David J. Gallo

Role of Toll-Like Receptors in Changes in Gene Expression and NF-κB Activation in Mouse Hepatocytes Stimulated with Lipopolysaccharide

ABSTRACT The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-κB activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.

THE ACUTE INFLAMMATORY RESPONSE IN DIVERSE SHOCK STATES

A poorly controlled acute inflammatory response can lead to organ dysfunction and death. Severe systemic inflammation can be induced and perpetuated by diverse insults such as the administration of toxic bacterial products (e.g., endotoxin), traumatic injury, and hemorrhage. Here, we probe whether these varied shock states can be explained by a universal inflammatory system that is initiated through different means and, once initiated, follows a course specified by the cellular and molecular mechanisms of the immune and endocrine systems. To examine this question, we developed a mathematical model incorporating major elements of the acute inflammatory response in C57Bl/6 mice, using input from experimental data. We found that a single model with different initiators including the autonomic system could describe the response to various insults. This model was able to predict a dose range of endotoxin at which mice would die despite having been calibrated only in nonlethal inflammatory paradigms. These results show that the complex biology of inflammation can be modeled and supports the hypothesis that shock states induced by a range of physiologic challenges could arise from a universal response that is differently initiated and modulated.

Characterization of the expression of inducible nitric oxide synthase in rat and human liver during hemorrhagic shock.

It has been previously shown that the inducible nitric oxide (NO) synthase (iNOS; NOS-2) is elevated after hemorrhage, and that iNOS-derived NO participates in the upregulation of inflammation as well as lung and liver injury postresuscitation from shock. The purpose of this study was to elucidate the time course of iNOS mRNA expression, as well as the cellular and subcellular localization of iNOS protein in the liver posthemorrhage in rats subjected to varying durations of hemorrhagic shock (HS; mean arterial blood pressure [MAP] = 40 mmHg) with or without resuscitation. Expression of iNOS mRNA in rat liver by real-time reverse transcriptase (RT)-PCR demonstrated iNOS upregulation in shocked animals as compared with their sham counterparts as early as 60 min after the initiation of hemorrhage. By 1 h of HS, iNOS protein was detectable in rat liver by immunofluorescence, and this expression increased with time. Immunofluorescence localized iNOS primarily to the hepatocytes, and in particular to hepatocytes in the centrilobular regions. This analysis, confirmed by immunoelectron microscopy, revealed that iNOS colocalizes with catalase, a peroxisomal marker. Furthermore, we determined that iNOS mRNA is detectable by RT-PCR in liver biopsies from human subjects with HS (MAP < 90 mmHg) associated with trauma (n = 18). In contrast, none of the seven nontrauma surgical patients studied had detectable iNOS mRNA in their livers. Collectively, these results suggest that hepatic iNOS expression, associated with peroxisomal localization, is an early molecular response to HS in experimental animals and possibly in human patients with trauma with HS.

Tissue Hypoxia Activates Jnk In The Liver During Hemorrhagic Shock

The earliest signaling pathways responsible for initiating the systemic response to hemorrhagic shock (HS) remain poorly characterized. We have investigated the involvement of the mitogen-activated protein (MAP) kinase C-JUN N-terminal kinase (JNK) and its activation in the liver as an early response to tissue hypoxia soon after the initiation of hemorrhage. In the present studies, hemorrhage of mice to 25 mmHg for 30 min resulted in a significant (2.1-fold) increase in JNK phosphorylation within the liver. Results were similar in rats hemorrhaged to 40 mmHg for 1 h. Hypoxia alone, replicated by warm isolated hepatic ischemia in vivo or hepatocytes cultured under 1% oxygen, also resulted in JNK phosphorylation. Finally, preservation of tissue perfusion and oxygenation by pretreatment with a blood-soluble drag-reducing polymer (DRP) in the rat HS model prevented phosphorylation of JNK in the liver. These results identify tissue hypoxia as a key factor in activating early signaling events in the liver following hemorrhage, as measured by JNK phosphorylation.

The role of hepatic type 1 plasminogen activator inhibitor (PAI‐1) during murine hemorrhagic shock

Hemorrhagic shock (HS) followed by resuscitation (HS‐R) is characterized by profound physiological changes. Even if the patient survives the initial blood loss, these poorly understood changes can lead to morbidity. One of the tissues most often affected is liver. We sought to recognize specific hepatic changes induced by this stressor to identify targets for therapeutic intervention. Gene array analyses using mouse liver mRNAs were used to identify candidate genes that contribute to hepatic damage. To verify the role of one of the genes identified using the arrays, mice were subjected to HS‐R, and multiple parameters were analyzed. A profound increase in plasminogen activator inhibitor type 1 (PAI‐1) mRNA was observed using hepatic mRNAs from C57Bl/6 mice after HS, both with and without resuscitation. Constitutive loss of PAI‐1 resulted in notable tissue preservation and lower (P < .05) alanine aminotransferase (ALT) levels. Fibrin degradation products (FDPs) and interleukins 6 and 10 (IL‐6 and IL‐10) were unaffected by loss of PAI‐1; however, enhanced urokinase activity, an elevation of active hepatocyte growth factor (HGF), an increase in unprocessed transforming growth factor‐β1 (TGF‐β1), and retention of ERK phosphorylation after HS‐R were associated with improved hepatic function. In conclusion, PAI‐1 protein is a negative effector of hepatic damage after HS‐R through its influence on classic regulators of hepatic growth, as opposed to its role in fibrinolysis. (HEPATOLOGY 2005;42:390–399.)

A role for angiotensin II in the activation of extracellular signal-regulated kinases in the liver during hemorrhagic shock.

Hemorrhagic shock (HS) is a complex process that initiates a global stress response. However, the earliest signaling pathways responsible for initiating this response remain unidentified. We have investigated the involvement of the extracellular signal-regulated kinases (ERK 1/2; also known as p42/44) and their activation in the liver by angiotensin II in the early signal transduction after HS. Hemorrhage of mice to 25 mmHg for 30 min was associated with the activation of ERK 1/2 in the liver, and this was accompanied by a 6.7-fold elevation of circulating angiotensin II levels. Similar results were obtained in rats. Both the angiotensin II levels and ERK 1/2 phosphorylation were suppressed by administration of an angiotensin-converting enzyme inhibitor peptide. Plasma from shocked rats, but not shocked rats treated with the angiotensin-converting enzyme inhibitor, increased ERK 1/2 phosphorylation in cultured hepatocytes. Together, these data suggest that angiotensin II is an important stimulus for ERK 1/2 activation in the liver during HS.