This study was undertaken to determine to what degree induced NO contributes to the inflammatory response and subsequent end organ damage after resuscitation from hemorrhagic shock. We have extended previous studies (7
) by showing that iNOS is not only upregulated during shock, but that it remains elevated after resuscitation. Using either the iNOS inhibitor L-NIL or iNOS knockout mice, we demonstrate that iNOS contributes to the induced expression of the cytokines IL-6 and G-CSF in the lung and liver after hemorrhagic shock. The iNOS-dependent increase in IL-6 and G-CSF mRNA levels is associated with an iNOS-dependent increase in NF-κB and Stat3 activation in these tissues. An association between the upregulation of these proinflammatory events by NO and organ injury is shown by our experiments, demonstrating reduced PMN accumulation and edema formation in the lungs, and reduced plasma levels of liver enzymes with iNOS suppression. These data provide compelling evidence, not only that iNOS is in part responsible for lung and liver damage after hemorrhage and resuscitation, but that the induction of NO synthesis is a key event in the subsequent activation of inflammatory cascades after resuscitation.
During hemorrhagic shock, iNOS is upregulated in several sites including the lung and liver (8
). The mechanism of upregulation is unclear, but could include hypoxia (6
) or the action of cytokines. We have shown that iNOS expression increases in parallel with the duration of shock (7
). Others have suggested that iNOS contributes to the initial vascular decompensation in hemorrhagic shock (8
) and we have suggested that the iNOS expression pattern is consistent with the possibility that iNOS may contribute to the progressive vascular dysfunction seen with sustained shock (7
). The current data support the idea that NO can increase cytokine expression through the activation of NF-κB, and that the activation of Stat3 may be the result of local cytokine expression (24
). Indirect evidence for this possibility is provided by the demonstration that NF-κB binding specific for the CK-1 element in the G-CSF promoter is present in hemorrhagic shock tissues. Furthermore, activation of Stat3 isoforms characteristic of IL-6 and G-CSF stimulation were identified as part of the activated Stat3 complex. Full resuscitation of the animals requires 20 min. However, as early as 1 h after the initiation of resuscitation, we found that the levels of IL-6 and G-CSF mRNA, as well as NF-κB and Stat3 activation in lungs and livers, were elevated to levels similar to those observed at 4 h (data not shown). Thus, it is likely that the NO-mediated signaling events that are initiated in early phases of resuscitation result in the rapid activation of downstream cascades. Although NO produced by the constitutive NO synthase has well-documented signaling functions in many systems, our novel observations provide strong evidence that induced NO also participates in lung cell signaling events in inflammation. That iNOS regulation of inflammatory gene expression is perhaps a more generalized phenomenon is supported by a recent observation that the upregulation of interferon γ and the response to IL-12 after Leishmania major
infection is iNOS dependent (31, and Bogdan, C., personal communication). Our results, however, do not exclude the possibility that the observed differences in both the L-NIL– treated rats and the iNOS knockout mice are due to some degree on changes in organ perfusion and oxygen delivery resulting from reduced NO availability.
NO is known to act as a signaling molecule in other circumstances either by activation of soluble guanylyl cyclase resulting in elevated cyclic guanosine 3′,5′ monophosphate (cGMP; reference 32
) or through S
-nitrosylation of proteins containing cysteine residues (33
). Significant differences in cytokine mRNA levels (14
) and transcriptional factor activation (25
) between the shock and sham groups was seen only after resuscitation, indicating that reperfusion was required. This suggests that redox-sensitive mechanisms were responsible for the NO-mediated signaling. Lander et al. (6
) have shown that NO activates p21ras
-nitrosylation and that this occurs more efficiently in human T cells subjected to oxidative stress. Downstream events include p38 kinase activation (35
) and NF-κB activation. In hemorrhagic shock, tissues are subjected to redox stress by hypoxia and oxygen radical production making S
-nitrosylation of p21ras
a reasonable candidate mechanism. A recent report demonstrates a role for increased phosphatidic acid in the signaling cascade involved in macrophage cytokine synthesis after hemorrhagic shock in mice (36
). A relationship between phosphatidic acid and NO is not apparent at this time.
We have previously shown that nonspecific NO synthase inhibition increases organ injury in hemorrhagic shock (37
), whereas here we show that the suppression of inflammation associated with selective iNOS inhibition results in a decrease in lung and liver injury. Taken together, the findings suggest that constitutive NOS is protective, but that the quantities of NO generated by iNOS cause injury. Our results suggest that organ damage in hemorrhagic shock is due, at least in part, to the proinflammatory action of NO. NO in combination with superoxide forms peroxynitrite, which could exert direct tissue toxicity. The results in hemorrhagic shock are contrasted by the observations in endotoxemia where the role of iNOS is less clear. iNOS knockout mice treated with high-dose endotoxin showed no difference in end organ damage when compared with their wild-type counterparts (11
). More recently, we have shown that iNOS inhibition during endotoxemia increases apoptosis in the liver without necrosis (38
). Difference between endotoxemia and hemorrhagic shock may be related to the higher levels of superoxide production and greater oxidant stress in the latter.
Approaches to remove NO in hemorrhagic shock could have therapeutic benefit. Patients suffering severe or sustained hemorrhagic shock after trauma or due to other causes of bleeding (e.g., ruptured aortic aneurysm) can develop organ injury and dysfunction. Our results indicate that selective inhibition of iNOS may be a reasonable approach. Alternatively, NO scavengers may also preserve adequate levels of NO needed to maintain perfusion while removing the excess NO which promotes inflammation and tissue injury.