Excessive production of proinflammatory mediators is observed in patients undergoing hemorrhagic and septic shock. Here, we report the detection of cold-inducible RNA-binding protein (CIRP) in the blood of surgical ICU individuals. In animal models of hemorrhage and sepsis, CIRP is up-regulated in several organs and released into the circulation. Under hypoxic stresses, CIRP in macrophages is translocated from the nucleus to the cytosol and actively released. Recombinant CIRP stimulates TNF-α and HMGB1 release in macrophages as well as induces inflammatory responses and causes tissue injury in animals. Antisera to CIRP attenuate shock-induced inflammation, tissue injury, and lethality. Extracellular CIRP's activity is mediated through the TLR4/MD2 complex. Surface plasmon resonance analysis indicates that CIRP binds to the TLR4/MD2 complex as well as to individual TLR4 and MD2. The human CIRP amino-acid segment 106-125 binds to MD2 with high affinity. Collectively, CIRP is a new proinflammatory mediator of shock.

Cold inducible RNA-binding protein (CIRP) is a nuclear protein which has been recently identified as a novel inflammatory mediator in hemorrhagic shock and sepsis. We hypothesized that CIRP acts as a potent inflammatory mediator in hepatic ischemia-reperfusion (I/R), and thus blocking CIRP protects against I/R-induced liver injury. Male C57BL/6 mice were subjected to 70% hepatic ischemia by microvascular clamping of the hilum of the left and median liver lobes for 60 min, followed by reperfusion. Anti-CIRP antibody (1 mg/kg body weight) or vehicle (normal saline) in 0.2 mL was injected via the internal jugular vein at the beginning of the reperfusion. Blood and liver tissues were collected 24 h after I/R for various measurements and a 10-day survival study was performed. CIRP released into the circulation was significantly increased 24 h after hepatic I/R. Anti-CIRP antibody treatment markedly reduced hepatocellular damage markers and significantly improved the liver microarchitecture. Anti-CIRP also reduced the systemic and local inflammation demonstrated by attenuation in both serum and hepatic levels of interleukin 6. The expression of neutrophil-attracting chemokine as well as liver neutrophil infiltration was reduced by anti-CIRP treatment. Anti-CIRP also dramatically decreased the amount of apoptosis and nitrosative stress, evidenced by decrease in TUNEL staining and inducible nitric oxide synthase and cyclooxygenase-2 levels, respectively. Finally, the 10-day survival rate was increased from 37.5% in the vehicle group to 75% in the anti-CIRP treatment group. Thus, targeting CIRP offers potential therapeutic implications in the treatment of hepatic I/R injury.

Excessive inflammation and apoptosis contribute to the pathogenesis of ischemic stroke. MFG-E8 is a 66-kDa glycoprotein that has shown tissue protection in various models of organ injury. However, the potential role of MFG-E8 in cerebral ischemia has not been investigated. We found that levels of MFG-E8 protein in the brain were reduced at 24 h after cerebral ischemia. To assess the potential role of MFG-E8 in cerebral ischemia, adult male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO). At 1 h post-stroke onset, an intravenous administration of 1 ml saline as vehicle or 160 μg/kg BW recombinant human MFG-E8 (rhMFG-E8) as treatment was given. The optimal dose of rhMFG-E8 was obtained from previous dose-response organ protection in rat sepsis studies. Neurological scores were determined at 24 h and 48 h post-MCAO. Rats were sacrificed thereafter and brains rapidly removed and analyzed for infarct size, histopathology, and markers of inflammation and apoptosis. Compared with saline vehicle, rhMFG-E8 treatment led to significant decreases in sensorimotor and vestibulomotor deficits, and infarct size at 24 h and 48 h post-MCAO. Measures associated with improved outcome included reduced microglial inflammatory cytokine secretion, adhesion molecules and neutrophil influx, cleaved caspase-3, and upregulation of peroxisome proliferator activated receptor-γ (PPAR-γ), and Bcl-2/Bax ratio leading to decreased apoptosis. Thus, rhMFG-E8 treatment is neuroprotective against cerebral ischemia through suppression of inflammation and apoptosis.

Sepsis and ischemia-reperfusion (I/R) injury are among the leading causes of death in critically ill patients at the surgical intensive care unit setting. Both conditions are marked by the excessive inflammatory response which leads to a lethal disease complex such as acute lung injury, systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Despite the advances in the understanding of the pathophysiology of those conditions, very little progress has been made toward therapeutic interventions. One of the key aspects of these conditions is the accumulation of apoptotic cells that have the potential to release toxic and proinflammatory contents due to secondary necrosis without appropriate clearance by phagocytes. Along with the prevention of apoptosis, that is reported to be beneficial in sepsis and I/R injury, thwarting the development of secondary necrosis through the active removal of apoptotic cells via phagocytosis may offer a novel therapy. Milk fat globule-EGF factor VIII (MFG-E8), which is mainly produced by macrophages and dendritic cells, is an opsonin for apoptotic cells and acts as a bridging protein between apoptotic cells and phagocytes. Recently, we have shown that MFG-E8 expression is decreased in experimental sepsis and I/R injury models. Exogenous administration of MFG-E8 attenuated the inflammatory response as well as tissue injury and mortality through the promotion of phagocytosis of apoptotic cells. In this review, we describe novel information available about the involvement of MFG-E8 in the pathophysiology of sepsis and I/R injury, and the therapeutic potential of exogenous MFG-E8 treatment for those conditions.