Aoudjehane Lynda, Pissaia Alcindo, Scatton Olivier, Podevin Philippe, Massault Pierre-Philippe, Chouzenoux Sandrine, Soubrane Olivier, Calmus Yvon, Conti Filomena
Laboratoire de Biologie Cellulaire, UPRES 1833, Université Paris 5, Paris, France.
Lab Invest. 2008 Sep;88(9):973-85. doi: 10.1038/labinvest.2008.61. Epub 2008 Jul 14.
Interleukin-4 (IL-4) is overexpressed in liver grafts in a context of severe recurrent hepatitis C, during which the development of fibrosis is dramatically accelerated. In this study, we examined the effects of IL-4 on the activation and collagen production of cultured human intrahepatic (myo)fibroblasts (hIHFs), and investigated the underlying mechanisms. The myofibroblastic nature of cells was evaluated morphologically using activation markers (smooth muscle alpha-actin, vimentin and prolyl 4-hydroxylase). Quiescent hIHFs were obtained by cell incubation in serum-free medium or cell culture on Matrigel. We first analyzed IL-4 receptor expression, STAT-6 activation by IL-4, and STAT-6 inhibition by an anti-IL-4 antibody or by STAT-6 small-interfering RNA (siRNA) transfection. We then focused on collagen production, using quantitative real-time PCR to analyze the effect of IL-4 on the mRNA expression of collagens I, III and IV, and on collagen levels in supernatants of hIHFs, using the Sircol collagen assay. hIHFs cultured in plastic wells appeared to be morphologically activated. The expression of activation markers was reduced by serum deprivation or culture on Matrigel, and restored by IL-4 incubation. The IL-4 receptor was expressed by hIHFs, and STAT-6 was activated following incubation with IL-4. Both anti-IL-4 antibody and STAT-6 siRNA transfection inhibited this activation. The treatment of hIHFs with IL-4 increased the mRNA expression of collagens I, III and IV (P<0.05) and elevated collagen levels in supernatants (P=0.01 vs untreated cells). Therefore, IL-4 exerts profibrotic effects by activating hIHFs and inducing collagen production and secretion. This effect requires IL4-R binding and STAT-6 activation. IL-4 may thus be involved in accelerated course of fibrogenesis during recurrent hepatitis C.
在严重复发性丙型肝炎的情况下，白细胞介素-4（IL-4）在肝移植中过度表达，在此期间纤维化的发展会显著加速。在本研究中，我们检测了IL-4对培养的人肝内（肌）成纤维细胞（hIHFs）活化和胶原蛋白产生的影响，并研究了其潜在机制。使用活化标记物（平滑肌α-肌动蛋白、波形蛋白和脯氨酰4-羟化酶）从形态学上评估细胞的肌成纤维细胞性质。通过在无血清培养基中孵育细胞或在基质胶上进行细胞培养来获得静止的hIHFs。我们首先分析了IL-4受体的表达、IL-4对STAT-6的激活以及抗IL-4抗体或STAT-6小干扰RNA（siRNA）转染对STAT-6的抑制作用。然后，我们专注于胶原蛋白的产生，使用定量实时PCR分析IL-4对I、III和IV型胶原蛋白mRNA表达的影响，并使用Sircol胶原蛋白测定法分析hIHFs上清液中的胶原蛋白水平。在塑料孔中培养的hIHFs在形态上似乎被激活。血清剥夺或在基质胶上培养可降低活化标记物的表达，而IL-4孵育可使其恢复。hIHFs表达IL-4受体，与IL-4孵育后STAT-6被激活。抗IL-4抗体和STAT-6 siRNA转染均抑制了这种激活。用IL-4处理hIHFs可增加I、III和IV型胶原蛋白的mRNA表达（P<0.05），并提高上清液中的胶原蛋白水平（与未处理细胞相比，P=0.01）。因此，IL-4通过激活hIHFs并诱导胶原蛋白的产生和分泌发挥促纤维化作用。这种作用需要IL4-R结合和STAT-6激活。因此，IL-4可能参与了复发性丙型肝炎期间纤维化形成的加速过程。
