Edward J. Pulido

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

In addition to stimulating IFN-γ synthesis, IL-18 also possesses inflammatory effects by inducing synthesis of the proinflammatory cytokines TNF and IL-1β and the chemokines IL-8 and macrophage inflammatory protein-1α. We hypothesized that neutralization of IL-18 would have a beneficial effect in lethal endotoxemia in mice. IL-1β converting enzyme (ICE)-deficient mice, lacking the ability to process mature IL-18 and IL-1β, were completely resistant to lethal endotoxemia induced by LPS derived from either Escherichia coli or Salmonella typhimurium. In contrast, both wild-type and IL-1β−/− mice were equally susceptible to the lethal effects of LPS, implicating that absence of mature IL-18 or IFN-γ but not IL-1β in ICE−/− mice is responsible for this resistance. However, IFN-γ-deficient mice were not resistant to S. typhimurium LPS, suggesting an IFN-γ-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-γ and macrophage inflammatory protein-2 in anti-IL-18-treated animals challenged with E. coli LPS, whereas IFN-γ 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-γ 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.

Thirty years of clinical trials in acute respiratory distress syndrome.

ObjectiveTo systematically review clinical trials in acute respiratory distress syndrome (ARDS). Data SourcesComputerized bibliographic search of published research and citation review of relevant articles. Study SelectionAll clinical trials of therapies for ARDS were reviewed. Therapies that have been compared in prospective, randomized trials were the focus of this analysis. Data ExtractionData on population, interventions, and outcomes were obtained by review. Studies were graded for quality of scientific evidence. Main ResultsLung 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. ConclusionsLung 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.

IL-1 regulates in vivo C—X—C chemokine induction and neutrophil sequestration following endotoxemia

The influx of neutrophils into tissues in response to inflammatory stimuli involves C—X—C chemokines. Interleukin-1 (IL-1) stimulates chemokine production in vitro , but its role in vivo on chemokine production is not as clearly understood. We hypothesized that IL-1 mediates in vivo tissue C—X—C chemokine production induced by systemic lipopolysaccharide (LPS). IL-1 activity was blockedbyIL-1 receptor antagonist (IL-1Ra). Rats were injected with Salmonella typhi LPS (0.5 mg/kg) with and without prior administration of IL-1Ra. Cytokine-induced neutrophil chemoattractant-1 (CINC-1) and macrophage inflammatory protein-2 (MIP-2) protein and mRNA levels, tissue neutrophil accumulation, and indices of organ injury were measured. LPS administration resulted in increased plasma, lung, and liver IL-1β that was decreased by IL-1Ra. LPS also induced an increase in plasma, lung, and liver CINC-1 and MIP-2 protein and mRNA. However, IL-1Ra had no effect on LPS-induced plasma or lung tissue CINC-1 levels. In contrast, IL-1Ra pretreatment did significantly decrease CINC-1 protein expression in the liver (45% decrease) and MIP-2 protein expression in plasma (100% decrease), lung (72% decrease) and liver (100% decrease) compared to LPS-treated controls. Steady-state mRNA levels by Northern blot analysis of both CINC-1 and MIP-2 in lung and liver were similar to the protein findings. Pretreatment with IL-1Ra also resulted in a 47% and 59% decrease in lung and liver neutrophil accumulation, respectively, following LPS. In addition, indices of both lung and liver injury were decreased in animals pretreated with IL-1Ra. In summary, LPS induces IL-1β and MIP-2 expression in the lung and liver, both of which are IL-1 dependent. Although lung neutrophil accumulation in both lung and liver after LPS is also IL-1 mediated, lung CINC-1 levels were unaffected by IL-1Ra. These data suggest that IL-1 regulates tissue chemokine expression and neutrophil accumulation after LPS.

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.

Interleukin-10 stabilizes inhibitory κB-α 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-α (TNF-α). Evidence suggests that IL-10 may inhibit activation of the transcription factor nuclear factor-kB (NF-kB) through an unknown mechanism. NF-kB activation in response to inflammatory signals is dependent upon degradation of its associated inhibitory peptide, inhibitory kB-α (IkB-α). We hypothesized that IL-10 prevents human monocyte NF-kB activation and resultant TNF-α production by stabilization of IkB-α. The purpose of this study was to determine the effect of IL-10 on lipopolysaccharide (LPS)-induced human monocyte TNF-α production, NF-kB activation, and IkB-α 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-α was measured in cell supernatants by ELISA, NF-kB activity by electrophoretic mobility shift assay, and IkB-α levels by Western blot. We observed that after LPS stimulation of human monocytes, TNF-α increased to 798 ± 67 pg/mL (p < .001 versus control). IL-10 attenuated LPS-stimulated TNF-α production (297 ± 54;p <.001 versus LPS alone). After LPS stimulation in human monocytes, IkB-α protein levels decreased, and NF-kB DNA binding increased. IL-10 pretreatment prevented LPS-induced decreases in IkB-α protein levels and attenuated NF-kB DNA binding. IL-10 appears to prevent activation of NF-kB by preserving IkB-α protein levels, leading to a reduction in TNF-α release.

Increased levels of myocardial IκB-α protein promote tolerance to endotoxin

Endotoxin [lipopolysaccharide (LPS)] causes tumor necrosis factor-α (TNF-α)-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 κB-α (IκB-α), releasing nuclear factor-κB (NF-κB) to activate TNF-α gene transcription. We hypothesized that LPS induces supranormal synthesis of myocardial IκB-α 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 IκB-α protein levels, activation of NF-κB, and increased TNF-α production. These events were followed by myocardial contractile depression. After the initial decrease in myocardial IκB-α, IκB-α protein levels rebounded to a level greater than control levels by 24 h. Dexamethasone pretreatment similarly increased myocardial IκB-α protein levels. In rats pretreated with either LPS or dexamethasone, myocardial IκB-α protein levels remained similar to control levels after LPS challenge. The preserved level of myocardial IκB-α protein was associated with diminished NF-κB activation, attenuated myocardial TNF-α production, and improved cardiac contractility. We conclude that LPS and dexamethasone upregulate myocardial IκB-α protein expression and that an increased level of myocardial IκB-α protein may promote cardiac tolerance to LPS by inhibition of NF-κB intranuclear translocation and myocardial TNF-α production.

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.

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.

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 α1-adrenoceptors, protein kinase C (PKC), and cardiac gene expression in cardiac protection. Rats were treated with NE (25 μg/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 α1-adrenoceptor antagonism and PKC inhibition on the protection were determined. Northern analysis was performed to examine cardiac expression of mRNAs encoding α-actin and myosin heavy chain (MHC) isoforms. Immunofluorescent staining was performed to localize PKC-βI 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-βI in cardiac myocytes at 10 min and increased skeletal α-actin and β-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. α1-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-βI may transduce the signal for reprogramming of cardiac gene expression.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.

Chemokines as mediators of diseases related to surgical conditions.

ABSTRACT— 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.