CXCR3 Ligands induce Expression of CXCL1 (KC/murine IL8 homolog) in Mouse Hepatic Stellate Cells

Abstract

Background and aim: Liver injury leads to infiltration of immune cells and a subsequent activation of hepatic stellate cells. Chemokines are ubiquitous chemotactic proteins which are involved in inflammatory pathways. It has been recently suggested that chemokines can also induce the expression of other chemokines and thereby indirectly regulate immune cell recruitment. We therefore investigated the ability of the chemokine CXCL9 to induce CXCL1, an important neutrophil chemoattractant. Methods:The ability of CXCR3 ligands CXCL9 and CXCL10 to induce CXCL1 expresssion was analyzed in immortalized (GRX) and primary hepatic stellate cells isolated from wild-type and CXCR3-/-mice. Cells were treated with different concentrations of chemokines in the absence and presence of pertussis toxin. Furthermore, mice were treated systemically with CXCL9 and hepatic CXCL1 levels as well as neutrophil infiltration were assessed. Results:Treatment of GRX cells with CXCL9 leads to a dose dependent induction of CXCL1 protein expression. This stimulatory effect was validated in primary hepatic stellate cells (P<0.01). In contrast, stimulation of stellate cells with CCL2 did not result in CXCL1 induction. Increased CXCL1 expression in response to CXCL9 was completely abolished by co-incubation with pertussis toxin and in stellate cells derived from CXCR3-/-mice, the canonical receptor for CXCL9 and CXCL10. Notably, systemic treatment of mice with CXCL9 led to elevated hepatic CXCL1 levels and was associated with enhanced neutrophil infiltration into the liver. Conclusions:The study describes a chemokine-chemokine pathway in hepatic stellate cells which leads to augmented infiltration of neutrophils into the liver during acute liver injury. laboratories (Sulzfeld, Germany). CXCR3 -/-mice were bred for more than 10 generations on the C57Bl/6 background as previously described [17]. Liver injury in wild-type mice was induced by administration of 0.8 ml/kg body weight carbon tetrachloride (CCl 4 ) i.p. Mice were treated concomitantly with 1µg recombinant murine CXCL9 (R&D systems) or vehicle i.p. daily. CXCR3 Ligands induce Isolation and culture of mice hepatic stellate cellsFor screening experiments the murine GRX hepatic stellate cell line was used for in vitro experiments [18,19]. Confirmation experiments were performed in primary murine hepatic stellate cells. These cells were isolated from wild-type C57Bl/6 or CXCR3 -/-mice by a collagenase-pronase two step perfusion method as described [3]. Cell purity was assessed by bright field microscopy and was estimated at 80 -90 %. Experiments were performed after 3 -4 day culturing of primary cells; medium was changed daily to clear cell debris and dead cells. Cells were cultured in DMEM with high-glucose; L-glutamine and sodium pyruvate (PAA Laboratories) supplemented with 10% fetal calf serum (Biochrom KG), 100 IU/ml penicillin, and 100 μg/ml streptomycin (PAA Laboratories). The medium was changed 1 day after initial plating, while cultures were maintained at 37°C, 5% CO 2 . Stim...

Cytokines, Chemokines, and ?Marshalling for War?

“…Two other reports discuss the impact of cytokines on disease-specific inflammation. Scholten et al [6] show that acute liver injury is combated by the recruitment of immune cells and neutrophils by the induction of chemokines in the leukocyte chemoattractant receptor network (CXC family) and ligands such as CXCL9 and CXCL1; their infiltration into liver tissue is associated with the fibrosis seen pathologically in many chronic liver diseases including viral hepatidites, steatohepatidites, or autoimmune disorders. In their study, mice treated with rCXCL9 had increased CXCL1 concentration in hepatic cells, and increased infiltration of neutrophils.…”