We next carried out experiments to determine if CD14 contributed to I/R-induced liver injury. CD14 KO mice and their wild-type (WT) counterparts were subjected to 1 h of ischemia followed by 6 or 24 h of reperfusion. There was no difference in ALT levels between WT and CD14 KO sham groups. However, circulating ALT levels were significantly higher in WT mice subjected to I/R at both 6 and 24 h than in CD14 KO mice (Fig. 3). Histological analysis confirmed that CD14 KO mice were protected from the extensive central lobular necrosis observed at 6 and 24 h in the WT mice following I/R (Fig. 4). Ischemia/reperfusion was also associated with apoptosis assessed by TUNEL staining at 24 h. Here again, CD14 deficiency was associated with the near absence of apoptosis (Fig. 5, A and B).
To confirm the importance of CD14 to I/R-induced injury, we treated WT mice with a neutralizing anti-CD14 antibody either before the onset of ischemia or just before reperfusion. As shown in Figure 6, pretreatment with anti-CD14 antibody significantly attenuated the injury as measured by circulating ALT levels. Administration of antibody just before reperfusion had minimal protective effects (data not shown). Thus, CD14 participates in the pathobiology of I/R-induced liver injury and most likely is engaged early in the ischemia insult.
Ischemia/reperfusion–induced liver injury is the result of both ischemic cell death and the inflammatory response that is induced by I/R. To determine if CD14 is involved in I/R-induced inflammation, we measured IL-6 in the liver and circulation as a marker of the inflammatory response. CD14 KO mice showed much lower elevations in IL-6 in the liver (Fig. 7A) and circulation (Fig. 7B) than seen in the WT mice subjected to I/R. Wild-type mice pretreated with anti-CD14 antibody also had lower circulating IL-6 levels following I/R (Fig. 7C). Thus, CD14 is involved in the regulation of I/R-induced inflammation.
High-mobility group box 1 is a prototypical danger-associated molecular pattern (DAMP) previously shown to contribute to liver damage in I/R through TLR4. Furthermore, rHMGB1 exacerbates I/R-induced liver damage via a TLR4-dependent mechanism (4). We found no difference in circulating HMGB1 levels between WT and CD14 KO mice at 6 h, suggesting that CD14 is not required for early HMGB1 release (Fig. 8C). We administered 55 μg/mouse rHMGB1 (dose based on a pilot study, not shown), which did not result in any detectable liver injury by itself, to both WT and CD14 KO mice intraperitoneally. This dose of HMGB1 did not induce liver injury in control mice. Whereas rHMGB1 worsened liver damage when administered at reperfusion to WT mice, no increase in damage was seen in CD14 KO mice receiving rHMGB1(Fig. 8D). Taken together, these data suggest that CD14 is involved in sensing extracellular HMGB1 in liver I/R.
This study was undertaken to determine if CD14 is involved in I/R-induced liver injury. This question is important because CD14 is known to participate in ligand recognition by the TLR4 receptor complex (6), and TLR4 is involved in the injury and inflammation induced by warm I/R in liver (4). Moreover, recently Luan and colleagues (8) reported that I/R could upregulate CD14 expression in Kupffer cells in rats. We show here that CD14 is involved in both the injury and the inflammation induced by liver I/R. Whereas the release of HMGB1 in liver I/R did not require CD14, the recognition of HMGB1 does. These findings suggest that DAMP recognition in liver I/R involves CD14.
CD14 is a 55-kd cell surface glycoprotein that together with TLR4 and MD-2 acts as a receptor for endotoxin (6). CD14 exists in two forms: one is anchored to the cellular membrane by a glycosylphosphatidylinositol tail, i.e., membrane CD14 (mCD14), and the other a soluble form (sCD14). Membrane CD14 is mainly expressed on macrophages, neutrophils, and dendritic cells, but epithelial cells (9), endothelial cells (10), and fibroblasts (11) also express low levels of mCD14. Although mononuclear phagocytes are known to shed their membrane-expressed CD14 (12) and liver can secrete CD14 (13), the origin of sCD14 in plasma is not yet totally clear. Soluble CD14 may confer LPS responsiveness to cells that do not express CD14 (14). Blood and other body fluids contain sCD14, and levels increase during inflammation and infection. Therefore, sCD14 can be regarded as an acute phase protein (15). Here we show that CD14 mRNA levels in the liver increase after the hepatic I/R and that this corresponds to an increase in circulating sCD14 levels. It is unclear if the source of this sCD14 is from the liver. These results do indicate that I/R is a potent stimulus for CD14 upregulation and release.
The proinflammatory properties of HMGB1 were first identified in sepsis models where HMGB1 was shown to be a late mediator of lethality (25). In contrast to sepsis, HMGB1 acts as an early mediator in models of sterile inflammation. For example, we have previously shown that HMGB1 contributes to TLR4-dependent signaling in liver I/R and that rHMGB1 exacerbates I/R-induced injury in the liver in a TLR4-dependent manner (4). Here, we show that CD14 is also required for the exacerbation in I/R injury induced by rHMGB1. We have also reported that CD14 is involved in the injury and inflammation in cold cardiac I/R, a model that would involve no pathogen-associated molecular pattern exposure (26). Therefore, CD14 is likely to be involved in the detection of DAMPs, such as HMGB1 released in I/R injury.
Whether I/R responses depend on sCD14 or mCD14 or both deserves further exploration. Because HMGB1 has been shown to bind to LPS and facilitate transfer of LPS to CD14 and enhance human monocyte activation (27), it is possible that HMGB1 can also bind other DAMPs. One potential candidate is heat shock protein (HSP) 70. Similar to HMGB1, it can be released actively from stressed cells (28) or passively from necrotic cells (29). Asea et al. (30) reported CD14 was a coreceptor for HSP70-mediated signaling in human monocytes. Exogenous HSP70 bound with high affinity to the plasma membrane of monocytes caused monocyte activation in both CD14-dependent and CD14-independent pathways.
Taken together, our findings provide new insights into a novel role for CD14 in I/R. A better understanding of its mechanisms may ultimately lead to the development of new methods to prevent warm hepatic I/R injury.
The authors thank Qingde Wang, Pei Zhou and Rick Shapiro for technical support in this study.
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