Interleukin-4 induces the activation and collagen production of cultured human intrahepatic fibroblasts via the STAT-6 pathway

Abstract

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 a-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 (Po0.05) and elevated collagen levels in supernatants (P ¼ 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.

LL-37 secreted by epithelium promotes fibroblast collagen production: a potential mechanism of small airway remodeling in chronic obstructive pulmonary disease

“…28 One milliliter of Sircol dye was added to 100 ml supernatant, followed by incubation under gentle rotation for 30 min at room temperature. After centrifugation for 10 min at 12000 rpm, the collagen-bound dye was redissolved with 250 ml Sircol alkali reagent, and the absorbance was measured at 540 nm.…”

Increased circulating IL-9-producing CD8+ T cells are associated with eosinophilia and high FeNO in allergic asthmatics

“…qPCR was performed on a Bio-Rad iQ5 real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) under the following conditions: Initial denaturation at 95˚C for 30 sec, followed by 35 cycles at 95˚C for 5 sec, 58˚C for 15 sec and 72˚C for 15 sec. Primer sequences were used as described previously (19)(20)(21) and are shown in Table II.…”

Development of an in vitro model to test antifibrotic drugs on primary human liver myofibroblasts

“…Cell isolation was accomplished in compliance with French ethical guidelines, using an established method. 12 The liver fragments (10-30 g) were initially perfused for 20 min with a prewarmed (37°C) calcium-free buffer supplemented with 5 mM ethylene glycol tetraacetic acid (EGTA) (Sigma Aldrich; Saint-Quentin Fallavier, France), followed by perfusion with a prewarmed (37°C) buffer containing 6 mM calcium (CaCl2) (Sigma Aldrich) and 0.05% collagenase (5 mg/ml) (Sigma Aldrich), for 15 min. The liver fragments were then gently shaken in wash medium to free the liver cells, and the resulting suspension was filtered through a gauze-lined funnel before the cells were centrifuged at low speed.…”

“…13 The HLMFs were then obtained by gradient centrifugation, as described elsewhere. 12,13 After 1 week of primary culture through several passages, all cells had a myofibroblast-like appearance and stained positive for vimentin and α-smooth muscle actin (Dako, Les Ulis, France). During all subsequent experiments, the HLMFs were used at passage 4.…”

“…In vitro cell models represent an alternative and complementary approach. We previously developed a culture model of human liver myofibroblasts (HLMFs), 12 which offers direct access to ECM-producing cells in the liver as the principal targets of antifibrotic drugs, and could enable the rapid testing of a large number of candidate drugs. Based on this HLMF model, we have now developed an in vitro test that could be used to predict the antifibrotic properties of new compounds for the use in human liver disease.…”