LPS未刺激组,PBMC培养4h、8h、12h、24h后,HLA-B27阳性患者TNF-α浓度分别为(1329.46±155.09)ng·L-1、(1926.76±163.28)ng·L-1、(1424.61±141.63)ng·L-1、(526.98±112.29)ng·L-1,正常人分别为(562.83±106.45)ng·L-1、(839.57±73.98)ng·L-1、(559.60±88.48)ng·L-1、(200.81±51.39)ng·L-1,两组各时间点相比差异均有统计学意义(均为P<0.05);HLA-B27阳性患者PBMC培养8h、12h、24h后,IL-10浓度分别为(145.51±26.91)ng·L-1、(259.16±32.71)ng·L-1、(435.98±134.54)ng·L-1,正常人分别为(63.57±17.28)ng·L-1、(123.21±15.73)ng·L-1、(247.82±32.10)ng·L-1,两组各时间点相比差异均有统计学意义(均为P<0.05)。LPS刺激后,正常人与HLA-B27阳性患者PBMC分泌TNF-α和IL-10水平在各时间点均比LPS刺激前明显升高,且HLA-B27阳性患者升高更明显。HLA-B27阳性患者PBMC在LPS刺激后与LPS+HTA125干预组相比,后者各时间点分泌TNF-α、IL-10的水平均显著降低,差异均有统计学意义(均为P<0.05),且HTA125干预组IL-6浓度在4h、8h时较单纯LPS刺激组低,差异均有统计学意义(均为P<0.05)。结论 HLA-B27阳性患者PBMC对LPS的刺激更加敏感,抗TLR4单克隆抗体能够抑制HLA-B27阳性患者PBMC分泌细胞因子的水平。

Objective To investigate the role of Toll like receptor 4（TLR4） in the pathogenesis of HLA-B27 associated anterior uveitis by comparing cytokines changes of peripheral blood mononuclear cells （PBMC） in normal and HLA-B27 positive patients stimulated with lipopolysaccharide （LPS） and blockage of anti-TLR4 Monoclonal antibodies （HTA125）. Methods Ten HLA-B27 associated anterior uveitis patients and 10 normal persons with similar ages and gender were selected.PBMC were isolated and divided into normal PBMC without LPS stimulation group （PBS with final concentration of 1 mg·L^-1 was added）,normal PBMC with LPS stimulation group （LPS with final concentration of 1 mg·L^-1 was added）,HLA-B27 positive PBMC without LPS stimulation group （PBS with final concentration of 1 mg·L^-1 was added）,HLA-B27 positive PBMC with LPS stimulation group （LPS with final concentration of 1 mg·L^-1 was added）,and HLA-B27 positive PBMC with LPS＋HTA125 stimulation group （HTA125 with final concentration of 5 mg·L^-1 was added at 30 minutes before adding LPS with final concentration of 1 mg·L^-1）.The tumor necrosis factor-α （TNF-α）,interleukin-10（IL-6） and interleukin-6（IL-6） in the supernatants were detected by ELISA at 4 hours,8 hours,12 hours,24 hours,respectively. Results In two groups without LPS stimulation,the concentration of TNF-α at each time point in HLA-B27 positive group were （1329.46±155.09）ng·L^-1,（1926.76±163.28）ng·L^-1,（1424.61± 141.63）ng·L^-1 and （526.98±112.29）ng·L^-1,respectively,which in normal PBMC group were （562.83±106.45）ng·L^-1,（839.57±73.98）ng·L^-1,（559.6±88.48）ng·L^-1 and （200.81±51.39）ng·L^-1,there were statistical differences between two groups （both P〈0.05）;The concentration of IL^-10 at 8 hours,12 hours,24 hours in HLA-B27 positive group were （145.51±26.91）ng·L^-1,（259.16±32.71）ng·L^-1,（435.98±134.54）ng·L^-1,which in normal PBMC group were （63.57±17.28）ng·L^-1,（123.21±15.73）ng·L^-1,（247.82±32.1）ng·L^-1,there were statistical differences between two groups （both P〈0.05）.The concentrations of TNF-α and IL^-10 were higher significantly both in normal and HLA-B27 positive groups after LPS stimulation than without LPS stimulation at all time points,especially the HLA-B27 positive group.The changes of TNF-α and IL^-10 after LPS stimulation were higher in B27 positive group than in normal group,there were statistical differences（both P〈0.05）.The concentration of TNF-α,IL^-10 and IL-6 were significantly decreased when blocked with HTA125 in B27 positive groups with LPS stimulation,there were statistical differences（both P〈0.05）. Conclusion HLA-B27 positive PBMC are more sensitive to LPS stimulation,and anti-TLR4 monoclonal antibodies can inhibit the secretion of cytokines in PBMC in HLA-B27 positive patients.