The superoxide anion radical and other reactive oxygen species (ROS) are formed in all aerobic organisms by enzymatic and nonenzymatic reactions. ROS arise in both physiological and pathological processes, but efficient mechanisms have evolved for their detoxification. Similarly, reactive nitrogen intermediates (RNI) have physiological activity, but can also react with different types of molecules, including superoxide, to form toxic products. ROS and RNI participate in the destruction of microorganisms by phagocytes, as in the formation of a myeloperoxidase-hydrogen peroxide-chloride/iodide complex which can destroy many cells, including bacteria. It is known that the cellular production of ROS and RNI is controlled by different mechanisms. These free radicals can react with key cellular structures and molecules, thus altering their biological function. An imbalance between the systems producing and removing ROS and RNI may result in pathological consequences.

The selective cyclooxygenase-2 (COX-2) inhibitor has been reported to have antiinflammatory, neuroprotective, and antioxidant effects in ischemia models. In this study, the authors examined whether a selective COX-2 inhibitor (celecoxib) reduces cerebral inflammation and edema after intracerebral hemorrhage (ICH), and whether functional recovery is sustained with longer treatment. ICH was induced using collagenase in adult rats. Celecoxib (10 or 20 mg/kg) was administered intraperitoneally 20 minutes, 6 hours, and 24 hours after ICH and then daily thereafter. Seventy-two hours after ICH induction, the rats were killed for histologic assessment and measurement of brain edema and prostaglandin E2. Behavioral tests were performed before and 1, 7, 14, 21, and 28 days after ICH. The brain water content of celecoxib-treated rats decreased both in lesioned and nonlesioned hemispheres in a dose-dependent manner. Compared with the ICH-only group, the number of TUNEL-positive, myeloperoxidase-positive, or OX42-positive cells was decreased in the periphery of hematoma and brain prostaglandin E2 level was reduced in the celecoxib-treated group. Celecoxib-treated rats recovered better by the behavioral tests at 7 days after ICH throughout the 28-day period, and the earlier the drug was administered, the better the functional recovery. Evidence of similar effects in an autologous blood-injected model showed that direct collagenase toxicity was not the major cause of inflammation or cell death. These data suggest that celecoxib treatment after ICH reduces prostaglandin E2 production, brain edema, inflammation, and perihematomal cell death in the perihematomal zone and induces better functional recovery.

Myeloperoxidase (MPO), a heme enzyme present in the primary granules of polymorphonuclear leukocytes (PMNs), has been demonstrated to participate in the oxygen-dependent microbicidal activity of these cells. Evidence for the importance of MPO in this role comes in part from studies of normal PMNs treated with the heme enzyme inhibitor, sodium azide. MPO has also been suggested to regulate the respiratory activity of PMNs during phagocytosis. The role of MPO in PMN oxygen metabolism was examined by studying parameters of the respiratory burst of PMNs from a number of unrelated MPO-deficient subjects; in addition, the ability of heme enzyme inhibitors to duplicate the MPO-deficient state was studied by treating normal and MPO-deficient cells with these compounds. MPO-deficient PMNs were found to have a time-dependent hypermetabolic response as assessed by measurement of oxygen consumption, superoxide generation, hydrogen peroxide release, and hexose monophosphate shunt activity. Catabolic pathways for hydrogen peroxide were normal, suggesting the increased recovery of oxygen metabolites reflects increased production rather than decreased catabolism of H2O2. These observations support the concept that MPO may play an important role in terminating the respiratory burst of normal PMNs. The three heme enzyme inhibitors studied--sodium azide, potassium cyanide, and 3-aminotriazole--differed greatly in the degree to which they inhibited various enzymatic systems in the PMN. Nonetheless, as a group, they exerted qualitatively similar effects on oxygen metabolism of normal and of MPO-deficient PMNs. This indicates that many of the mechanisms by which heme enzyme inhibitors influence PMN metabolism are independent of the inhibition of MPO. Conclusions from studies using such treatment of PMNs should be interpreted with caution.

The genes encoding Hoxa9 and Meis1 are transcriptionally coactivated in a subset of acute myeloid leukemia (AML) in mice. In marrow reconstitution experiments, coexpression of both genes produces rapid AML, while neither gene alone generates overt leukemia. Although Hoxa9 and Meis1 can bind DNA as heterodimers, both can also heterodimerize with Pbx proteins. Thus, while their coactivation may result from the necessity to bind promoters as heterodimers, it may also result from the necessity of altering independent biochemical pathways that cooperate to generate AML, either as monomers or as heterodimers with Pbx proteins. Here we demonstrate that constitutive expression of Hoxa9 in primary murine marrow immortalizes a late myelomonocytic progenitor, preventing it from executing terminal differentiation to granulocytes or monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3. This immortalized phenotype is achieved in the absence of endogenous or exogenous Meis gene expression. The Hoxa9-immortalized progenitor exhibited a promyelocytic transcriptional profile, expressing PU.1, AML1, c-Myb, C/EBP alpha, and C/EBP epsilon as well as their target genes, the receptors for GM-CSF, G-CSF, and M-CSF and the primary granule proteins myeloperoxidase and neutrophil elastase. G-CSF obviated the differentiation block of Hoxa9, inducing neutrophilic differentiation with accompanying expression of neutrophil gelatinase B and upregulation of gp91phox. M-CSF also obviated the differentiation block, inducing monocytic differentiation with accompanying expression of the macrophage acetyl-low-density lipoprotein scavenger receptor and F4/80 antigen. Versions of Hoxa9 lacking the ANWL Pbx interaction motif (PIM) also immortalized a promyelocytic progenitor with intrinsic biphenotypic differentiation potential. Therefore, Hoxa9 evokes a cytokine-selective block in differentiation by a mechanism that does not require Meis gene expression or interaction with Pbx through the PIM.

Neurogenic mechanisms have been implicated in the induction of inflammatory bowel disease (IBD). Vanilloid receptor type 1 (TRPV1) has been visualized on nerve terminals of intrinsic and extrinsic afferent neurones innervating the gastrointestinal tract and local administration of a TRPV1 antagonist, capsazepine, reduces the severity of dextran sulphate sodium (DSS)-induced colitis in rats (Gut 2003; 52: 713-9(1)). Our aim was to test whether systemically or orally administered TRPV1 antagonists attenuate experimental colitis induced by 5% DSS in Balb/c mice. Intraperitoneal capsazepine (2.5 mg kg(-1), bid), significantly reduced the overall macroscopic damage severity compared with vehicle-treated animals (80% inhibition, P < 0.05); however, there was no effect on myeloperoxidase (MPO) levels. An experimental TRPV1 antagonist given orally was tested against DSS-induced colitis, and shown to reverse the macroscopic damage score at doses of 0.5 and 5.0 mg kg(-1). Epithelial damage assessed microscopically was significantly reduced. MPO levels were attenuated by approximately 50%, and diarrhoea scores were reduced by as much as 70%. These results suggest that pharmacological modulation of TRPV1 attenuates indices of experimental colitis in mice, and that development of orally active TRPV1 antagonists might have therapeutic potential for the treatment of IBD.

BACKGROUND

Chorioamnionitis frequently associates with preterm delivery and increased amniotic fluid IL-1, and causes fetal lung and systemic inflammation. However, chorioamnionitis is also associated with a paradoxical reduction in the incidence of surfactant deficiency-related respiratory distress syndrome in preterm infants.

OBJECTIVE

To identify the role of IL-1 signaling in the mediation of pulmonary and systemic inflammation and lung maturation in a fetal sheep model of lipopolysaccharide (LPS) induced chorioamnionitis.

METHODS

After confirming the efficacy of recombinant human IL-1 receptor antagonist (rhIL-1ra), fetal sheep were exposed to intraamniotic (IA) injections of Escherichia coli LPS with or without prior IA injections of rhIL-1ra. Preterm lambs were delivered at 82% of term gestation.

RESULTS

rhIL-1ra decreased IA LPS-induced lung inflammation assessed by decreased lung neutrophil and monocyte influx, inducible nitric oxide synthase expression, lung IL-6 and IL-1beta mRNA expression, and airway myeloperoxidase concentrations. rhIL-1ra inhibited IA LPS-induced fetal systemic inflammation assessed by decreased plasma IL-8, protein carbonyls, blood neutrophilia, and the expression of serum amyloid A3 mRNA in the liver. rhIL-1ra also partially blocked the lung maturational effects of IA LPS. Therefore blockade of IL-1 signaling in the amniotic compartment inhibited fetal lung and systemic inflammation and lung maturation in response to LPS-induced chorioamnionitis.

CONCLUSIONS

IL-1 plays a central role in the pathogenesis of chorioamnionitis-induced fetal inflammatory responses.

Inflammation has been recognized as an important factor in cancer development. For the lung, experimental studies with rats, as well as molecular epidemiological studies in humans, have provided evidence that the influx of neutrophils into the airways may be an important process linking inflammation with carcinogenesis. Currently it is believed that the genotoxic capacity of neutrophils is a crucial aetiological factor in this carcinogenic response. In the present review we discuss two major pathways of neutrophil-induced genotoxicity: (i) induction of oxidative DNA damage through release of reactive oxygen species (ROS) and (ii) myeloperoxidase (MPO)-related metabolic activation of chemical carcinogens. So far, direct evidence for a role of neutrophils in pulmonary genotoxicity has largely been derived from in vitro studies using co-cultures of activated neutrophils and target cells. Current evidence from in vivo studies is primarily indirect and additional animal studies are needed to substantiate causality. A further challenge will be to extrapolate results from such studies to humans. Taken together, this will provide a better insight into the role of neutrophils in pulmonary carcinogenicity and may, hence, lead to novel approaches for cancer prevention strategies.

Leukocyte differential counting by flow cytochemistry has shown 28 subjects with partial or complete neutrophil myeloperoxidase (MPO) deficiency in a population of about 60 000 patients screened at a general hospital. Partial (13 patients) or complete (13 patients) MPO deficiency was confirmed by examination of cytochemical stains in 26, biochemical measurement of total enzymatic activity in eight, and flow cytometry in six patients. None had apparent hematologic disorders. Only four patients had infections; of these, two had major systemic infections (one, candidiasis; one, bacteremia). In assays of leukocyte function only minor defects in killing of Staphylococcus aureus by MPO-deficient cells were noted whereas killing of Candida albicans was much more impaired. Family studies in eight patients have shown various degrees of partial or complete MPO deficiency in first-degree relatives of six. The findings indicate that the incidence of MPO deficiency is much higher than previously suspected. Although MPO appears to be necessary for killing of Candida species by neutrophils, the importance of its role in normal antibacterial defense must be re-evaluated.

The present study was designed to examine the effects of lifestyle modification on key contributing factors to atherogenesis, including oxidative stress, inflammation, chemotaxis, and cell adhesion. Obese men (n = 31), 15 of whom had metabolic syndrome, were placed on a high-fiber, low-fat diet in a 3-wk residential program where food was provided ad libitum and daily aerobic exercise was performed. In each subject, pre- and postintervention fasting blood was drawn for circulating levels of serum lipids, glucose and insulin (for estimation of insulin sensitivity), oxidative stress-generating enzyme myeloperoxidase and marker 8-isoprostaglandin F2alpha, the inflammatory protein C-reactive protein, soluble ICAM-1 as an indicator of endothelial activation, sP-selectin as a marker of platelet activation, the chemokine macrophage inflammatory protein-1alpha, and total matrix metalloproteinase-9. Using subject sera and human aortic endothelial cell culture systems, we measured VCAM-1 cell surface abundance and monocyte chemotactic protein-1, nitric oxide, superoxide, and hydrogen peroxide production in vitro by fluorometric detection. Also determined in vitro was serum-induced, monocyte adhesion and monocyte chemotactic activity. After 3 wk, significant reductions (P < 0.05) in body mass index, all serum lipids and lipid ratios, fasting glucose, insulin, homeostasis model assessment for insulin resistance, myeloperoxidase, 8-isoprostaglandin F2alpha, C-reactive protein, soluble ICAM-1, soluble P-selectin, macrophage inflammatory protein-1alpha, and matrix metalloproteinase-9 were noted. In vitro, serum-stimulated cellular VCAM-1 expression, monocyte chemotactic protein-1 production, and fluorometric detection of superoxide and hydrogen peroxide production decreased, whereas a concomitant increase in NO production was noted (all P < 0.01). Additionally, both monocyte adhesion (P < 0.05) and MCA (P < 0.01) decreased. Nine of 15 were no longer positive for metabolic syndrome postintervention. Intensive lifestyle modification may ameliorate novel coronary artery disease risk factors in men with metabolic syndrome factors before reversal of obesity.

Most humans with microscopic polyarteritis and anti-myeloperoxidase (anti-MPO), anti-neutrophil cytoplasmic antibodies (ANCA) develop "pauci-immune" crescentic glomerulonephritis. For dissection of the roles of ANCA and cell-mediated effectors in microscopic polyarteritis, experimental autoimmune anti-MPO glomerulonephritis was induced by immunizing C57BL/6 mice with human MPO. Autoimmunity to mouse MPO (ANCA and CD4+ cell reactivity) was induced. Challenge with anti-glomerular basement membrane globulin resulted in accumulation of neutrophils, CD4+ cells and macrophages, and significant numbers of crescentic glomeruli compared with similarly challenged control-immunized mice. MPO-deficient (Mpo(-/-)) mice immunized with MPO developed similar immune responses to MPO but failed to recruit effector cells to glomeruli or develop significant crescent formation, suggesting that MPO is acting as a planted glomerular autoantigen. Effector CD4+ cell depletion in this model attenuated crescentic glomerulonephritis and effector cell influx without altering ANCA titers. However, B cell-deficient mice, with no ANCA, still developed severe crescentic glomerulonephritis with accumulation of effector cells. Intravital microscopy studies demonstrated that passive transfer of sera from MPO-immunized Mpo(-/-) mice to LPS-primed mice rapidly induced glomerular neutrophil accumulation and release of MPO. These studies provide in vivo evidence in a relevant vascular bed for both humoral and cellular anti-MPO responses as key inducers of injury. ANCA induces glomerular neutrophil infiltration and MPO deposition. Subsequently, anti-MPO CD4+ cells recognize MPO as a planted glomerular antigen and act with macrophages to amplify severe glomerular injury.

Microscopic polyangiitis (MPA) is an ANCA-associated vasculitis that affects small vessels, especially renal glomeruli. We recently demonstrated that the abnormal formation and impaired degradation of neutrophil extracellular traps (NETs) may be crucially involved in the generation of myeloperoxidase (MPO)-ANCA and subsequent development of MPA. This study assessed the formation and regulation of NETs in patients with MPO-ANCA-associated MPA. Peripheral blood samples were obtained from 38 patients with MPO-ANCA-associated MPA, 23 patients with systemic lupus erythematosus (SLE), and 8 healthy controls. IgG eluted from MPO-ANCA-associated MPA sera demonstrated the highest ability to induce NETs, and this ability correlated with disease activity and paralleled ANCA affinity for MPO. Moreover, addition of recombinant human MPO to these IgG samples reduced NET induction. Additionally, MPO-ANCA-associated MPA sera exhibited lower rates of NET degradation that recovered partially upon depletion of IgG. The activity of DNase I, an important regulator of NETs, was also lower in MPO-ANCA-associated MPA and SLE sera. IgG depletion from MPO-ANCA-associated MPA sera partially restored the rate of NET degradation, and addition of DNase I synergistically enhanced this restoration. Addition of anti-MPO antibodies did not inhibit DNase I activity, and some MPO-ANCA-associated MPA sera contained anti-NET antibodies at levels not correlated with MPO-ANCA titers, suggesting the involvement of unidentified autoantibodies as well. The collective evidence suggests a vicious cycle involving MPO-ANCA and the regulation of NETs could be critically involved in the pathogenesis of MPO-ANCA-associated MPA.

Recent studies show that morbid obesity is associated with activation of the innate immune response. Neutrophil activation is a fundamental process in the innate immune response. Therefore, the activation state of neutrophils in severely obese subjects and the effect of bariatric surgery on neutrophil activation was evaluated. Neutrophil activation was assessed by measuring circulating concentrations of myeloperoxidase (MPO) and calprotectin in 37 severely obese and 9 control subjects (enzyme-linked immunosorbent assay). Moreover, membrane expression of CD66b on circulating neutrophils was measured using flow cytometry in a group of seven severely obese and six control subjects. Immunohistochemical detection of MPO was performed in adipose and muscle tissue. Plasma MPO and calprotectin levels were significantly increased in severely obese subjects as compared to healthy controls, 27.1 +/- 10.8 vs. 17.3 +/- 5.5 ng/ml (P < 0.001) and 115.5 +/- 43.5 vs. 65.1 +/- 23.1 ng/ml (P < 0.001) for MPO and calprotectin, respectively. In line, CD66b expression was significantly increased in severely obese individuals, 177.3 +/- 43.7 vs. 129.7 +/- 9.2 (mean fluorescence intensity) (P < 0.01). Bariatric surgery resulted in decreased calprotectin, but MPO plasma levels remained elevated. Adipose and muscle tissue did not contain increased numbers of MPO expressing cells in severely obese individuals. These results point out that circulating neutrophils are activated to a greater extent in severely obese subjects. Our data support the finding that the innate immune system is activated in severely obese individuals. Moreover, because neutrophils have a short life span, this indicates that the chronic inflammatory condition associated with morbid obesity is characterized by a continuous activation of the innate immune system.

The administration of bone mesenchymal stem cells (BMSCs) could reverse experimental colitis, and the predominant mechanism in tissue repair seems to be related to their paracrine activity. BMSCs derived extracellular vesicles (BMSC-EVs), including mcirovesicles and exosomes, containing diverse proteins, mRNAs and micro-RNAs, mediating various biological functions, might be a main paracrine mechanism for stem cell to injured cell communication. We aimed to investigate the potential alleviating effects of BMSC-EVs in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model. Intravenous injection of BMSC-EVs attenuated the severity of colitis as evidenced by decrease of disease activity index (DAI) and histological colonic damage. In inflammation response, the BMSC-EVs treatment significantly reduced both the mRNA and protein levels of nuclear factor kappaBp65 (NF-κBp65), tumor necrosis factor-alpha (TNF-α), induciblenitric oxidesynthase (iNOS) and cyclooxygenase-2 (COX-2) in injured colon. Additionally, the BMSC-EVs injection resulted in a markedly decrease in interleukin-1β (IL-1β) and an increase in interleukin-10 (IL-10) expression. Therapeutic effect of BMSC-EVs associated with suppression of oxidative perturbations was manifested by a decrease in the activity of myeloperoxidase (MPO) and Malondialdehyde (MDA), as well as an increase in superoxide dismutase (SOD) and glutathione (GSH). BMSC-EVs also suppressed the apoptosis via reducing the cleavage of caspase-3, caspase-8 and caspase-9 in colitis rats. Data obtained indicated that the beneficial effects of BMSC-EVs were due to the down regulation of pro-inflammatory cytokines levels, inhibition of NF-κBp65 signal transduction pathways, modulation of anti-oxidant/ oxidant balance, and moderation of the occurrence of apoptosis.

In proteins released from quiescent human neutrophils during incubation, 21% of the methionine (Met) residues were found to be oxidized. However, the portion of oxidized Met in extracellular proteins increased to 66% after stimulating the cells with zymosan and to 75% after stimulation with phorbol myristate acetate (PMA). Generation of such high levels of oxidized Met in native proteins by activated neutrophils has, so far, not been observed. The presence of superoxide dismutase during incubation of PMA-stimulated cells produced a negligible effect on methionine oxidation, while the presence of catalase resulted in a methionine sulfoxide (Met(O)) content of only 28% in the released proteins. It is proposed that the conversion of Met to Met(O) in these proteins predominantly occurs by action of the myeloperoxidase/H2O2/Cl- system in the extracellular space.

Actively phagocytizing polymorphonuclear leukocytes (PMN) emit light or chemiluminescence (CL) which has been shown to be linked to the oxidative activity of the PMN. The measurement of CL has been demonstrated to be a useful tool for the in vitro assessment of intracellular and opsonophagocytic function of PMN. We have increased the sensitivity of the CL measurement by the addition of luminol to the in vitro reaction of PMN, bacteria, and serum. The presence of luminol, which can be oxidized to emit light, amplifies the detection of CL and PMN cellular activity. This amplification effectively reduces the number of PMN that are necessary for assessment of PMN function from 1 x 10(7) to as low as 2 x 10(4) PMN/assay and permits the evaluation of PMN function in severely neutropenic patients (100 PMN/mm3) in whom cellular PMN function has been heretofore extremely difficult to assess by other methodology. When this luminol-dependent CL method was used, three of eight neutropenic leukemic patients with gram-negative septicemia were found to have deficient opsonic activity and/or increased or depressed cellular oxidative activity. Because the initial slope of CL is dependent on the amount of serum and heat-labile factors, this method can also be used effectively as a simple technique for the analysis of specific rates of opsonophagocytosis of various microorganisms. Additionally, this method can detect the cellular PMN abnormalities of chronic granulomatous disease and myeloperoxidase deficiency. The luminol-dependent CL method is a simple, sensitive, reproducible technique that provides useful information about PMN metabolic activity, particularly in studies in which the number of PMN is limited.

OBJECTIVE

The transient receptor potential (TRP) channels TRPV1 and TRPA1 have each been associated with regulation of efferent properties of primary afferent neurons that initiate neurogenic inflammation and are required for the development of inflammatory hyperalgesia. To evaluate the role of these channels in producing pain during pancreatic inflammation, we studied pancreatic nodose ganglion (NG) and dorsal root ganglion (DRG) sensory neurons (identified by content of retrograde tracer) and behavioral outcomes in a mouse model of acute pancreatitis.

METHODS

Pancreatic inflammation was induced by 8 hourly injections of cerulein (50 μg/kg). The extent of inflammation, pancreatic neuron TRP channel expression and function and excitability, and pain-related behaviors were evaluated over the course of the following week.

RESULTS

Histology and myeloperoxidase activity confirmed pancreatic inflammation that was associated with increased excitability and messenger RNA expression of the TRP channels in NG and DRG pancreatic neurons. Calcium imaging of pancreatic NG and DRG neurons from mice given cerulein revealed increased responses to TRP agonists. TRPV1 and TRPA1 antagonists attenuated cerulein-induced pain behaviors and pancreatic inflammation; they had a synergistic effect.

CONCLUSIONS

Pancreatic inflammation significantly increased the expression and functional properties of TRPV1 and TRPA1, as well as the excitability of pancreatic sensory neurons in vagal and spinal pathways. TRP channel antagonists acted synergistically to reverse pancreatic inflammation and associated pain behaviors; reagents that target interactions between these channels might be developed to reduce pain in patients with acute pancreatitis.

Inflammation causes vascular dysfunction and perpetuates proatherosclerotic processes. We hypothesized that a broad panel of inflammatory biomarkers and single nucleotide polymorphisms in inflammatory genes is associated with vascular stiffness. We assessed 12 circulating inflammatory biomarkers (C-reactive protein, fibrinogen, interleukin-6, intercellular adhesion molecule-1, lipoprotein-associated phospholipase-A2 [mass and activity], monocyte chemoattractant protein-1, myeloperoxidase, CD40 ligand, osteoprotegerin, P-selectin, and tumor necrosis factor receptor-II) in relation to tonometry variables (central pulse pressure, mean arterial pressure, forward pressure wave, reflected pressure wave, carotid-femoral pulse wave velocity, and augmentation index) measured in 2409 Framingham Heart Study participants (mean age: 60 years; 55% women; 13% ethnic/racial minorities). Single nucleotide polymorphisms (n=2195) in 240 inflammatory candidate genes were related to tonometry measures in 1036 white individuals. In multivariable analyses, biomarkers explained <1% of any tonometry measure variance. Applying backward elimination, markers related to tonometry (P<0.01) were as follows: tumor necrosis factor receptor-II (inversely) with mean arterial pressure; C-reactive protein (positively) and lipoprotein-associated phospholipase-A2 (inversely) with reflected pressure wave; and interleukin-6 and osteoprotegerin (positively) with carotid-femoral pulse wave velocity. In genetic association analyses, lowest P values (false discovery rate <0.50) were observed for rs10509561 (FAS), P=6.6x10(-5) for central pulse pressure and rs11559271 (ITGB2), P=1.1x10(-4) for mean arterial pressure. These data demonstrate that, in a community-based sample, circulating inflammatory markers tumor necrosis factor receptor-II (mean arterial pressure), C-reactive protein, lipoprotein-associated phospholipase-A2 activity (reflected pressure wave), interleukin-6, and osteoprotegerin (carotid-femoral pulse wave velocity) were significantly but modestly associated with measures of arterial stiffness and wave reflection. Additional studies are needed to determine whether variation in inflammatory marker genes is associated with tonometry measures.

Hydrogen sulfide (H2S) is a naturally occurring gaseous transmitter, which may play important roles in normal physiology and disease. Here, we investigated the role of H2S in the organ injury caused by severe endotoxemia in the rat. Male Wistar rats were subjected to acute endotoxemia (Escherichia coli lipopolysaccharide (LPS) 6 mg kg(-1) intravenously (i.v.) for 6 h) and treated with vehicle (saline, 1 ml kg(-1) i.v.) or DL-propargylglycine (PAG, 10-100 mg kg(-1) i.v.), an inhibitor of the H2S-synthesizing enzyme cystathionine-gamma-lyase (CSE). PAG was administered either 30 min prior to or 60 min after the induction of endotoxemia. Endotoxemia resulted in circulatory failure (hypotension and tachycardia) and an increase in serum levels of alanine aminotransferase and aspartate aminotransferase (markers for hepatic injury), lipase (indicator of pancreatic injury) and creatine kinase (indicator of neuromuscular injury). In the liver, endotoxemia induced a significant increase in the myeloperoxidase (MPO) activity, and in the expression and activity of the H2S-synthesizing enzymes CSE and cystathionine-beta-synthase. Administration of PAG either prior to or after the injection of LPS dose-dependently reduced the hepatocellular, pancreatic and neuromuscular injury caused by endotoxemia, but not the circulatory failure. Pretreatment of rats with PAG abolished the LPS-induced increase in the MPO activity and in the formation of H2S and in the liver. These findings support the view that an enhanced formation of H2S contributes to the pathophysiology of the organ injury in endotoxemia. We propose that inhibition of H2S synthesis may be a useful therapeutic strategy against the organ injury associated with sepsis and shock.

Sepsis is associated with a widespread production of proinflammatory cytokines and various oxidant species. Activation of the enzyme poly(ADP-ribose) polymerase (PARP) has been shown to contribute to cell necrosis and organ failure in various diseases associated with inflammation and reperfusion injury. The aim of the current study was to elucidate the role of PARP activation in the multiple organ dysfunction complicating sepsis in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Mice genetically deficient in PARP (PARP-/-) and their wild-type littermates (PARP+/+) were subjected to CLP. After 12 and 24 h, the proinflammatory cytokines TNF-alpha and IL-6, as well as the anti-inflammatory cytokine IL-10, and nitrite/nitrate were measured in plasma samples. Organs were harvested for the measurement of myeloperoxidase (MPO) and malondialdehyde (MDA) levels, and immunohistochemical staining for nitrotyrosine and poly(ADP ribose) was performed in gut sections. PARP-/- mice, and their wild-type littermate showed a similar time-dependent increase in plasma nitrite/nitrate and in gut and lung MDA content, as well as the presence of nitrotyrosine in the gut. In contrast to wild-type mice showing a PARP activation in the gut, PARP-/- mice had no staining for poly(ADP ribose). PARP-/- mice had significantly lower plasma levels of TNF-alpha, IL-6, and IL-10, and they exhibited a reduced degree of organ inflammation, indicated by decreased MPO activity in the gut and lung. These effects were associated with a significant improvement in the survival of CLP in PARP-/- mice. Thus, PARP activation has an important role in systemic inflammation and organ damage in the present model of polymicrobial septic shock.

Acute renal failure (ARF) in response to ischemia-reperfusion is thought to be associated with neutrophil infiltration. Neutrophil recruitment depends on adhesion molecules, including P-selectin. Our study sought to characterize the role of P-selectin in ischemia-reperfusion (I/R) -induced acute renal failure (ARF). In wild-type (wt) and P-selectin-deficient (P-/-) mice (both C57BL/6), ARF was induced by 32 min bilateral renal ischemia, followed by reperfusion (I/R). Wt showed a 12- and 20-fold increase in creatinine at 24 and 48 h after I/R, respectively. Similar changes were seen in blood urea nitrogen (BUN). By contrast, in P-/- creatinine and BUN increased only moderately (fourfold over sham). In wt, renal myeloperoxidase activity, indicating neutrophil infiltration, peaked after 24 h (19-fold over sham). This was significantly attenuated in P-/- (fivefold over sham). Western blot analysis revealed maximum P-selectin expression 12 h after I/R in wt. Immunostaining detected P-selectin in glomerular endothelium and in platelets adherent in glomerular and peritubular vessels. Postischemic injection of P-selectin antibody at 10 min after reperfusion, but not isotype control antibody, protected wt from ARF similar to the protection seen in P-/-. We conclude that blocking P-selectin even after onset of reperfusion protects mice from I/R-induced ARF, suggesting potential therapeutic strategies aimed at blocking P-selectin.

Enhanced nitric oxide (NO) generation by stimulated NO synthase (NOS) activity may, through its oxidative metabolism contribute to tissue injury in experimental colitis. In this study the possible amelioration of experimental colitis by NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS activity, was evaluated. Colitis was induced in rats by intracolonic administration of 30 mg trinitrobenzene sulphonic acid (TNB) dissolved in 0.25 ml 50% ethanol or by flushing the colon of capsaicin pretreated rats with 2 ml of 5% acetic acid. In several experiments, L-NAME 0.1 mg/ml was added to the drinking water at the time of colitis induction with TNB or seven days before acetic acid treatment. Rats were killed at various time intervals after induction of colitis. A 10 cm distal colonic segment was isolated, weighed, lesion area measured, and explants organ cultured for 24 hours for determination of NO generation by the Greiss reaction. The rest of the mucosa was scraped for determination of myeloperoxidase and NOS activities and leukotriene generation. In TNB treated rats mean arterial pressure was also determined up to 72 hours after damage induction, with or without cotreatment with nitroprusside. L-NAME significantly decreased the extent of tissue injury in TNB treated rats. Seven days after TNB treatment lesion area was reduced by 55%, colonic weight by 37%, and myeloperoxidase and NOS activity by 59% and 42%, respectively. Acetic acid induced colitis in capsaicin pretreated rats was also significantly decreased by L-NAME. Twenty four hours after acetic acid treatment lesion area was reduced by 61%, colonic weight by 21% and NOS activity by 39%. Mean (SEM) arterial blood pressure in TNB+L-NAME treated rats was 37.6 (8.1) mm Hg higher than in TNB treated rats, an effect that was only partially abolished by nitroprusside. These results show that inhibition of NO synthesis by an L-arginine analogue significantly ameliorates the extent of tissue injury in two models of experimental colitis, an effect that is not due only to its vasoconstrictor properties. Modulation of NO generation may be a novel therapeutic approach in inflammatory bowel disease.

Although neutrophils are strongly implicated in eliminating pathogens, excessive recruitment may cause tissue damage. Therefore, reducing cell influx during an inflammatory process may be a potential target for treating inflammatory bowel diseases (IBD). As CXCR2 is involved in neutrophil migration, this study aimed to evaluate whether the systemic therapeutic treatment with selective CXCR2 antagonist SB225002 ameliorates experimental colitis, which was induced in mice by 2,4,6-trinitrobenzene sulfonic acid (TNBS). After colitis establishment (24 h), mice were treated with SB225002. At later time-points, up to 72 h, mice were monitored for body weight loss and overall mortality. At the time of sacrifice, colonic tissues were scored for macro- and microscopic damage, and cytokine levels, myeloperoxidase (MPO) activity, and protein expression were analyzed. TNBS administration induced macro- and microscopic damage in colon tissue, leading in most cases to animal death. Curative treatment with SB225002 significantly reduced all of the parameters analyzed, leading to an improvement of inflammatory signs. SB225002 reduced neutrophil influx, MPO activity, IL-1beta, MIP-2, and keratinocyte-derived chemokine (KC) levels and the expression of vascular endothelial growth factor, inducible NO synthase, and cyclooxygenase-2 proteins into the colon tissue. Levels of IL-4 and IL-10 were increased significantly in the colons of animals treated with SB225002. Additionally, curative treatment with mouse anti-KC significantly reduced MPO activity and colonic damage. These results taken together demonstrate that a selective blockade of CXCR2 consistently reduced TNBS-induced colitis, suggesting that the use of SB225002 is a potential therapeutic approach for the treatment of IBD and other related inflammatory disorders.

BACKGROUND

Evidence is emerging that the endoplasmic reticulum (ER) participates in initiation of apoptosis induced by the unfolded protein response and by aberrant Ca(++) signaling during cellular stress such as ischemia/reperfusion injury (I/R injury). ER-induced apoptosis involves the activation of caspase-12 and C/EBP homologous protein (CHOP), and the shutdown of translation initiated by phosphorylation of eIF2alpha. Sodium 4-phenylbutyrate (PBA) is a low molecular weight fatty acid that acts as a chemical chaperone reducing the load of mutant or unfolded proteins retained in the ER during cellular stress and also exerting anti-inflammatory activity. It has been used successfully for treatment of urea cycle disorders and sickle cell disease. Thus, we hypothesized that PBA may reduce ER-induced apoptosis triggered by I/R injury to the liver.

METHODS

Groups of male C57BL/6 mice were subjected to warm ischemia (70% of the liver mass, 45 minutes). Serum aspartate aminotransferase was assessed 6 hours after reperfusion; apoptosis was evaluated by enzyme-linked immunosorbent assays of caspase-12 and plasma tumor necrosis factor alpha, Western blot analyses of eIF2alpha, and reverse transcriptase-polymerase chain reaction of CHOP expression.

RESULTS

A dose-dependent decrease in aspartate aminotransferase was demonstrated in mice given intraperitoneal PBA (1 hour before and 12 hours after reperfusion), compared with vehicle-treated controls; this effect was associated with reduced pyknosis, parenchymal hemorrhages, and neutrophil infiltrates in PBA-treated mice, compared with controls. In a lethal model of total liver I/R injury, all vehicle-treated controls died within 3 days after reperfusion. In contrast, 50% survival (>30 days) was observed in animals given PBA. The beneficial effects of PBA were associated with a greater than 45% reduction in apoptosis, decreased ER-mediated apoptosis characterized by significant reduction in caspase-12 activation, and reduced levels of both phosphorylated eIF2alpha and CHOP. Significant reductions in plasma levels of tumor necrosis factor alpha and liver myeloperoxidase content were demonstrated after PBA treatment.

CONCLUSIONS

Reduction in ER stress-induced hepatocellular injury was achieved by the administration of PBA. Targeting the ER-associated cell death pathway might offer a novel approach to reduce I/R injury to the liver.

Recent studies demonstrated that activation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) by oxidant-mediated DNA damage is an important pathway of tissue injury in conditions associated with oxidative stress. Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with the phenanthridinone PARP inhibitor PJ-34, we now demonstrate an essential role of PARP-1 in the development of pulmonary inflammation induced by lipopolysaccharide (LPS). PARP-1+/+ and PARP-1-/- mice received an intratracheal instillation of LPS (50 microg), followed after 24 h by bronchoalveolar lavage to measure the cytokines TNF-alpha, IL-1beta, and IL-6, the chemokines MIP-1alpha and MIP-2, leukocyte counts and myeloperoxidase activity (neutrophil accumulation), protein content (high permeability edema), and nitrite/ nitrate (nitric oxide production). Malondialdehyde (an index of lipid peroxidation) was measured in lung tissue. Similar experiments were conducted in BALB/c mice treated with PJ-34 or vehicle. The absence of functional PARP-1 reduced LPS-induced increases of cytokines and chemokines, alveolar neutrophil accumulation, lung hyperpermeability, NO production, and lipid peroxidation. Histological analysis revealed attenuated lung damage after PARP inhibition. Our findings support a mechanistic role of PARP-1 in the regulation of LPS-induced lung inflammation. Pharmacological inhibition of PARP may be useful in clinical conditions associated with overwhelming lung inflammation.