Expression of regulated oncogen-alpha by primary hepatocytes following isolation and heat shock stimulation

High levels of regulated oncogen-alpha [GRO-a] expression have been observed in the liver. GRO-a stimulates proliferation of epithelial cells and induction of rolling and extravascular migration of neutrophils and mononuclear cells. Given the above observations, this chemokine was chosen to be analyzed in freshly isolated and cultured hepatocytes. In this study, hepatocytes [2_106 cell/ml] were isolated from male Sprague Dawley rat liver and cultured on plates that were pre-coated with collagen type-I matrix. The western and northern blot analyses were employed to detect GRO-a at the protein and mRNA levels in freshly isolated and cultured hepatocytes in response to isolation and heat shock stresses. GRO-a was shown to be expressed by isolated rat hepatocytes immediately after isolation and early culture and decreased with time. mRNA was also expressed in freshly isolated cells [0 h] and did not decrease after 48h of culture and further time points [P<0.01]. These results also demonstrated that expression of GRO-a by hepatocytes increased in response to heat shock at different time points in comparison with the control [P<0.01]. These results demonstrated that the isolation and heat shock stresses induced the expression of GRO-a in hepatocytes in a time-dependent manner. Thus, it seems that hepatocytes mimic the experiences that the liver encounters after injury in vivo. In such a situation, liver produces stress related agents like chemokines to overcome injurious conditions

Expression of stromal derived factor alpha [SDF-1a] by primary hepatocytes following isolation and heat shock stimulation

Stromal derived factor-alpha [SDF-1-alpha] is a CXC chemokine which has been demonstrated as a recruitment factor for leukocytes to the site of inflammation, infection, injury and following stress. This chemokine has been shown to be expressed by liver cells and in liver diseases. Hence, the aim of this study was to examine the expression of SDF-1 by hepatocytes in responses to the stress imposed during isolation by collagenase perfusion and under heat shock stimulation. In this study hepatocytes [2-5 x 10[6]] were isolated from male Sprague Dawley rat liver and cultured in plates that were pre-coated with collagen Type-I matrix. The western and northern blotting analysis were employed to detect SDF-1 at protein and mRNA levels in isolated and cultured hepatocytes in response to isolation and heat shock stresses. The SDF-1 is expressed by isolated rat hepatocytes immediately after isolation and early culture and decreased with time. SDF-1 protein was highly expressed in freshly isolated cells and decreased by time [27h] [P < 0.05]. mRNA was also expressed in freshly isolated cells [0h] but decreased after 24h of culture [P < 0.01]. This results also demonstrated that expression of SDF-1 by hepatocytes was increased in response to heat shock at different time points comparing with control [P < 0.01]. These results demonstrated that the isolation and heat shock stresses induced the expression of SDF-1 in hepatocytes in a time-dependent manner. Accordingly, it seems that hepatocytes mimic the experiences that liver experience after injury in vivo and therefore, produce stress related agents like chemokines to overcome such a injurious condition

Expression of IP-10 chemokine is regulated by pro-inflammatory cytokines in cultured hepatocytes

Chemokines are classified in four distinct groups as CXC, CC, CX3C and C, depending on the presence or absence of a motif called ELR [Arg-Leu-Glu] before the first cysteine residue in their structure. CXC chemokines are also subdivided into ELR+and ELR-. Increasing evidence has indicated the existence of a chemokine network in the liver which is involved in both physiological responses and, under certain circumstances, pathological and repair processes following hepatic injury. The CXC chemokines play a major role in both these processes, and much attention has been focused on their therapeutic applications to liver disease. The aim of this study was to examine the response of cultured hepatocytes to exogenous inflammatory cytokines [TNF-alpha and IFN-gamma] regarding expression of IP-10 and growth regulatory oncogen [Gro] chemokines. In this study we employed western and northern analysis to measure chemokines at the level of protein and mRNA by hepatocytes in response to pro-inflammatory cytokines. We found that, the pro-inflammatory cytokines, TNF-alpha and IFN-gamma, selectively stimulated expression of IP-10 but were without effect on Gro. This confirms a potential direct involvement of these cytokines in chemokine production by hepatocytes. Thus, IFN-gamma and TNF-alpha may play a role in hepatic injury and inflammation and produce some of their biological effects by localized induction of chemokines by hepatocytes. Given the similarity to an acute phase response, we were able to show that IFN-gamma and TNF-alpha mimicked the effects of cell isolation and culture on induction of IP-10 expression. Further, evidence for linkages between IFN-gamma and TNF-alpha and liver injuries is seen in hepatitis C and hepatitis B in which increased levels of TNF-alpha and its soluble receptor were reported

Expression of cxc chemokines Gro/Kc and SDF-1 alpha in rat H4 h epatomacells inresponse to different stimuli

It is now well established that several environmental stress factors cause activation of p38 MAP kinase and JNK in various cell types to produce chemokines. To investigate the expression of CXC chemokines Gro/KC and SDF- 1alpha in rat's H4 hepatoma cells in response to heat shock, hyperosmolarity and oxidative stress. Hepatoma cells were maintained in MEM medium. Cells were subjected to different stresses [[H[2]O[2] 0.15% [w/v], manitol and NaCl [160 mM] and heat shock [42 °C for 20 minutes]]. Cells were harvested and RNA was extracted, purified and the CXC chemokine Gro/KC and SDF-1alpha expression was analysed by RT-PCR. cDNA was separated by gel electrophoresis on a 1% [w/v] agarose gel and visualized under a UV transilluminator. There was detectable but low expression of both SDF-1 alpha and Gro/KC in H4 hepatoma cells. Heat shock failed to induce expression of SDF-1alpha and Gro/KC in H4 hepatoma cells of rat. Hyperosmolarity also did not stimulate SDF-1alpha and Gro/KC expression. In this study we have also shown that oxidative stress did not induce expression of SDF-1alpha and Gro/KC. Overall, although detection is possible but regulatory responses were not observed in H4 hepatoma cells. Several known injurious conditions cause recruitment of macrophages, neutrophils and other immune cells to the liver. Immune cells are recruited to the hepatic vasculature following local liver injury and subsequent chemokine production. Our results demonstrated that failure to produce chemokines by hepatoma cells may be a way to escape from mechanism of immune surveillance