Activation of hepatic stellate cells after phagocytosis of... : Hepatology (original) (raw)

Liver Biology and Pathobiology

Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis

Muhanna, Nidal1,2,3,4,5; Doron, Sarit1; Wald, Ori1; Horani, Amjad1; Eid, Ahmed2; Pappo, Orit3; Friedman, Scott L.4; Safadi, Rifaat5*

1_Liver and Gastroenterology Units; Division of Medicine, Hadassah Hospital, Jerusalem, Israel_

2_Liver and Gastroenterology Units; Division of Surgery, Hadassah Hospital, Jerusalem, Israel_

3_Liver and Gastroenterology Units; Division of Pathology Department, Hadassah Hospital, Jerusalem, Israel_

4_Division of Liver Diseases, New York, NY_

5_Mount Sinai School of Medicine, New York, NY_

* Address reprint requests to: Liver and Gastroenterology Units, Division of Medicine Hadassah University Hospital and Hebrew University-Hadassah Medical School, POB 12000, IL-91120 Jerusalem, Israel

Email:[email protected]

Received 25 November 2007; Accepted 1 May 2008

Published online in Wiley InterScience (www.interscience.wiley.com).

Supported by NIH; Grant Number: DK 56621; Grant sponsor: US-Israel Binational Scientific Foundation Awards; Grant Numbers: 8033603 8033605.

Potential conflict of interest: Nothing to report.

fax: 972-2-6420338

Abstract

Increased CD8-T lymphocytes and reduced natural killer (NK) cells contribute to hepatic fibrosis. We have characterized pathways regulating the interactions of human hepatic stellate cells (HSCs) with specific lymphocyte subsets in vivo and in vitro . Fluorescence-activated cell sorting (FACS) was used to characterize human peripheral blood lymphocytes (PBLs) and intrahepatic lymphocytes (IHLs) obtained from healthy controls and from patients with either hepatitis B virus (HBV) or hepatitis C virus (HCV) with advanced fibrosis. Liver sections were analyzed by immunohistochemistry and confocal microscopy. To investigate in vitro interactions, PBLs from healthy controls or patients with HCV cirrhosis were co-cultured with an immortalized human HSC line (LX2 cells) or with primary HSCs. Significant alterations in lymphocyte distribution were identified in IHLs but not PBLs. The hepatic CD4/CD8 ratio and NK cells were significantly reduced in HBV/HCV patients. Expression of alpha-smooth muscle actin and infiltration of CD4, CD8, and NK cells were readily apparent in liver sections from patients with cirrhosis but not in healthy controls. Lymphocytes from each subset were in proximity to HSCs primarily within the periportal regions, and some were directly attached or engulfed. In culture, HSC activation was stimulated by HCV-derived CD8-subsets but attenuated by NK cells. Confocal microscopy identified lymphocyte phagocytosis within HSCs that was completely prevented by blocking intracellular adhesion molecule 1 (ICAM-1) and integrin molecules, or by irradiation of HSCs. LX2 knockdown of either Cdc42 or Rac1 [members of the Rho-guanosine triphosphatase (GTPase) family] prevented both phagocytosis and the activation of HSC by HCV-derived lymphocytes. Conclusion: The CD4/CD8 ratio and NK cells are significantly decreased in livers with advanced human fibrosis. Moreover, disease-associated but not healthy lymphocytes are engulfed by cultured HSCs, which is mediated by the Rac1 and Cdc42 pathways. Ingestion of lymphocytes by HSCs in hepatic fibrosis is a novel and potentially important pathway regulating the impact of lymphocytes on the course of hepatic fibrosis. (Hepatology 2008.)

Abbreviations: α-SMA, alpha-smooth muscle actin; DiOC, dioctadecyloxacarbocyanine perchlorate; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GTPase; guanosine triphosphatase; HBV, hepatitis B virus; HCV, hepatitis C virus; HSC, hepatic stellate cell; ICAM-1, intracellular adhesion molecule 1; IFN-γ, interferon gamma; IHL, intrahepatic lymphocyte; MHC, major histocompatibility complex; mRNA, messenger RNA; NK, natural killer; PBL, peripheral blood lymphocyte; PBS, phosphate-buffered saline; PE, phycoerythrin; PI, propidium iodide; siRNA, small interfering RNA.