Higher neutrophil infiltration mediated by osteopontin is a likely contributing factor to the increased susceptibility of females to alcoholic liver disease

Abstract

Alcoholic liver disease (ALD) is a major public health problem in the United States and women are known to be more susceptible to ALD. However, the precise mechanism for increased susceptibility of females to ALD is not completely understood. The present study is based on the hypothesis that induction of osteopontin (OPN), a matricellular protein, is the likely contributing factor for higher neutrophil recruitment in females during alcoholic steatohepatitis (ASH). ASH was induced in male and female Sprague-Dawley rats by feeding them a Lieber-DeCarli diet containing ethanol (EtOH) for 6 weeks, followed by a single injection of lipopolysaccharide (LPS, 10 mg/kg, ip). Liver injury, measured by plasma transaminase elevations and confirmed by haematoxylin and eosin-stained liver sections, revealed approximately 25-fold higher liver injury in the female ASH model compared with the males. Although steatosis, necrosis, and neutrophil infiltration were evident in both male and female rats, hepatic neutrophilic necrotic foci were noted as early as 2 h after LPS injection in the EtOH-treated female rats. Hepatic neutrophil infiltration correlated with higher expression of cleaved (cOPN) and uncleaved OPN in the EtOH + LPS-treated female rats compared with the males. OPN secretion was localized predominantly in the biliary epithelium and females had significantly higher OPN mRNA than their male counterparts in the ASH model. The ability of OPN to attract neutrophils was further confirmed in vivo, in a peritonitis rat model, and by neutralizing OPN (nOPN) antibody experiments. Hepatic neutrophil infiltration was largely inhibited ( approximately 50%) by nOPN antibody. Flow cytometry experiments revealed OPN-mediated up-regulation of the CD11b neutrophil adhesion molecule. In conclusion, these data suggest that higher hepatic expression of OPN is the likely reason for higher and early hepatic neutrophil infiltration making females more susceptible to ALD during ASH.

The Temporal Expression of Osteopontin (SPP-1) in the Rodent Model of Alcoholic Steatohepatitis: A Potential Biomarker

“…It has been shown to be a chemoattractant for macrophages and neutrophils both in vitro and in vivo (Bayless et al, 1998, Apte et al, 2005. Recent studies from our laboratory have shown that OPN acts as a chemokine in attracting neutrophils to the liver, making it more susceptible to injury in alcoholic steatohepatitis (ASH) (Apte et al, 2005;Banerjee et al, 2006). We have also reported that neutralizing antibodies directed against OPN significantly decrease neutrophil infiltration into the hepatic parenchyma in our rodent model of ASH (unpublished data).…”

“…A recent study from our laboratory has clearly shown that administration of neutralizing antibody against OPN abolishes hepatic neutrophilic infiltration and prevents ethanol-induced liver injury (Banerjee et al, 2006). Measurement of OPN protein in the liver to assess neutrophil infiltration has potential limitations.…”

“…Histochemical detection of neutrophils was performed on paraffin-embedded liver sections. The H&E staining was employed to identify the neutrophils, based on the segmented morphology of the nucleus followed by quantification with Naphthol AS-D Chloroacetate Esterase Staining (Sigma Diagnostics, St. Louis, MO, USA) as described previously (Banerjee et al, 2006). Briefly, 4 µm thick formalin-fixed paraffin-embedded liver sections were stained in a mixture of Sodium nitrite, Fast Red Violet LB Base solution, prewarmed deionized water, Trizma buffer and Naphthol AS-D Chloroacetate solution.…”

Osteopontin: Role in immune regulation and stress responses

“…Neutrophils (the majority of PMNs) are the primary responder to exogenous and endogenous stimuli and rapidly infiltrate the injured tissue to initiate an innate host defense. OPN is a chemoattractant for neutrophils (Weiss et al, 2001;Banerjee et al, 2006;Koh et al, 2007). Activated neutrophils release proteases and cytotoxic mediators in combination with phagocytosis and respiratory bursts to eliminate invading infectious agents.…”

“…However, cytotoxic products released by neutrophils may also cause host tissue destruction such as observed in inflammatory bowel disease . OPN has been found to play an important role in neutrophil recruitment in alcoholic liver disease (Apte et al, 2005;Banerjee et al, 2006). The expression and localization of CD44 in neutrophils is independent of OPN, although CD44 is required for the polarization and chemotaxis of neutrophils (Alstergren et al, 2004).…”