S. M. Coyle

Epinephrine inhibits tumor necrosis factor-alpha and potentiates interleukin 10 production during human endotoxemia.

Short-term preexposure of mononuclear cells to epinephrine inhibits LPS-induced production of TNF, whereas preexposure for 24 h results in increased TNF production. To assess the effects of epinephrine infusions of varying duration on in vivo responses to LPS, the following experiments were performed: (a) Blood obtained from eight subjects at 4-24 h after the start of a 24-h infusion of epinephrine (30 ng/kg per min) produced less TNF after ex vivo stimulation with LPS compared with blood drawn before the start of the infusion, and (b) 17 healthy men who were receiving a continuous infusion of epinephrine (30 ng/kg per min) started either 3 h (EPI-3; n = 5) or 24 h (EPI-24; n = 6) were studied after intravenous injection of LPS (2 ng/kg, lot EC-5). EPI-3 inhibited LPS-induced in vivo TNF appearance and also increased IL-10 release (both P < 0.005 versus LPS), whereas EPI-24 only attenuated TNF secretion (P = 0.05). In separate in vitro experiments in whole blood, epinephrine increased LPS-induced IL-10 release by a combined effect on alpha and beta adrenergic receptors. Further, in LPS-stimulated blood, the increase on IL-10 levels caused by epinephrine only marginally contributed to concurrent inhibition of TNF production. Epinephrine, either endogenously produced or administered as a component of sepsis treatment, may have a net antiinflammatory effect on the cytokine network early in the course of systemic infection.

Effects of drotrecogin alfa (activated) in human endotoxemia

In a phase III clinical trial, drotrecogin alfa (activated) was shown to improve survival and promote faster improvement of cardiovascular and respiratory dysfunction in patients with severe sepsis. To further examine mechanisms involved in the action of this drug, a healthy human endotoxin model was used. Healthy volunteers (eight per group) received drotrecogin alfa (activated) or placebo intravenously for 8 h in a randomized, double-blind, controlled manner. After 2 h of study drug infusion, endotoxin (2 ng/kg) was infused and measurement of physiologic responses and biomarkers continued for 24 h. Consistent with results from severe sepsis clinical trials, drotrecogin alfa (activated) improved mean arterial pressure during the period of infusion after endotoxin exposure. In contrast to severe sepsis clinical trials using drotrecogin alfa (activated) but similar to another human endotoxin study, no significant antithrombotic, profibrinolytic, or anti-inflammatory effects were observed. These results suggest a novel role for drotrecogin alfa (activated) in the human endotoxin model.

Modulation of the lipopolysaccharide receptor complex (CD14, TLR4, MD-2) and toll-like receptor 2 in systemic inflammatory response syndrome-positive patients with and without infection: relationship to tolerance.

The lipopolysaccharide (LPS) receptor complex consists of two interacting receptors (CD14 and TLR4) and an associated protein (MD-2). When engaged by LPS, as in gram-negative infection, this complex transduces a signal detected by MyD88 and passed onward by a cascade of the IRAKs, TRAF6, and NIK, resulting in activation of NF-&kgr;B. A similar cascade, mediated by TLR2, occurs with ligands derived from gram-positive bacteria. In vitro studies of human monocytes have shown that TLR4 mRNA is paradoxically upregulated in response to “tolerizing” doses of LPS. This study evaluated changes in vivo of blood monocyte CD14, TLR4, TLR2, and MD-2 mRNA by reverse transcription followed by real-time polymerase chain reaction in surgical intensive care unit patients and in normal controls. In addition cell-surface receptor expression of TLR2, TLR4, and CD14 was assessed by flow cytometry in patients and normal controls. Inflammation-induced acute tolerance to LPS was evaluated by ex vivo whole blood tumor necrosis factor &agr; production and was significantly reduced in patients compared with controls, confirming LPS hyporesponsiveness. Monocyte mRNA and cell-surface receptor expression of TLR4 were increased 2.4-fold (P < 0.05) and 1.7-fold (P < .002), respectively, in patients compared with normal controls. Monocyte TLR2 mRNA, MD-2 mRNA and CD14 and TLR2 cell-surface expression were not significantly changed compared with controls. The present study suggests that the acute inflammatory condition associated with peripheral cellular LPS hyporesponsiveness is neither specific to prior infectious challenge nor can be ascribed to significant alterations in expression of the cell-surface LPS binding complex proteins.

Neutralization of TNF does not influence endotoxininduced changes in thyroid hormone metabolism in humans.

To determine the role of tumor necrosis factor (TNF) in endotoxin-induced changes in plasma thyroid hormone and thyroid-stimulating hormone (TSH) concentrations, 24 healthy postabsorptive humans were studied on a control study day (n = 6), after infusion of a recombinant TNF receptor IgG fusion protein (TNFR:Fc; 6 mg/m2; n = 6) after intravenous injection of endotoxin (2 ng/kg; n = 6), or after administration of endotoxin with TNFR:Fc (n = 6). Administration of TNFR:Fc alone did not affect thyroid hormone or TSH levels when compared with the control day. Endotoxin induced a transient rise in plasma TNF activity (1.5 h: 219 +/- 42 pg/ml), which was completely prevented by TNFR:Fc (P < 0.05). After endotoxin administration, plasma L-thyroxine (T4), free T4, 3,5, 3-triiodothyronine (T3), and TSH were lower and 3,3, 5-triiodothyronine was higher than on the control day (all P < 0. 05). Coinfusion of TNFR:Fc with endotoxin did not influence these endotoxin-induced changes. Our results suggest that endogenous TNF does not play an important role in the alterations in plasma thyroid hormone and TSH concentrations induced by mild endotoxemia in healthy humans.To determine the role of tumor necrosis factor (TNF) in endotoxin-induced changes in plasma thyroid hormone and thyroid-stimulating hormone (TSH) concentrations, 24 healthy postabsorptive humans were studied on a control study day ( n= 6), after infusion of a recombinant TNF receptor IgG fusion protein (TNFR:Fc; 6 mg/m2; n = 6) after intravenous injection of endotoxin (2 ng/kg; n = 6), or after administration of endotoxin with TNFR:Fc ( n = 6). Administration of TNFR:Fc alone did not affect thyroid hormone or TSH levels when compared with the control day. Endotoxin induced a transient rise in plasma TNF activity (1.5 h: 219 ± 42 pg/ml), which was completely prevented by TNFR:Fc ( P < 0.05). After endotoxin administration, plasmal-thyroxine (T4), free T4, 3,5,3-triiodothyronine (T3), and TSH were lower and 3,3,5-triiodothyronine was higher than on the control day (all P < 0.05). Coinfusion of TNFR:Fc with endotoxin did not influence these endotoxin-induced changes. Our results suggest that endogenous TNF does not play an important role in the alterations in plasma thyroid hormone and TSH concentrations induced by mild endotoxemia in healthy humans.

DIFFERENTIAL CELL DEATH GENE EXPRESSION IN MONOCYTES FROM LPS-CHALLENGED HEALTHY HUMAN VOLUNTEERS PRE-TREATED WITH CORTISOL

GHRELIN INHIBITS SYMPATHETIC NERVOUS ACTIVITY IN SEPSIS. R. Wu, M. Zhou, P. Das*, W. Dong*, Y. Ji*, D. Yang*, M. Miksa, T.S. Ravikumar*, P. Wang. North Shore University Hospital-LIJ Medical Center, Manhasset, NY 11030. Our previous studies have shown that norepinephrine (NE) upregulates proinflammatory cytokines by activating !2A-adrenoceptor. Therefore, modulation of the sympathetic nervous system represents a novel treatment for sepsis. We have also shown that a novel stomach-derived peptide, ghrelin, is downregulated in sepsis and that its intravenous (IV) administration decreases proinflammatory cytokines and improves survival. This beneficial effect appears to be mediated through the central nervous system, as evidenced by the inhibitory effect of intracerebroventricular administration of ghrelin on LPS-induced TNF-! release in rats. However, it remains to be determined whether ghrelin inhibits sympathetic activity through central ghrelin receptors (i.e., GHSR-la) in sepsis. To study this, sepsis was induced in male rats by cecal ligation and puncture (CLP). First, ghrelin was given IV (4 nmol/rat) infused for 2.5h starting 0.5h prior to CLP) and plasma levels of NE was measured by ELISA. Ghrelin significantly reduced the elevated NE levels at 2h post-CLP. Second, the effect of ghrelin on c-fos expression (i.e., a measure of neuronal activations) in a sympathostimulatory nucleus in the brain [i.e., the periventricular hypothalamic nucleus (PHN)] was examined. A bolus IV injection of 2 nmol ghrelin at 5h after CLP was followed by a continuous infusion of 12 nmol ghrelin via an osmotic pump for 15h. The expression of c-fos increased markedly 20h after CLP. Ghrelin administration reduced c-fos expression in the PHN. Third, to determine whether the inhibitory effect of ghrelin on NE is mediated by ghrelin receptors, a GHSR-la antagonist [D-Arg D-Phe D-Trp Leu]-substance P was injected to normal animals (210 nmol/rat) for 1h and plasma NE and TNF-! levels were assessed. GHSR-la inhibition significantly elevated both NE and TNF-! levels even in normal animals. Finally, to determine whether ghrelin can directly affect NE production, a neuron catecholaminergic cell line, CAD cells, was incubated with various doses of ghrelin. Ghrelin reduced NE production from CAD cells in a dose-dependent manner. These results suggest that ghrelin has sympathoinhibitory properties which are likely mediated by central ghrelin receptors (NIH R01 GM053008).