Fetuin, a negative acute phase protein, attenuates TNF synthesis and the innate inflammatory response to carrageenan.

Activation of the innate immune system, even by relatively innocuous stimuli, stimulates the release of cytokines (e.g. TNF) that can injure or kill the host. To maintain homeostasis, mammals have evolved a counter-regulatory response that suppresses the development of excessively robust inflammation. Fetuin, a 66-kD negative acute phase glycoprotein, was first identified in 1944. We recently discovered an anti-inflammatory role for fetuin, because it suppressed the release of TNF from lipopolysaccharide- (LPS) stimulated macrophages. Here the anti-inflammatory effects of fetuin were studied in vivo in an LPS-independent model of acute inflammation caused by administration of carrageenan. Administration of fetuin (5-500 mg/kg intraperitoneally) dose-dependently attenuated the development of paw edema as compared to either asialofetuin (500 mg/kg) or bovine albumin (500 mg/kg). TNF production in the carrageenan-injected paws was significantly inhibited by administration of fetuin (586+/-98 pg TNF/paw) as compared to either asialofetuin (1018+/-186 pg TNF/paw) or saline (1,005+/-172 pg TNF/paw). When specific anti-fetuin IgG was administered into the paw prior to the application of carrageenan, the development of edema formation was significantly increased as compared to irrelevant IgG, indicating that endogenous fetuin normally attenuates the inflammatory response. These results now reveal a previously unrecognized anti-inflammatory role of fetuin in counter-regulating the innate immune response, and suggest that it may be possible to use fetuin as an experimental anti-inflammatory agent.

Proinflammatory cytokines (tumor necrosis factor and interleukin 1) stimulate release of high mobility group protein-1 by pituicytes.

BACKGROUND: Cytokines mediate the metabolic and physiologic responses to injury and infection. Anterior pituitary cells express receptors for tumor necrosis factor (TNF) and interleukin 1 (IL-1), which can signal these cells to release corticotropin, growth hormone, and cytokines such as IL-1 and macrophage migration inhibitory factor. This interaction provides an important link between the immune system and the neuroendocrine system. We reasoned that pituicytes activated with TNF or IL-1 might release previously unrecognized factors that could participate in this signaling from the neuroendocrine to the immune system. METHODS: Proteins released from rat pituicytes (GH3) after stimulation with proinflammatory cytokines were identified by N-terminal amino acid sequencing. Polyclonal antibodies against a peptide corresponding to the N-terminal amino acid sequence were generated and used to determine the kinetics of protein release. RESULTS: Cytokine stimulation induced the release of a 30-kd protein from rat pituicytes. After the protein was isolated and the N-terminal amino acid sequence determined, a protein database analysis revealed that it is high mobility group-1 (HMG-1) protein. TNF and IL-1 induced the release of HMG-1 from pituicytes in a time- and dose-dependent manner. Interferon gamma alone did not induce the release of HMG-1, but it enhanced TNF-induced HMG-1 release. CONCLUSION: Stimulation of pituicytes by TNF or IL-1 induces the release of HMG-1, which may participate in the regulation of neuroendocrine and immune responses to infection or injury.

HMG-1 as a late mediator of endotoxin lethality in mice.

Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release large quantities of tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can precipitate tissue injury and lethal shock (endotoxemia). Antagonists of TNF and IL-1 have shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis. Here a potential late mediator of lethality is identified and characterized in a mouse model. High mobility group-1 (HMG-1) protein was found to be released by cultured macrophages more than 8 hours after stimulation with endotoxin, TNF, or IL-1. Mice showed increased serum levels of HMG-1 from 8 to 32 hours after endotoxin exposure. Delayed administration of antibodies to HMG-1 attenuated endotoxin lethality in mice, and administration of HMG-1 itself was lethal. Septic patients who succumbed to infection had increased serum HMG-1 levels, suggesting that this protein warrants investigation as a therapeutic target.

Hypophysectomy, high tumor necrosis factor levels, and hemoglobinemia in lethal endotoxemic shock.

Experimental models of lethal endotoxemia in rodents are widely used to delineate pathogenic mechanisms of inflammation, sepsis, and septic shock. One long-standing but poorly understood observation is that removal of the pituitary gland (hypophysectomy) renders experimental animals 1,000-fold more sensitive to the lethal sequelae of lipopolysaccharide (LPS). Previous explanations for this phenomenon focused on hypophysectomy-induced deficiencies of corticosteroids, because glucocorticoids effectively suppress the synthesis of tumor necrosis factor (TNF), which is a primary mediator of LPS lethality. We measured LPS-stimulated macrophage TNF release in the presence of serum from hypophysectomized rats to detect the appearance of an inducible 65 kDa protein that enhances TNF release. Surprisingly, the N-terminal amino acid sequence analysis of the isolated, purified protein revealed its identity as hemoglobin. Hypophysectomy significantly increases serum hemoglobin levels (control hemoglobin = 103+/-18 microg/mL versus hypophysectomized serum hemoglobin = 279+/-13 microg/mL; p < .05). Purified hemoglobin enhances TNF synthesis in LPS-stimulated macrophages by at least 1,000-fold, which is specifically inhibited by antihemoglobin antibodies. Thus, hemoglobin mediates increased TNF synthesis in endotoxemic, hypophysectomized rats. This mechanism of increased TNF release has potential implications for patients with hemoglobinemia following blood transfusion, surgery, injury, infection, or other conditions that can be associated with endotoxemia and sepsis.

Tumor necrosis factor is a brain damaging cytokine in cerebral ischemia.

Two contrasting roles, one beneficial and the injurious, have been proposed for tumor necrosis factor (TNF) in the pathogenesis of cerebral ischemia. Reported here are results obtained in a standard model of permanent focal cortical ischemia in rats, in which the volume of cerebral infarction is measured after permanent occlusion of the middle cerebral artery. Administration of neutralizing anti-rat TNF antibodies (P114) into the brain cortex significantly reduced ischemic brain damage (85% reduced infarct volume as compared with preimmune-treated controls). Similar results were achieved by systemic administration of CNI-1493, a recently described tetravalent guanylhydrazone compound, which effectively inhibited endogenous brain TNF synthesis and conferred significant protection against the development of cerebral infarction (80% reduced infarct volume as compared with vehicle controls treated 1 h postischemia with 10 mg/kg). P114 anti-TNF and CNI-1493 were each cerebroprotective when given within a clinically relevant time window for up to 2 h after the onset of ischemia. These findings establish an important, pathophysiological role of TNF in mediating the progression of ischemic brain damage, and suggest that inhibiting TNF with CNI-1493 may be beneficial in the future treatment of stroke.