Macrophages have been implicated as key cellular effectors in the pathogenesis of AA-induced hepatotoxicity. These cells, which consist of resident and infiltrating macrophages, are rapidly activated following AA intoxication to release cytotoxic and proinflammatory mediators which contribute to liver damage (Gardner and Laskin, 2007
; Laskin, 2009
; Laskin et al., 2011
). Previous studies have suggested that macrophage activation is mediated in part, by factors released from AA-injured hepatocytes (Laskin et al., 1986
). Characterization of these factors and their biological effects may provide a better mechanistic understanding of pathways leading to hepatotoxicity.
The present studies demonstrate that AA-induced cytotoxicity in primary mouse hepatocytes was associated with the release of macrophage activating factors. Thus, in response to CM from AA-injured hepatocytes, macrophage production of ROS and expression of COX-2, 12/15-LOX, MIP-1α, MIP-2 and RAGE were increased, indicating macrophage activation to a proinflammatory phenotype. Surprisingly, CM-AA had no effect on expression of TNFα or IL-1, two proinflammatory mediators generated by classically activated cytotoxic/proinflammatory macrophages (Mosser and Edwards, 2008
). These findings suggest that there are multiple pathways regulating macrophage activation in the liver during the early stages of AA-induced hepatotoxicity.
Evidence suggests that ROS including superoxide anion, hydrogen peroxide and hydroxyl radicals derived from inflammatory phagocytes contribute to hepatic injury induced by diverse xenobiotics (reviewed in Laskin et al., 2011
). ROS can induce membrane, protein and DNA damage leading to cytotoxicity and this may be important in the pathogenesis of AA-induced hepatotoxicity (Jaeschke et al., 2003
). ROS generation is a characteristic feature of classically activated macrophages (Mosser and Edwards, 2008
). We found that CM from AA-injured mouse hepatocytes stimulated macrophage ROS production. These results are in accord with previous reports that AA-treated rat hepatocytes release factors that induce a respiratory burst in Kupffer cells (Laskin et al., 1986
). These data support the idea that hepatocytes contribute to the proinflammatory environment in the liver following AA intoxication. CM from AA-injured hepatocytes was also found to upregulate catalase and HO-1 expression in macrophages. These antioxidants play an important role in protecting against AA-induced hepatotoxicity (Chen et al., 2002
; Ferret et al., 2001
; Chiu et al., 2002
). Increased expression of antioxidants by macrophages may be a compensatory response to oxidative stress induced by hepatocyte-derived proinflammatory mediators.
COX-2 is the major enzyme mediating macrophage biosynthesis of proinflammatory prostaglandins, including PGE2. It is induced in macrophages by inflammatory stimuli and activation is associated with tissue injury (Stables and Gilroy, 2011
). The present studies show that CM from AA-injured hepatocytes upregulated COX-2 expression in macrophages, which is consistent with a cytotoxic/proinflammatory phenotype of these cells (Mosser and Edwards, 2008
). Treatment of animals with AA results in increased expression of COX-2 in the liver (Reilly et al., 2001
; Oz and Chen, 2008
; Gardner et al., 2010
). Surprisingly, loss of COX-2 is associated with increased susceptibility of mice to AA-induced liver injury (Reilly et al., 2001
). This may be due to reduced generation of the anti-inflammatory prostanoids PGD2 and 15d-PGJ2 by COX-2. Recent studies suggest that activation of COX-2 and the generation of PGE2 during the onset phase of inflammation may also indirectly elicit pro-resolution effects by inducing the transcription of enzymes involved in the generation of anti-inflammatory lipoxins (Serhan et al., 2008
). It is possible that early activation of COX-2 is important in converting macrophages into anti-inflammatory/wound repair cells and initiating the resolution of the inflammatory response to AA.
Macrophages activated by inflammatory stimuli are also known to synthesize leukotrienes via a family of LOX enzymes including 5-LOX, 12-LOX, and 15-LOX. Whereas 5-LOX induces the generation of proinflammatory leukotrienes such as leukotriene B4, and is thought to be a pathogenic factor in acute liver injury, 12-LOX and 15-LOX are mainly involved in the production of anti-inflammatory lipoxins which serve a protective function, initiating the resolution of inflammation (Stables and Gilroy, 2011
). Interestingly, CM from AA-treated hepatocytes was found to upregulate expression of 12/15-LOX, but to downregulate expression of 5-LOX. These data suggest a potential mechanistic pathway, in addition to COX-2, involved in the transition of classically activated proinflammatory macrophages into alternatively activated immunosuppressive cells, an important step in wound repair.
Expression of the proinflammatory chemokines MIP-1α and MIP-2 is increased in the liver after AA administration to rodents (Lawson et al., 2000
; Liu et al., 2004
; Bourdi et al., 2007
). These chemokines are important in macrophage and neutrophil trafficking into inflamed tissues and have been implicated in hepatotoxicity (Hogaboam et al., 1999
). Moreover, increased expression of chemokines, including MIP-1α and MIP-2, is a feature of classically activated macrophages (Mosser and Edwards, 2008
; Mantovani et al., 2004
). We found that hepatocytes injured by AA release factors that upregulate macrophage expression of both MIP-1α and MIP-2. These data are in accord with reports that hepatocytes treated with cytotoxic doses of AA or with ethanol release chemotactic factors for rat Kupffer cells, as well as neutrophils, and provide additional support for the idea that injured hepatocytes aggravate hepatotoxicity via activation of phagocytic leukocytes (Laskin et al., 1986
; Takada et al., 1995
; Horbach et al., 1997
; Perez et al., 1984
; Shiratori et al., 1993
). Interestingly, AA by itself upregulated expression of MIP-2 in macrophages. These results were unexpected since previous studies showed that AA has no effect on macrophage functional responses (Laskin et al., 1986
). It may be that gene expression is a more sensitive marker of macrophage activation than functional analyses. In contrast to its effects on MIP-1α and MIP-2, CM from AA-injured hepatocytes had no effect on expression of MIP-1β. These data suggest the presence of multiple factors in hepatocyte CM that differentially modulate macrophage accumulation in the liver. We also found that mRNA expression of MCP-1 was downregulated by CM-AA in macrophages. MCP-1 expression is increased in the liver after AA administration and it has been shown to be required for emigration of anti-inflammatory/repair macrophages into the tissue (Dambach et al., 2002
). The fact that CM-AA suppresses MCP-1 supports the idea that hepatocytes damaged by AA release factors that predominantly induce proinflammatory macrophage accumulation and activation.
In further studies we assessed potential biochemical mechanisms mediating the effects of hepatocyte-derived activating factors on macrophages. MAP kinases comprise a family of protein-serine/threonine kinases that transduce extracellular signals into a variety of cellular activities including proliferation, differentiation, survival and inflammation (Dong et al., 2002
). Following treatment of macrophages with CM from AA-injured hepatocytes, activation of the p44/42 MAP kinase pathway was noted within 30 min. Additionally, CM-AA induced expression of COX-2, MIP-1α, and MIP-2 was dependent on p44/42. Thus, pretreatment of macrophages with the MEK1/2 inhibitor U0126, blocked the effects of CM-AA on expression of these genes. Evidence suggests that the biological actions of a number of classical macrophage activators, including lipopolysaccharide (LPS) and HMGB1, involve activation of the p44/42 MAP kinase signaling pathway (Kokkola et al., 2005
; Pedrazzi et al., 2007
). These data suggest a common biochemical mechanism mediating macrophage proinflammatory activity.
The precise identity of the hepatocyte-derived factors that activate macrophages for proinflammatory activity in the liver during the pathogenesis of AA-induced toxicity is unknown. The present studies suggest that HMGB1 may be responsible, at least in part, for this activity. This is based on our findings that AA-induced hepatocyte cytotoxicity was associated with the release of HMGB1, a response that was blocked by pretreatment of hepatocytes with ethyl pyruvate, which prevents HMGB1 release (Tang et al., 2010
). Additionally, both CM-AA and purified HMGB1 upregulate expression of COX-2, MIP-1α, MIP-2, RAGE, and phospho-p44/42 (Pedrazzi et al., 2007
; Li et al
. 1998; Kokkola et al. 2005
). Recent studies have shown that AA treatment of immortalized hepatocytes leads to HMGB1 release (Martin-Murphy et al., 2010
). However, higher doses of AA were required to induce this response, which may reflect the reduced sensitivity of transformed cells, relative to primary cells, to the cytotoxic effects of AA, potentially as a result of diminished metabolic capacity.
Ethyl pyruvate pretreatment of hepatocytes also blocked CM-AA-induced macrophage ROS production, a response that was also suppressed by immunoprecipitation of CM-AA with anti-HMGB1 antibody; these data suggest that the ROS-inducing activity in CM-AA is due to HMGB1. These findings are in accord with previous reports that HMGB1 released from necrotic cells induces a respiratory burst in eosinophils, and that purified HMGB1 stimulates neutrophil ROS production (Lotfi et al., 2009
; Fan et al., 2007
). Pretreatment of hepatocytes with ethyl pyruvate was also found to suppress CM-AA-induced expression of COX-2, as well as HO-1. These results are consistent with reports that HO-1 deficiency is associated with enhanced release of HMGB1 and decreased survival during endotoxemia The fact that inhibition of HO-1 and COX-2 was not complete suggests that CM-AA contains factors, in addition to HMGB1, that contribute to macrophage activation. This is supported by our findings that ethyl pyruvate pretreatment of hepatocytes had no effect on CM-AA-induced macrophage chemokine expression. The mechanisms underlying the ability of ethyl pyruvate to block HMGB1 release have not been established. In lung epithelial cells, ethyl pyruvate-mediated inhibition of HMGB1 release appears to be due to a switch from necrotic to apoptotic cell death (Lim et al., 2007
). It remains to be determined if a similar mechanism is involved in the inhibitory actions of ethyl pyruvate on AA-injured hepatocytes.
A major macrophage receptor for HMGB1 is RAGE, which is reported to be activated during inflammation and in response to oxidative stress (Sims et al., 2010
). RAGE activation is detrimental in various models of inflammatory injury including sepsis, LPS-induced lung injury, arthritis, partial hepatectomy, and liver ischemia-reperfusion injury (Herold et al., 2007
; Zhang et al., 2008
; Zeng et al., 2004
). Blockade of RAGE has also been reported to protect against lethal doses of AA (Ekong et al., 2006
). We found that macrophage expression of RAGE is upregulated in response to CM-AA. Consequences of RAGE activation include the generation of ROS, and increased expression of COX-2, HO-1 and chemokines, such as MIP-2 (Lotfi et al., 2009
; Wautier et al., 2001
; Shanmugam et al., 2003
; Zeng et al., 2009
). Our findings that CM-AA induced similar responses in macrophages, and that this correlated with increased RAGE expression, provide additional support for a role of HMGB1 in inducing these activities.
A question arises on potential differences in responsiveness of RAW 264.7 macrophages and primary liver macrophages to CM-AA. RAW 264.7 macrophages exhibit many functional characteristics of primary mouse macrophages, including phagocytosis and pinocytosis, antibody-dependent killing, responsiveness to bacterial products, and high secretory profile [Raschke et al., 1978
]. Moreover, in previous studies we demonstrated that primary cultures of Kupffer cells respond to hepatocyte CM with increased chemotaxis and oxidative metabolism [Laskin et al., 1986
]. These findings suggest that RAW 264.7 macrophages responses are indeed reflective of Kupffer cells.
In conclusion, the present studies suggest that factors released from AA-injured hepatocytes, including HMGB1, activate macrophages to produce cytotoxic and proinflammatory mediators known to be involved in hepatotoxicity (Laskin, 2009
; Laskin et al., 2011
). Inhibition of HMGB1 release or neutralization of its biological activity may represent a potential approach to downregulating activated macrophages, a key step in mitigating AA-induced hepatotoxicity.