Suppression of tumor necrosis factor alpha production by cAMP in human monocytes: dissociation with mRNA level and independent of interleukin-10.

BACKGROUND: Elevation of cellular cAMP inhibits lipopolysaccharide(LPS)-stimulated tumor necrosis factor alpha (TNF-alpha) production and increases the expression of interleukin (IL)-10 in mononuclear cells. TNF-alpha gene expression obligates activation of the transcription factor nuclear factor kappaB (NF-kappaB). Exogenous IL-10 inhibits NF-kappaB in monocytes and thus attenuates TNF-alpha production. We examined the role of endogenous IL-10 in the regulation of NF-kappaB activation and TNF-alpha production in human monocytes by cAMP. METHODS: Human monocytes were stimulated with Escherichia coli LPS (100 ng/ml) with and without forskolin (FSK, 50 microM) or dibutyryl cyclic AMP (dbcAMP, 100 microM). Cytokine (TNF-alpha and IL-10) release was measured by immunoassay. TNF-alpha mRNA was measured by reverse transcription polymerase chain reaction, and NF-kappaB DNA binding activity was assessed by gel mobility shift assay. RESULTS: cAMP-elevating agents inhibited LPS-stimulated TNF-alpha release (0.77 +/- 0.13 ng/10(6) cells in LPS + dbcAMP and 0.68 +/- 0.19 ng/10(6) cells in LPS + FSK, both P < 0.05 vs 1.61 +/- 0.34 ng/10(6) cells in LPS alone). Conversely, cAMP enhanced LPS-stimulated IL-10 release (100 +/- 21.5 pg/10(6) cells in LPS + dbcAMP and 110 +/- 25.2 pg/10(6) cells in LPS + FSK, both P < 0.05 vs 53.3 +/- 12.8 pg/10(6) cells in LPS alone). Neither TNF-alpha mRNA expression nor NF-kappaB activation stimulated by LPS was inhibited by the cAMP-elevating agents. Neutralization of IL-10 with a specific antibody did not attenuate the effect of cAMP-elevating agents on TNF-alpha production. CONCLUSION: The results indicate that cAMP inhibits LPS-stimulated TNF-alpha production through a posttranscriptional mechanism that is independent of endogenous IL-10.

L-arginine attenuates lipopolysaccharide-induced lung chemokine production.

Chemokines stimulate the influx of leukocytes into tissues. Their production is regulated by nuclear factor-kappaB (NF-kappaB), an inducible transcription factor under the control of inhibitory factor kappaB-alpha (IkappaB-alpha). We have previously demonstrated that L-arginine (L-Arg) attenuates neutrophil accumulation and pulmonary vascular injury after administration of lipopolysaccharide (LPS). We hypothesized that L-Arg would attenuate the production of lung chemokines by stabilizing IkappaB-alpha and preventing NF-kappaB DNA binding. We examined the effect of L-Arg on chemokine production, IkappaB-alpha degradation, and NF-kappaB DNA binding in the lung after systemic LPS. To block nitric oxide (NO) production, a NO synthase inhibitor was given before L-Arg. LPS induced the production of chemokine protein and mRNA. L-Arg attenuated the production of chemokine protein and mRNA, prevented the decrease in IkappaB-alpha levels, and inhibited NF-kappaB DNA binding. NO synthase inhibition abolished the effects of L-Arg on all measured parameters. Our results suggest that L-Arg abrogates chemokine protein and mRNA production in rat lung after LPS. This effect is dependent on NO and is mediated by stabilization of IkappaB-alpha levels and inhibition of NF-kappaB DNA binding.

Thirty years of clinical trials in acute respiratory distress syndrome.

OBJECTIVE: To systematically review clinical trials in acute respiratory distress syndrome (ARDS). DATA SOURCES: Computerized bibliographic search of published research and citation review of relevant articles. STUDY SELECTION: All clinical trials of therapies for ARDS were reviewed. Therapies that have been compared in prospective, randomized trials were the focus of this analysis. DATA EXTRACTION: Data on population, interventions, and outcomes were obtained by review. Studies were graded for quality of scientific evidence. MAIN RESULTS: Lung protective ventilator strategy is supported by improved outcome in a single large, prospective trial and a second smaller trial. Other therapies for ARDS, including noninvasive positive pressure ventilation, inverse ratio ventilation, fluid restriction, inhaled nitric oxide, almitrine, prostacyclin, liquid ventilation, surfactant, and immune-modulating therapies, cannot be recommended at this time. Results of small trials using corticosteroids in late ARDS support the need for confirmatory large clinical trials. CONCLUSIONS: Lung protective ventilator strategy is the first therapy found to improve outcome in ARDS. Trials of prone ventilation and fluid restriction in ARDS and corticosteroids in late ARDS support the need for large, prospective, randomized trials.

Chemokines as mediators of diseases related to surgical conditions.

Chemokines are important mediators of inflammation. Animal studies suggest that inhibition of chemokine action results in a decrease in inflammation. Novel anti-inflammatory agents directed against chemokines are now available. Surgeons are uniquely positioned to treat multiple chemokine-mediated diseases. In this article, we review the biology and nomenclature of chemokines as well as their role in neutrophil migration. Further, the potential role of chemokines in various diseases related to surgical conditions, including adult respiratory distress syndrome, atherosclerosis, inflammatory bowel disease, and solid organ rejection, is reviewed. Finally, the idea that chemokines could be targets for novel therapeutic agents is discussed.

Differential inducible nitric oxide synthase expression in systemic and pulmonary vessels after endotoxin.

Inducible nitric oxide synthase (iNOS) is associated with vascular hypocontractility in systemic vessels after endotoxin lipopolysaccharide (LPS) administration. Although lung iNOS is increased after LPS, its role in the pulmonary circulation is unclear. We hypothesized that whereas iNOS upregulation is responsible for LPS-induced vascular dysfunction in systemic vessels, iNOS does not play a significant role in the pulmonary artery (PA). Using isolated aorta (AO) and PA rings, we examined the effect of nonselective NOS inhibition [N(G)-monomethyl-L-arginine (L-NMMA); 100 micromol/l] and selective iNOS inhibition (aminoguanidine, AG; 100 micromol/l) on alpha(1)-adrenergic-mediated vasoconstriction (phenylephrine; 10(-9) to 10(-3) M) after LPS (Salmonella typhimurium, 20 mg/kg ip). We also determined the presence of iNOS using Western blot and immunohistochemistry. LPS markedly impaired AO contractility (maximal control tension 1,076 +/- 33 mg vs. LPS 412 +/- 39 mg, P < 0.05), but PA contractility was unchanged (control 466 +/- 29 mg vs. LPS 455 +/- 27 mg, P > 0.05). Selective iNOS inhibition restored the AO's response to vasoconstriction (LPS + AG 1,135 +/- 54 mg, P > 0.05 vs. control and P < 0.05 vs. LPS), but had no effect on the PA (LPS + AG 422 +/- 38 mg, P > 0.05 vs. control and LPS). Western blot and immunohistochemistry revealed increased iNOS expression in the AO after LPS, but iNOS was not detected in the PA. Our results suggest that differential iNOS expression after LPS in systemic and pulmonary vessels contributes to the phenomenon of sepsis/endotoxemia-induced systemic hypotension and pulmonary hypertension.

Neutralization of IL-18 reduces neutrophil tissue accumulation and protects mice against lethal Escherichia coli and Salmonella typhimurium endotoxemia.

In addition to stimulating IFN-gamma synthesis, IL-18 also possesses inflammatory effects by inducing synthesis of the proinflammatory cytokines TNF and IL-1beta and the chemokines IL-8 and macrophage inflammatory protein-1alpha. We hypothesized that neutralization of IL-18 would have a beneficial effect in lethal endotoxemia in mice. IL-1beta converting enzyme (ICE)-deficient mice, lacking the ability to process mature IL-18 and IL-1beta, were completely resistant to lethal endotoxemia induced by LPS derived from either Escherichia coli or Salmonella typhimurium. In contrast, both wild-type and IL-1beta-/- mice were equally susceptible to the lethal effects of LPS, implicating that absence of mature IL-18 or IFN-gamma but not IL-1beta in ICE-/- mice is responsible for this resistance. However, IFN-gamma-deficient mice were not resistant to S. typhimurium LPS, suggesting an IFN-gamma-independent role for IL-18. Anti-IL-18 Abs protected mice against a lethal injection of either LPS. Anti-IL-18 treatment also reduced neutrophil accumulation in liver and lungs. The increased survival was accompanied by decreased levels of IFN-gamma and macrophage inflammatory protein-2 in anti-IL-18-treated animals challenged with E. coli LPS, whereas IFN-gamma and TNF concentrations were decreased in treated mice challenged with S. typhimurium. In conclusion, neutralization of IL-18 during lethal endotoxemia protects mice against lethal effects of LPS. This protection is partly mediated through inhibition of IFN-gamma production, but mechanisms involving decreased neutrophil-mediated tissue damage due to the reduction of either chemokines (E. coli LPS) or TNF (S. typhimurium LPS) synthesis by anti-IL-18 treatment may also be involved.

Inhibition of PARS attenuates endotoxin-induced dysfunction of pulmonary vasorelaxation.

Endotoxin (Etx) causes excessive activation of the nuclear repair enzyme poly(ADP-ribose) synthase (PARS), which depletes cellular energy stores and leads to vascular dysfunction. We hypothesized that PARS inhibition would attenuate injury to mechanisms of pulmonary vasorelaxation in acute lung injury. The purpose of this study was to determine the effect of in vivo PARS inhibition on Etx-induced dysfunction of pulmonary vasorelaxation. Rats received intraperitoneal saline or Etx (Salmonella typhimurium; 20 mg/kg) and one of the PARS inhibitors, 3-aminobenzamide (3-AB; 10 mg/kg) or nicotinamide (Nic; 200 mg/kg), 90 min later. After 6 h, concentration-response curves were determined in isolated pulmonary arterial rings. Etx impaired endothelium-dependent (response to ACh and calcium ionophore) and -independent (sodium nitroprusside) cGMP-mediated vasorelaxation. 3-AB and Nic attenuated Etx-induced impairment of endothelium-dependent and -independent pulmonary vasorelaxation. 3-AB and Nic had no effect on Etx-induced increases in lung myeloperoxidase activity and edema. Lung ATP decreased after Etx but was maintained by 3-AB and Nic. Pulmonary arterial PARS activity increased fivefold after Etx, which 3-AB and Nic prevented. The beneficial effects were not observed with benzoic acid, a structural analog of 3-AB that does not inhibit PARS. Our results suggest that PARS inhibition with 3-AB or Nic improves pulmonary vasorelaxation and preserves lung ATP levels in acute lung injury.

Exogenous calcium preconditions myocardium from patients taking oral sulfonylurea agents.

We have previously reported that atrial trabeculae from patients taking oral sulfonylurea hypoglycemic agents cannot be preconditioned by transient ischemia, which may, in part, explain the increased cardiovascular mortality historically associated with the use of these agents (J. C. Cleveland et al., 1997, Circulation 96, 29-32). Recently, we reported that clinically accessible and acceptable exogenous Ca(2+) pretreatment protects human atrial trabeculae from subsequent ischemia (B. S. Cain et al., 1998, Ann. Thoracic Surg. 65, 1065-1070). It remains unknown whether this preconditioning strategy could confer protection to trabeculae from patients taking oral sulfonylurea drugs. We therefore hypothesized that exogenous Ca(2+) confers ischemic protection to trabeculae from patients taking oral sulfonylureas. Human atrial trabeculae were suspended in organ baths and field stimulated at 1 Hz, and force development was recorded. Following 90 min equilibration, trabeculae from patients taking oral sulfonylurea agents (n = 6 patients) were subjected to ischemia/reperfusion (I/R; 45/120 min) with or without Ca(2+) (1 mM increase x 5 min) 10 min prior to I/R. I/R decreased postischemic human myocardial contractility in trabeculae from patients on oral hypoglycemics to 15.3 +/- 2.0% baseline developed force (%BDF). Ca(2+) pretreatment increased postischemic human myocardial developed force to 35.3 +/- 2.9 %BDF in these patients (P < 0.05 vs I/R, ANOVA and Bonferroni/Dunn). We conclude that atrial muscle from patients taking oral hypoglycemic agents can be preconditioned with exogenous Ca(2+). This therapy may offer a clinically relevant means to precondition the myocardium of diabetics taking oral hypoglycemic agents prior to clinical interventions such as coronary angioplasty or cardiac bypass.

Cardiotrophin-1 attenuates endotoxin-induced acute lung injury.

Cardiotrophin-1 (CT-1) is a recently discovered member of the gp130 cytokine family, which includes IL-6, IL-11, leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. Recent evidence suggests that, like other members of this family, CT-1 may possess anti-inflammatory properties. We hypothesized that in vivo CT-1 administration would attenuate endotoxin (ETX)-induced acute lung injury. We studied the effects of CT-1 (100 microgram/kg ip, 10 min prior to ETX) in a rat model of ETX-induced acute lung injury (Salmonella typhimurium lipopolysaccharide, 20 mg/kg ip). Six hours after ETX, lungs were harvested for determination of neutrophil accumulation (myeloperoxidase, MPO, assay) and lung edema (wet-to-dry weight ratio). Mechanisms of pulmonary vasorelaxation were examined in isolated pulmonary artery rings at 6 h by interrogating endothelium-dependent (response to acetylcholine) and endothelium-independent (response to sodium nitroprusside) relaxation following alpha-adrenergic (phenylephrine)-stimulated preconstriction. CT-1 abrogated the endotoxin-induced lung neutrophil accumulation: 2.3 +/- 0.2 units MPO/g wet lung (gwl) vs 6. 3 +/- 0.3 units MPO/gwl in the ETX group (P < 0.05 vs ETX, P > 0.05 vs control). Similarly, CT-1 prevented ETX-induced lung edema: wet-to-dry-weight ratio, 4.473 +/- 0.039 vs 4.747 +/- 0.039 in the ETX group (P < 0.05 vs ETX, P > 0.05 vs control). Endotoxin caused significant impairment of both endothelium-dependent and -independent pulmonary vasorelaxation, and CT-1 attenuated this injury. Thus, cardiotrophin-1 possesses significant anti-inflammatory properties in a model of endotoxin-induced acute lung injury.

Adrenergic induction of bimodal myocardial protection: signal transduction and cardiac gene reprogramming.

This study tested the hypothesis that in vivo norepinephrine (NE) treatment induces bimodal cardiac functional protection against ischemia and examined the roles of alpha1-adrenoceptors, protein kinase C (PKC), and cardiac gene expression in cardiac protection. Rats were treated with NE (25 micrograms/kg iv). Cardiac functional resistance to ischemia-reperfusion (25/40 min) injury was examined 30 min and 1, 4, and 24 h after NE treatment with the Langendorff technique, and effects of alpha1-adrenoceptor antagonism and PKC inhibition on the protection were determined. Northern analysis was performed to examine cardiac expression of mRNAs encoding alpha-actin and myosin heavy chain (MHC) isoforms. Immunofluorescent staining was performed to localize PKC-betaI in the ventricular myocardium. NE treatment improved postischemic functional recovery at 30 min, 4 h, and 24 h but not at 1 h. Pretreatment with prazosin or chelerythrine abolished both the early adaptive response at 30 min and the delayed adaptive response at 24 h. NE treatment induced intranuclear translocation of PKC-betaI in cardiac myocytes at 10 min and increased skeletal alpha-actin and beta-MHC mRNAs in the myocardium at 4-24 h. These results demonstrate that in vivo NE treatment induces bimodal myocardial functional adaptation to ischemia in a rat model. alpha1-Adrenoceptors and PKC appear to be involved in signal transduction for inducing both the early and delayed adaptive responses. The delayed adaptive response is associated with the expression of cardiac genes encoding fetal contractile proteins, and PKC-betaI may transduce the signal for reprogramming of cardiac gene expression.

LPS-Induced NF-kappaB activation and TNF-alpha release in human monocytes are protein tyrosine kinase dependent and protein kinase C independent.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-kappaB) and TNF-alpha release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappaB activation and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappaB DNA binding and TNF-alpha release in human monocytes. MATERIALS AND METHODS: Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-alpha release in culture supernatants was measured by an ELISA. NF-kappaB DNA binding was evaluated by electrophoretic mobility shift assay. RESULTS: LPS increased NF-kappaB DNA binding and TNF-alpha release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-kappaB activation and TNF-alpha release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-kappaB DNA binding or TNF-alpha release. PTK inhibition with Gen attenuated both LPS-induced NF-kappaB DNA binding and TNF-alpha production in human monocytes. Direct activation of PKC with PMA induced both NF-kappaB activation and TNF-alpha production by human monocytes. CONCLUSIONS: These results suggest that LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes and thus could be a potential therapeutic target in inflammatory states.

Therapeutic implications of interleukin-10 in surgical disease.

Pharmacological therapy of surgical disease often involves manipulating the physiologic balance between pro- and anti-inflammatory responses. Many agents target only one aspect of the inflammatory cascade. Originally identified as a protein elaborated by T-lymphocytes, IL-10 appears to globally inhibit cytokine production. The purpose of this manuscript is to examine the immunomodulatory and anti-inflammatory effects of interleukin-10 (IL-10) in an attempt to define the clinical utility of IL-10, both as a marker of and as a therapeutic strategy for intervention in inflammatory and immune-mediated diseases. IL-10 is elaborated from multiple sources and has diverse cellular effects to regulate immune and inflammatory responses. Accumulating evidence suggests that the anti-inflammatory influence of IL-10 observed at the cellular level may be manipulated to impact the immune and inflammatory-mediated responses associated with injury and sepsis, gastrointestinal and cardiovascular disease, and transplantation. In conclusion, IL-10 is an important mediator of immune and anti-inflammatory responses in surgical disease and, as such, has therapeutic promise as an immunomodulator and as an anti-inflammatory agent.

Endotoxin differentially impairs receptor-mediated relaxation in rat isolated pulmonary and thoracic aortic vessels.

PURPOSE: The purpose of this study was to determine the effect of endotoxin on vasorelaxation in the pulmonary and systemic circulations in response to the following agonists that require generation of cyclic adenosine monophosphate: (1) beta-adrenergic receptor stimulation with isoproterenol; (2) H2 receptor stimulation with dimaprit; and (3) adenylate cyclase stimulation with forskolin. METHODS: Male Sprague-Dawley rats weighing 250 to 350 g were injected with endotoxin (20 mg/kg intraperitoneal) or saline. Six hours later, the cumulative dose response to beta-adrenergic receptor stimulation (isoproterenol), H2 receptor stimulation (dimaprit), and adenylate cyclase stimulation (forskolin) was determined in isolated rat pulmonary artery and thoracic aortic rings preconstricted with phenylephrine. RESULTS: Endotoxin caused significant impairment of relaxation to isoproterenol in the pulmonary artery, but the response in the aorta was not different from the control response. In the pulmonary circulation, endotoxin converted the response to dimaprit from vasorelaxation to vasoconstriction. On the other hand, dimaprit resulted in vasorelaxation in the thoracic aorta after endotoxin; however, the response was impaired compared with the control response. Endotoxin did not affect the dose response to forskolin in either the pulmonary artery or the thoracic aorta. CONCLUSION: From these data, we conclude that endotoxin causes regional specific changes in vascular reactivity. These changes in vascular reactivity result in preserved vasorelaxation in the systemic circulation and impairment of vasorelaxation in the pulmonary circulation in response to endotoxin.

Increased levels of myocardial IkappaB-alpha protein promote tolerance to endotoxin.

Endotoxin [lipopolysaccharide (LPS)] causes tumor necrosis factor-alpha (TNF-alpha)-mediated myocardial contractile depression. Tolerance to the cardiac toxicity of LPS can be induced by a prior exposure to LPS or by pretreatment with glucocorticoids. The mechanisms by which the myocardium acquires tolerance to LPS remain unknown. LPS causes phosphorylation and degradation of inhibitory kappaB-alpha (IkappaB-alpha), releasing nuclear factor-kappaB (NF-kappaB) to activate TNF-alpha gene transcription. We hypothesized that LPS induces supranormal synthesis of myocardial IkappaB-alpha protein and thus renders the myocardium tolerant to subsequent LPS. Rats were challenged with LPS after pretreatment with LPS, dexamethasone, or saline. In saline-pretreated rats, LPS caused a rapid decrease in myocardial IkappaB-alpha protein levels, activation of NF-kappaB, and increased TNF-alpha production. These events were followed by myocardial contractile depression. After the initial decrease in myocardial IkappaB-alpha, IkappaB-alpha protein levels rebounded to a level greater than control levels by 24 h. Dexamethasone pretreatment similarly increased myocardial IkappaB-alpha protein levels. In rats pretreated with either LPS or dexamethasone, myocardial IkappaB-alpha protein levels remained similar to control levels after LPS challenge. The preserved level of myocardial IkappaB-alpha protein was associated with diminished NF-kappaB activation, attenuated myocardial TNF-alpha production, and improved cardiac contractility. We conclude that LPS and dexamethasone upregulate myocardial IkappaB-alpha protein expression and that an increased level of myocardial IkappaB-alpha protein may promote cardiac tolerance to LPS by inhibition of NF-kappaB intranuclear translocation and myocardial TNF-alpha production.

Therapeutic antidysrhythmic and functional protection in human atria.

Approximately 30% of patients suffer supraventricular dysrhythmias after cardiac bypass. While the heart can be constructively preconditioned to maintain function against subsequent ischemic insult using a variety of stimuli across many species, preconditioning in experimental animals is associated with decreased postischemic reperfusion cardiac dysrhythmias. This mode of therapeutic preconditioning has not been previously examined in human atrial myocardium. We therefore hypothesized that preconditioning provides both antidysrhythmic and functional protection to human atria. To study this, human atrial trabeculae were suspended in organ baths, paced at 1 Hz, while force development and ectopy were recorded before and after simulated ischemia. The study consisted of five groups: (1) control trabeculae (n = 12), (2) trabeculae exposed to dysrhythmogenic stimuli (phenylephrine 50 microM and isoproterenol 25 microM (n = 8)), (3) trabeculae exposed to ischemia-reperfusion (I/R) injury and then drug stimulated (n = 10), (4) trabeculae preconditioned with adenosine (ADO 125 microM) then drug stimulated (n = 10), and (5) trabeculae preconditioned with ischemic preconditioning (IPC) then drug stimulated (n = 6) each at end reoxygenation. Differences between groups were assessed using X2 analysis and ANOVA (Bonferroni/Dunn). Results demonstrated that human atrial trabeculae did not exhibit dysrhythmia at baseline or when stimulated with alpha and beta agonists. After I/R, control trabeculae exhibited stimulated reperfusion dysrhythmia, while trabeculae preconditioned with either ADO or transient ischemia exhibited decreased stimulated dysrhythmia (each P < 0.05 vs. I/R). Functionally, I/R decreased developed force (DF) to 16 +/- 2% of baseline (%BDF) while ADO pretreatment increased postischemic DF to 41 +/- 3% BDF (P < 0.05 vs. I/R) while IPC increased DF to 49 +/- 3% BDF (P < 0.05 vs. I/R). We conclude that (1) human atrial trabeculae can ve functionally preconditioned with either ADO or IPC, and (2) protective preconditioning/ cardioprotection does extend to dysrhythmia control and is therapeutically accessible in human atrial myocardium.

The biology of estrogen-mediated repair of cardiovascular injury.

Women appear to be protected from cardiovascular disease until the onset of menopause. Considerable evidence supports the atheroprotective effects of endogenous and supplemental estrogens. The beneficial effects of estrogens on lipid metabolism cannot wholly explain this phenomenon. Accumulating data suggest that estrogen may act at the cellular and molecular level to influence atherogenesis. The purpose of this review is to examine lipid-independent mechanisms of estrogen-mediated atheroprotection after cardiovascular injury.

Interleukin-10 stabilizes inhibitory kappaB-alpha in human monocytes.

Interleukin-10 (IL-10) protects animals from lethal endotoxemia. This beneficial effect is mediated, in part, by inhibition of inflammatory cytokine production, including tumor necrosis factor-alpha (TNF-alpha). Evidence suggests that IL-10 may inhibit activation of the transcription factor nuclear factor-kappaB (NF-kappaB) through an unknown mechanism. NF-kappaB activation in response to inflammatory signals is dependent upon degradation of its associated inhibitory peptide, inhibitory kappaB-alpha (IkappaB-alpha). We hypothesized that IL-10 prevents human monocyte NF-kappaB activation and resultant TNF-alpha production by stabilization of IkappaB-alpha. The purpose of this study was to determine the effect of IL-10 on lipopolysaccharide (LPS)-induced human monocyte TNF-alpha production, NF-kappaB activation, and IkappaB-alpha degradation. Monocytes were isolated from human donors. Cells were stimulated with endotoxin (LPS, 100 ng/mL) with and without human IL-10 (10 ng/mL). Following stimulation, TNF-alpha was measured in cell supernatants by ELISA, NF-kappaB activity by electrophoretic mobility shift assay, and IkappaB-alpha levels by Western blot. We observed that after LPS stimulation of human monocytes, TNF-alpha increased to 798+/-67 pg/mL (p < .001 versus control). IL-10 attenuated LPS-stimulated TNF-alpha production (297+/-54; p < .001 versus LPS alone). After LPS stimulation in human monocytes, IkappaB-alpha protein levels decreased, and NF-kappaB DNA binding increased. IL-10 pretreatment prevented LPS-induced decreases in IkappaB-alpha protein levels and attenuated NF-kappaB DNA binding. IL-10 appears to prevent activation of NF-kappaB by preserving IkappaB-alpha protein levels, leading to a reduction in TNF-alpha release.