Hepatic ischemia-reperfusion results in an acute inflammatory response culminating in the recruitment of activated neutrophils that directly injure hepatocytes. Recent evidence suggests that CD4+ lymphocytes may regulate this neutrophil-dependent injury, but the mechanisms by which this occurs remain to be elucidated. In the present study, we sought to determine the type of CD4+ lymphocytes recruited to the liver after ischemia-reperfusion and the manner in which these cells regulated neutrophil recruitment and tissue injury. Wild-type and CD4 knockout (CD4-/-) mice were subjected to hepatic ischemia-reperfusion. CD4+ lymphocytes were recruited in the liver within 1 h of reperfusion and remained for at least 4 h. These cells were comprised of conventional (alphabetaTCR-expressing), unconventional (gammadeltaTCR-expressing), and natural killer T cells. CD4-/- mice were then used to determine the functional role of CD4+ lymphocytes in hepatic ischemia-reperfusion injury. Compared with wild-type mice, CD4-/- mice had significantly greater liver injury, yet far less neutrophil accumulation. Adoptive transfer of CD4+ lymphocytes to CD4-/- mice recapitulated the wild-type response. In wild-type mice, neutralization of interleukin (IL)-17, a cytokine released by activated CD4+ lymphocytes, significantly reduced neutrophil recruitment in association with suppression of MIP-2 expression. Finally, oxidative burst activity of liver-recruited neutrophils was higher in CD4-/- mice compared with those from wild-type mice. These data suggest that CD4+ lymphocytes are rapidly recruited to the liver after ischemia-reperfusion and facilitate subsequent neutrophil recruitment via an IL-17-dependent mechanism. However, these cells also appear to attenuate neutrophil activation. Thus the data suggest that CD4+ lymphocytes have dual, opposing roles in the hepatic inflammatory response to ischemia-reperfusion.

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The liver exhibits an exquisitely controlled cell cycle, wherein hepatocytes are maintained in quiescence until stimulated to proliferate. The retinoblastoma tumor suppressor, pRB, plays a central role in proliferative control by inhibiting inappropriate cell cycle entry. In many cases, liver cancer arises due to aberrant cycles of proliferation, and correspondingly, pRB is functionally inactivated in the majority of hepatocellular carcinomas. Therefore, to determine how pRB loss may provide conditions permissive for deregulated hepatocyte proliferation, we investigated the consequence of somatic pRB inactivation in murine liver. We show that liver-specific pRB loss results in E2F target gene deregulation and elevated cell cycle progression during postnatal growth. However, in adult livers, E2F targets are repressed and hepatocytes become quiescent independent of pRB, suggesting that other factors may compensate for pRB loss. Therefore, to probe the consequences of acute pRB inactivation in livers of adult mice, we gave adenoviral-Cre by i.v. injection. We show that acute pRB loss is sufficient to elicit E2F target gene expression and cell cycle entry in adult liver, demonstrating a critical role for pRB in maintaining hepatocyte quiescence. Finally, we show that liver-specific pRB loss results in the development of nuclear pleomorphism associated with elevated ploidy that is evident in adult mice harboring both acute and chronic pRB loss. Together, these results show the crucial role played by pRB in maintaining hepatocyte quiescence and ploidy in adult liver in vivo and underscore the critical importance of delineating the consequences of acute pRB loss in adult animals. (Cancer Res 2005; 65(11): 4568-77)

The current study explored the concept that adult and pediatric populations differ in their response to major injury. Male C57BL/6 mice of a "young adult" (8-12 weeks) or "mature adult" (12-13 months) age were subjected to partial hepatic ischemia and reperfusion. Mature adult mice displayed significantly more liver injury than young adult mice as assessed histologically and by serum levels of alanine aminotransferase. Interestingly, there was far less neutrophil accumulation in the livers of mature adult mice. However, liver-recruited neutrophils from mature adult mice had a higher activation state than those from young adult mice. Activation of the inflammatory transcription factor, NF-kappaB, was suppressed in whole livers from mature adult mice. In isolated liver cells, Kupffer cells showed no difference in NF-kappaB activation, but hepatocytes from mature adult mice had delayed NF-kappaB activation in response to TNF. Furthermore, isolated hepatocytes from young adult mice produced abundant amounts of the chemokine, macrophage inflammatory protein-2, whereas hepatocytes from mature adult mice produced little, if any macrophage inflammatory protein-2. Mature adult mice had much lower hepatic expression of the cytoprotective protein, heat shock protein 70, than did young adult mice. In contrast, serum heat shock protein 70 levels, which has been linked to subsequent tissue injury, were higher in mature adult mice than in young adult mice. These data suggest that there are multiple alterations at the cellular and molecular levels that contribute to enhanced postischemic liver injury in mature adult mice.

The present study examined the role of hepatocyte NF-kappaB activation during ischemia-reperfusion injury. Second, we evaluated the effects of ischemic hypothermia on NF-kappaB activation and liver injury. C57BL/6 mice underwent 90 min of partial hepatic ischemia and up to 8 h of reperfusion. Body temperature was regulated during the ischemic period between 35 and 37 degrees C, 33 and 35 degrees C, 29 and 33 degrees C or unregulated, where temperature fell to <29 degrees C. Liver injury, as measured by serum alanine aminotransferase as well as liver histopathology, was inversely proportional to regulated body temperature, with the unregulated group (<29 degrees C) being highly protected and the normothermic group (35-37 degrees C) displaying the greatest injury. Inflammation, as measured by production of TNF-alpha and liver recruitment of neutrophils, was greatest in the normothermic groups and lowest in the ischemic hypothermia groups. Interestingly, hepatocyte NF-kappaB activation was highest in the hypothermic group and least in the normothermic group. Paradoxically, degradation of IkappaB proteins, IkappaB-alpha and IkappaB-beta, was greatest in the normothermic group, suggesting an alternate NF-kappaB regulatory mechanism during ischemia-reperfusion injury. Subsequently, we found that NF-kappaB p65 protein was increasingly degraded in normothermic versus hypothermic groups, and this degradation was specific for hepatocytes and was associated with decreased expression of the peptidyl-prolyl isomerase Pin1. The data suggest that NF-kappaB activation in hepatocytes is a protective response during ischemia-reperfusion and can be augmented by ischemic hypothermia. Furthermore, it appears that Pin1 promotes NF-kappaB p65 protein stability such that decreased expression of Pin1 during ischemia-reperfusion results in p65 degradation, reduced nuclear translocation of NF-kappaB, and enhanced hepatocellular injury.