Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NF-κB p65 signal pathway.

Tea polyphenols (TP) was investigated in rats for its protective effect on renal ischemia/reperfusion injury (RIRI). Rats were randomized into groups as follows: (I) sham group (n=10); (II) RIRI group (n=10); (III) RIRI+TP (100mg/kg) group (n=5); (IV) RIRI+TP (200mg/kg) group (n=5); (V) RIRI+TP+ Astragalus mongholicus aqueous extract (AMAE) (300 mg/kg+100mg/kg) group (n=5). For the IRI+TP groups, rats were orally given with tea polyphenols (100, 200 and 300 mg/kg body weight) once daily 10 days before induction of ischemia, followed by renal IRI. For the sham group and RIRI group, rats were orally given with equal volume of saline once daily 10 days before induction of ischemia, followed by renal IRI. Results showed that tea polyphenol pretreatment significantly suppressed ROS level and MDA release. On the other hand, in rats subjected to ischemia-reperfusion, the activities of endogenous antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) showed recovery, whereas the levels of urea nitrogen and serum creatinine were reduced by administration of tea polyphenols orally for 10 days prior to ischemia-reperfusion. Moreover, tea polyphenol pretreatment significantly decreased TLR4 and NF-κB p65 protein expression levels in RIRI rats. At the same time, tea polyphenol pretreatment attenuated the increased level of serum IL-1ß, IL-6, ICAM-1 and TNF-α, and enhanced IL-10 production in RIRI rats. Furthermore, tea polyphenol pretreatment significantly decreased renal epithelial tubular cell apoptosis induced by renal ischemia/reperfusion, alleviating renal ischemia/reperfusion injury. These results cumulatively indicate that tea polyphenol pretreatment could suppress the TLR4/NF-κB p65 signaling pathway, protecting renal tubular epithelial cells against ischemia/reperfusion-induced apoptosis, which implies that antioxidants may be a potential and effective agent for prevention of the ischemic/reperfusion injury through the suppression extrinsic apoptotic signal pathway induced by TLR4/NF-κB p65 signal pathway. Moreover, supplement of AMAE can increased renal protection effect of TP.

Baccharis trimera improves the antioxidant defense system and inhibits iNOS and NADPH oxidase expression in a rat model of inflammation.

Acetaminophen is a common analgesic and antipyretic compound which, when administered in high doses, has been associated with significant morbidity and mortality, secondary to hepatic toxicity. Although this may be due to a direct interaction of reactive acetaminophen metabolites with hepatocyte proteins, recent studies have suggested that reactive species produced by neutrophils also contribute to the pathophysiological process. Researches on the chemical composition of B. trimera show that this plant has bioactive compounds such as flavonoids, related to the organism's protection against free radicals. Therefore, in the present study, using Fischer rats, the effect of B. trimera on the antioxidant defense system, the production of nitric oxide (NO) and on the expression of nitric oxide synthase (iNOS), superoxide dismutase (SOD), catalase (CAT) and of the subunits of the NADPH oxidase in neutrophils was evaluated in a model of phagocytosis induced by zimosan (ZC3b) and in a model of inflammation induced by acetaminophen. The results show that the treatment with B. trimera improves the defense system of antioxidant and restores the balance ROS / NO that is altered in the inflammatory process induced by APAP. In conclusion, B. trimera extracts exert antioxidant properties by scavenging ROS and decrease the expression of genes responsible by reactive species production in neutrophils.

Effects of p38 mitogen-activated protein kinase inhibition on anti-neutrophil cytoplasmic autoantibody pathogenicity in vitro and in vivo.

OBJECTIVE: To determine whether inhibition of p38 mitogen-activated protein kinase (p38MAPK) reduces the pathogenicity of anti-neutrophil cytoplasmic autoantibodies (ANCAs) in vitro and in vivo. METHODS: The effects of the p38MAPK-specific inhibitor AR-447 were studied in vitro using neutrophil respiratory burst and degranulation assays, and in lipopolysaccharide (LPS)-stimulated human glomerular endothelial cells. In vivo, p38MAPK inhibition was investigated in a mouse anti-myeloperoxidase (MPO) IgG/LPS glomerulonephritis model. Mice were treated orally with AR-447 daily, starting before (pretreatment group) or 24 h after disease onset (treatment group), and killed after 1 or 7 day(s). RESULTS: In vitro, AR-447 diminished neutrophil respiratory burst and degranulation induced by patient-derived MPO-ANCA and proteinase 3 (Pr3)-ANCA. In glomerular endothelial cells, AR-447 reduced LPS-induced secretion of IL-6 and IL-8, but not of MCP-1. In mice, pretreatment with AR-447 reduced albuminuria 1 day after induction of glomerulonephritis. After 7 days, no effects on urinary abnormalities were observed upon AR-447 pretreatment or treatment. Also, glomerular neutrophil accumulation was not diminished. In contrast, glomerular macrophage accumulation and the formation of glomerular crescents was significantly reduced by AR-447 pretreatment (vehicle: 12.5 ± 5.6% crescentic glomeruli; AR-447: 7.7 ± 2.7%) and treatment (vehicle 14.6 ± 1.8%; AR-447 6.0 ± 3.4%) at 7 days. CONCLUSION: This study shows that p38MAPK inhibition markedly reduces ANCA-induced neutrophil activation in vitro. In vivo, p38MAPK inhibition partly reduced crescent formation when the drug was administered prior to disease induction and after disease onset, suggesting that besides p38MAPK activity other signalling pathways contribute to the disease activity.

Sphingosine 1-phosphate enhances Fc gamma receptor-mediated neutrophil activation and recruitment under flow conditions.

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that is released by platelets and endothelial cells and has been implicated in diverse biological functions. We hypothesized that S1P may influence immune complex-mediated polymorphonuclear neutrophil activation. Using flow cytometry and fluorescence spectrometry, we found that exogenous addition of S1P led to an enhanced polymorphonuclear neutrophil Fcgamma receptor-mediated rise in intracellular Ca(2+) and reactive oxygen species generation in a pertussis toxin-independent manner, while having only a small effect by itself. Thus, S1P amplifies a positive feedback loop where Fcgamma receptor-mediated rises in Ca(2+) and reactive oxygen species are interdependent, with reactive oxygen species acting to increase tyrosine phosphorylation and activity of upstream signaling intermediates. S1P augmentation of Fcgamma receptor signaling translates to downstream functional consequences, including shape change and recruitment to endothelial surfaces coated with suboptimal levels of immune complexes. Taken together, S1P from activated platelets or endothelial cells may serve to amplify leukocyte recruitment and tissue injury at sites of immune complex deposition in vasculitis.

Inhibitory effect of glycyrrhizin on the neutrophil-dependent increase of R5 HIV replication in cultures of macrophages.

It has been described that polymorphonuclear neutrophils (PMNs) enhance the replication of CC-chemokine receptor 5/macrophage-tropic (R5) HIV in cultures of monocyte-derived macrophages (MDMs). In this study, the inhibitory effect of glycyrrhizin (GL) on R5 HIV replication influenced by PMNs was investigated in MDM cultures. The replication of R5 HIV in MDMs was greatly enhanced when cells were co-cultured with freshly isolated PMNs (syngeneic to MDMs). When GL was added to this culture, however, the viral replication enhanced by PMNs was completely inhibited. CCL2 and interleukin 10 (IL-10) were produced in cultures of PMNs exposed to R5 HIV, and the replication of R5 HIV was greatly enhanced in MDM cultures supplemented with a mixture of recombinant CCL2 and IL-10. However, CCL2 and IL-10 were not produced by PMNs exposed to R5 HIV, when GL was added to the cultures. In the presence of GL, these soluble factors were not detected in co-cultures of MDMs and PMNs exposed to R5 HIV. In addition, the replication of R5 HIV in MDMs stimulated with CCL2 and IL-10 was not directly influenced by GL. These results indicated that GL suppresses the PMN-dependent increase of R5 HIV replication in MDMs through inhibiting CCL2/IL-10 production by PMNs stimulated with R5 HIV.

Potent inhibition of superoxide anion production in activated human neutrophils by isopedicin, a bioactive component of the Chinese medicinal herb Fissistigma oldhamii.

Fissistigma oldhamii is widely used in traditional Chinese medicine to treat rheumatoid arthritis. Activation of neutrophils is a key feature of inflammatory diseases. Herein, the anti-inflammatory functions of isopedicin, a flavanone derived from F. oldhamii, and its underlying mechanisms were investigated in human neutrophils. Isopedicin potently and concentration-dependently inhibited superoxide anion (O(2)(*)(-)) production in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils with an IC(50) value of 0.34+/-0.03 microM. Furthermore, isopedicin displayed no superoxide-scavenging ability, and it failed to alter subcellular NADPH oxidase activity. The inhibitory effect of isopedicin on O(2)(*)(-) production was reversed by protein kinase A (PKA) inhibitors. Moreover, isopedicin increased cAMP formation and PKA activity in FMLP-activated human neutrophils, which occurred through the inhibition of phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function. In addition, isopedicin reduced FMLP-induced phosphorylation of extracellular regulated kinase and c-Jun N-terminal kinase, which was reversed by the PKA inhibitor. In contrast, isopedicin failed to alter FMLP-induced phosphorylation of p38 mitogen-activated protein kinase and calcium mobilization. In summary, these results demonstrate that inhibition of O(2)(*)(-) production in human neutrophils by isopedicin is associated with an elevation of cellular cAMP and activation of PKA through its inhibition of cAMP-specific PDE.

Hypothalamic proline-rich polypeptide is a regulator of oxidative burst in human neutrophils and monocytes.

OBJECTIVE: The effects of proline-rich polypeptide (PRP) isolated from neurosecretory granules of bovine neurohypophysis produced by nuclei supraopticus and paraventricularis on phagocytosis, bacterial intracellular killing and oxidative burst induction in normal human cells and inflammatory cells from patients with Behçet's disease (BD), i.e. peripheral blood neutrophils and monocytes, were investigated. METHODS: Intracellular killing of Staphylococcus aureus by neutrophils and monocytes of normal controls and BD patients, phagocytic activity as well as spontaneous and N-formyl-Met-Leu-Phe (fMLP)- or phorbol 12-myristate 13-acetate (PMA)-induced activation of their respiratory burst were determined by quantitative flow cytometry using highly specific fluorescence probes. RESULTS: PRP does not affect human peripheral blood neutrophil and monocyte phagocytosis but dramatically enhances spontaneous or fMLP- and PMA-induced oxidative burst as well as the intracellular killing of S. aureus. PRP induced the upregulation of the spontaneous or fMLP- and PMA-induced oxidative burst in normal PMNs and monocytes; the number of inflammatory BD cells did neither increase further nor undergo spontaneous or PMA-stimulated oxidative burst. In BD patients, increased spontaneous production of reactive oxygen intermediates (ROIs) by neutrophils and monocytes is characterized by impaired intracellular protein-kinase-C (PKC)-dependent oxidative burst regulation as well as over-regulation of chemotaxis/inflammation-mediated respiratory burst induction. PRP restores rather the impaired intracellular PKC-dependent regulation of ROI production in inflammatory diseased cells than the chemotaxis/induction of the inflammation-mediated respiratory burst. CONCLUSION: We demonstrated the regulatory role for PRP on oxidative burst in neutrophils and monocytes from normal controls and BD patients. Our results suggest that PRP differentially affects both chemotaxis- and PKC-dependent oxidative burst in normal and inflammatory cells from patients.

Regulation of human polymorphonuclear leukocytes functions by the neuropeptide pituitary adenylate cyclase-activating polypeptide after activation of MAPKs.

Anti-inflammatory activities of pituitary adenylate cyclase-activating protein (PACAP) are mediated in part through specific effects on lymphocytes and macrophages. This study shows that in human polymorphonuclear neutrophils (PMNs), PACAP acts as a proinflammatory molecule. In PMNs, vaso-intestinal peptide/PACAP receptor 1 (VPAC-1) was the only receptor found to be expressed by RT-PCR. Using VPAC-1 Ab, we found that VPAC-1 mRNA was translated into proteins. In PMNs, PACAP increases cAMP, inositol triphosphate metabolites, and calcium. It activates two of the three members of the MAPK superfamily, the ERK and the stress-activated MAPK p38. U73122, an inhibitor of phospholipase C (PLC), inhibits PACAP-induced ERK activation, whereas p38 MAPK phosphorylation was unaffected. Using specific pharmalogical inhibitors of ERK (PD098059) and p38 MAPK (SB203580), we found that PACAP-mediated calcium increase was ERK and PLC dependent and p38 independent. PACAP primes fMLP-associated calcium increase; it also primes fMLP activation of the respiratory burst as well as elastase release, these last two processes being ERK and PLC dependent and p38 MAPK independent. PACAP also increases membrane expression of CD11b and release of lactoferrin and metallo proteinase-9 (MMP-9). These effects were PLC dependent (CD 11b, lactoferrin, MMP-9), ERK dependent (CD 11b, lactoferrin, MMP-9), and p38 dependent (CD11b, lactoferrin). We conclude that PACAP is a direct PMN activator as well as an effective PMN priming agent that requires PLC, ERK, and p38 MAPK activities.

A taurine-supplemented vegan diet may blunt the contribution of neutrophil activation to acute coronary events.

Neutrophils are activated in the coronary circulation during acute coronary events (unstable angina and myocardial infarction), often prior to the onset of ischemic damage. Moreover, neutrophils infiltrate coronary plaque in these circumstances, and may contribute to the rupture or erosion of this plaque, triggering thrombosis. Activated neutrophils secrete proteolytic enzymes in latent forms which are activated by the hypochlorous acid (HOCl) generated by myeloperoxidase. These phenomena may help to explain why an elevated white cell count has been found to be an independent coronary risk factor. Low-fat vegan diets can decrease circulating leukocytes--neutrophils and monocytes--possibly owing to down-regulation of systemic IGF-I activity. Thus, a relative neutropenia may contribute to the coronary protection afforded by such diets. However, vegetarian diets are devoid of taurine - the physiological antagonist of HOCl--and tissue levels of this nutrient are relatively low in vegetarians. Taurine has anti-atherosclerotic activity in animal models, possibly reflecting a role for macrophage-derived myeloperoxidase in the atherogenic process. Taurine also has platelet-stabilizing and anti-hypertensive effects that presumably could reduce coronary risk. Thus, it is proposed that a taurine-supplemented low-fat vegan diet represents a rational strategy for diminishing the contribution of activated neutrophils to acute coronary events; moreover, such a regimen would work in a number of other complementary ways to promote cardiovascular health. Moderate alcohol consumption, the well-tolerated drug pentoxifylline, and 5-lipoxygenase inhibitors--zileuton, boswellic acids, fish oil--may also have potential in this regard.

Inhibition of enteral enzymes by enteroclysis with nafamostat mesilate reduces neutrophil activation and transfusion requirements after hemorrhagic shock.

BACKGROUND: The gut origin of the inflammatory response in trauma patients has been difficult to define. "In vivo" generation of neutrophil-activating factors by gut proteases may be a cause of multiorgan failure after hemorrhagic shock, and can be prevented with the serine protease inhibitor nafamostat mesilate (Futhan). The objective of this study was to determine the effect of nafamostat mesilate given by enteroclysis on enteric serine protease activity, neutrophil activation, and transfusion requirements during hemorrhagic shock. METHODS: Sixteen pigs weighing 21 to 26 kg were divided into control and treatment groups. A laparotomy was performed under anesthesia, and catheters were placed in the duodenum, midjejunum, and terminal ileum. Pigs were bled 30 mL/kg over 30 minutes and maintained at a mean arterial pressure of 30 mm Hg for 60 minutes. Shed blood was then used to maintain a mean arterial pressure of 45 mm Hg for another 3 hours. Treated animals received 100 mL/kg of 0.37 mmol/L nafamostat mesilate in GoLYTELY through the duodenal catheter at 1 L/h. Control animals received GoLYTELY only. Samples of enteral content and blood were taken at baseline, after shock, and at 30-minute intervals during resuscitation. Animals were killed after 3 hours of resuscitation. Enteral trypsin-like activity at the three gut sites was measured by spectrophotometry. Activation of naive human neutrophils by pig plasma was measured by the percentage of cells having pseudopods larger than 1 microm on microscopy. Lung, liver, and small bowel were analyzed by histology and myeloperoxidase assay. RESULTS: Both control and nafamostat mesilate-treated groups had significant reductions in protein and protease levels in the duodenum during enteroclysis; however, only nafamostat mesilate-treated animals had persistent suppression of protease activity throughout the experiment. Nafamostat mesilate-treated animals had a lower transfusion requirement of shed blood, 18.1 +/- 4.5 mL/kg versus 30 +/- 0.43 mL/kg (p = 0.002). Nafamostat mesilate-treated animals had significantly less neutrophil activation than controls at 150 minutes after resuscitation (33.7 +/- 6.48% vs. 42.4 +/- 4.57%,p = 0.01) and 180 minutes after resuscitation (31.1 +/- 3.31% vs. 46.9 +/- 4.53%, p = 0.0002). Lung myeloperoxidase activity was lower in nafamostat mesilate-treated animals (0.31 +/- 0.14) than in control animals (0.16 +/- 0.04, p = 0.04). Histology of liver and small intestine showed less injury in nafamostat mesilate-treated animals. CONCLUSION: Nafamostat mesilate given by means of enteroclysis with GoLYTELY significantly reduces enteral protease levels, leukocyte activation, and transfusion requirements during resuscitation from hemorrhagic shock. This strategy may have clinical promise.

Triggering receptor expressed on myeloid cells-1 in neutrophil inflammatory responses: differential regulation of activation and survival.

Polymorphonuclear neutrophils (PMN) are crucial in the innate host defense by their ability to rapidly accumulate in inflamed tissues and clear a site of infection from microbial pathogens by their potent effector mechanisms. The triggering receptor expressed on myeloid cells (TREM)-1 is a recently described activating receptor on PMN with an important role in inflammation. However, the effects of TREM-1 stimulation on a cellular level remain to be further defined. To characterize TREM-1-mediated activation of human PMN, we evaluated the effect of receptor ligation on PMN effector functions. Activation via TREM-1 induces immediate degranulation of neutrophilic granules resulting in the release of IL-8, respiratory burst, and phagocytosis. TREM-1 ligation synergizes with the activation by the Toll-like receptors (TLR) ligands LPS, Pam(3)Cys, and R-848. In contrast, no synergy between TREM-1- and TLR-mediated stimulation was observed concerning PMN survival, whereas TLR-mediated stimuli protect PMN from apoptosis, concurrent TREM-1 activation neutralizes these anti-apoptotic effects. These results give a new perspective for the regulation of neutrophil inflammatory responses emphasizing the importance of TREM-1 in innate immunity.

Differential response of lymphocytes and neutrophils to high intensity physical activity and to vitamin C diet supplementation.

We have determined the effects of chronic vitamin C intake on neutrophil and lymphocyte antioxidant defences during the acute phase immune response induced by intense exercise. Blood samples were taken from 16 voluntary athletes in basal conditions, both immediately after and 1 h after a duathlon competition. Sportsmen's nutrient intakes were determined before the competition. After determining the basal plasmatic ascorbate levels, the results were analysed taking into account the vitamin C intake and their plasmatic levels. Two groups were constituted, the vitamin C supplemented group and the control group, with the dietary vitamin C intake as the only statistical difference between groups. The duathlon competition induced a significant neutrophilia, which was higher in the supplemented group. Lymphocyte antioxidant enzyme activities increased after the competition, with a higher increase in SOD activity in the control group than in the supplemented one. The competition decreased neutrophil antioxidant enzyme activities and neutrophil ascorbate concentration. The decrease in the SOD activity in the supplemented group was higher than in the control group. Finally, the duathlon competition increased the expression of MAC-1 neutrophil adhesion molecule in the supplemented group. High vitamin C intake influenced the response of neutrophils and lymphocytes to oxidative stress induced by exercise, increasing the neutrophil activation.

A low-molecular-weight fraction of bovine colostrum and milk enhances the oxidative burst activity of polymorphonuclear leukocytes.

Bovine colostrum and milk contain many immunomodulatory components. The low-molecular-weight fraction (< 10 kDa) was separated from colostrum and milk by gel filtration chromatography, and its effect on the oxidative burst of bovine polymorphonuclear leukocytes (PMNL) was investigated in vitro. The oxidative burst activity induced by Staphylococcus aureus was considerably enhanced when PMNLs were incubated with this low-molecular-weight fraction. However, phorbol 12-myristate 13-acetate did not trigger a burst after priming with this fraction. The oxidative burst activity enhanced by this fraction was reduced after heating. These results confirmed that a low-molecular-weight substance(s) of less than 10 kDa, present in bovine milk and colostrum, enhances the oxidative burst activity of PMNL.

A novel anionic modification of N-glycans on mammalian endothelial cells is recognized by activated neutrophils and modulates acute inflammatory responses.

We previously reported an unusual carboxylated modification on N:-glycans isolated from whole bovine lung. We have now raised IgG mAbs against the modification by immunization with biotinylated aminopyridine-derivatized glycans enriched for the anionic species and screening for Abs whose reactivities were abrogated by carboxylate neutralization of bovine lung glycopeptides. One such Ab (mAb GB3.1) was inhibited by carboxylated bovine lung glycopeptides and other multicarboxylated molecules, but not by glycopeptides in which the carboxylate groups were modified. The Ab recognized an epitope constitutively expressed on bovine, human, and other mammalian endothelial cells. Stimulated, but not resting, neutrophils bound to immobilized bovine lung glycopeptides in a carboxylate-dependent manner. The binding of activated neutrophils to immobilized bovine lung glycopeptides was inhibited both by mAb GB3.1 and by soluble glycopeptides in a carboxylate-dependent manner. The Ab also inhibited extravasation of neutrophils and monocytes in a murine model of peritoneal inflammation. This inhibition of cell trafficking correlated with the increased sequestration but reduced transmigration of leukocytes that were found to be adherent to the endothelium of the mesenteric microvasculature. Taken together, these results indicate that these novel carboxylated N:-glycans are constitutively expressed on vascular endothelium and participate in acute inflammatory responses by interaction with activated neutrophils.

ATP acts as an agonist to promote stimulus-induced secretion of IL-1 beta and IL-18 in human blood.

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1beta, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1beta, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X(7) receptor involvement. Quantities of IL-1beta generated by an individual donor's blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-alpha levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1beta and IL-18, and suggest that circulating blood monocytes constitutively express a P2X(7)-like receptor.

Activation of human leukocytes reduces surface P-selectin glycoprotein ligand-1 (PSGL-1, CD162) and adhesion to P-selectin in vitro.

P-selectin glycoprotein ligand-1 (PSGL-1), the primary ligand for P-selectin, is constitutively expressed on the surface of circulating leukocytes. The objective of this study was to examine the effect of leukocyte activation on PSGL-1 expression and PSGL-1-mediated leukocyte adhesion to P-selectin. PSGL-1 expression was examined via indirect immunofluorescence and flow cytometry before and after leukocyte stimulation with platelet activating factor (PAF) and PMA. Human neutrophils, monocytes, and eosinophils were all demonstrated to have significant surface expression of PSGL-1 at baseline, which decreased within minutes of exposure to PAF or PMA. PSGL-1 was detected in the supernatants of PAF-activated neutrophils by immunoprecipitation. Along with the expression data, this suggests removal of PSGL-1 from the cell surface. Soluble PSGL-1 was also detected in human bronchoalveolar lavage fluids. Down-regulation of PSGL-1 was inhibited by EDTA. However, inhibitors of L-selectin shedding and other sheddase inhibitors did not affect PSGL-1 release, suggesting that PSGL-1 may be shed by an as yet unidentified sheddase or removed by some other mechanism. Functionally, PSGL-1 down-regulation was associated with decreased neutrophil adhesion to immobilized P-selectin under both static and flow conditions, with the most profound effects seen under flow conditions. Together, these data indicate that PSGL-1 can be removed from the surface of activated leukocytes, and that this decrease in PSGL-1 expression has profound effects on leukocyte binding to P-selectin, especially under conditions of flow.

Early post-operative hyperbaric oxygen therapy modifies neutrophile activation.

BACKGROUND/AIMS: To investigate the effect of acute hyperbaric oxygen therapy (HBOT) on post-operative sinusoidal endothelial cell (SEC) damage caused by activated neutrophils. METHODOLOGY: 12 non-cirrhotic patients (Group H), who underwent elective hepatectomy for liver cancer, were given 2 courses of HBOT: 2.0 atm with inhalation of 100% oxygen, for 60 min, at 3 hours and 24 hours after hepatectomy; they were then compared with the 12 patients (Group C) who had been treated to maintain normal hemodynamic values. RESULTS: In group H, peak levels of polymorphonuclear leukocyte elastase (PMNE) and thrombomodulin (TM) were clearly diminished and delayed compared to Group C. All subjects in Group C showed more than a 10% increase in CD18 12 hours after surgery; however, in Group H, the elevation of CD18 expression was clearly suppressed compared to Group C. No patient in Group H had post-operative hyperbilirubinemia or hepatic failure; however, 3 had post-operative hyperbilirubinemia and 1 had intraperitoneal infection in Group C. CONCLUSIONS: Our results provide direct evidence that HBOT, especially at 3 hours after hepatectomy, has favorable effects on the activation of neutrophiles decreasing SEC injury.

Anti-inflammatory effects of U74389F in a rat model of intestinal ischemia/reperfusion injury.

OBJECTIVE: To investigate the role of eicosanoid generation and neutrophilic infiltration in the protective effects of U74389F against ischemia/reperfusion injury in the small intestines of rats. DESIGN: Prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Adult, male Sprague-Dawley rats weighing between 200 and 300 g. INTERVENTIONS: Groups (5-8) of rats treated with U74389F or vehicle were subjected to a sham operation and 30 mins of ischemia by occlusion of the superior mesenteric artery or 30 mins of ischemia followed by 60 or 120 mins of reperfusion. U74389F (2.5 mg/kg i.v.) or vehicle (citrate buffer) were slowly injected 2 mins before ischemia. MEASUREMENTS AND MAIN RESULTS: Ischemia significantly (p < .05) increased mucosal injury (0 [normal] to 5) in both U74389F and untreated rats. In contrast, U74389F significantly (p < .05) attenuated the severity of injury after reperfusion. In vehicle-treated rats, ischemia/reperfusion significantly reduced villus height in both U74389F and untreated groups. However, the surface epithelial layer was intact in the U74389F but not in the vehicle-treated group. In addition, compared with the vehicle-treated group, U74389F significantly reduced neutrophil infiltration and prevented the increase in leukotriene B4 and prostaglandin E2 in response to ischemia and reperfusion. CONCLUSIONS: This study demonstrates that the mechanism of U74389F against mesenteric ischemia/reperfusion includes a delay and reduction of neutrophilic infiltrate, an inhibition of leukotriene B4 production, and a facilitation of mucosal restitution.

Extracellular acidification induces human neutrophil activation.

In the current work, we evaluated the effect of extracellular acidification on neutrophil physiology. Neutrophils suspended in bicarbonate-buffered RPMI 1640 medium adjusted to acidic pH values (pH 6.5-7.0) underwent: 1) a rapid transient increase in intracellular free calcium concentration levels; 2) an increase in the forward light scattering properties; and 3) the up-regulation of surface expression of CD18. By contrast, extracellular acidosis was unable to induce neither the production of H2O2 nor the release of myeloperoxidase. Acidic extracellular pH also modulated the functional profile of neutrophils in response to conventional agonists such as FMLP, precipiting immune complexes, and opsonized zymosan. It was found that not only calcium mobilization, shape change response, and up-regulation of CD18 expression but also production of H2O2 and release of myeloperoxidase were markedly enhanced in neutrophils stimulated in acidic pH medium. Moreover, extracellular acidosis significantly delayed neutrophil apoptosis and concomitantly extended neutrophil functional lifespan. Extracellular acidification induced an immediate and abrupt fall in the intracellular pH, which persisted over the 240-s analyzed. A similar abrupt drop in the intracellular pH was detected in cells suspended in bicarbonate-supplemented PBS but not in those suspended in bicarbonate-free PBS. A role for intracellular acidification in neutrophil activation is suggested by the fact that only neutrophils suspended in bicarbonate-buffered media (i.e., RPMI 1640 and bicarbonate-supplemented PBS) underwent significant shape changes in response to extracellular acidification. Together, our results support the notion that extracellular acidosis may intensify acute inflammatory responses by inducing neutrophil activation as well as by delaying spontaneous apoptosis and extending neutrophil functional lifespan.

Granulocyte-macrophage colony-stimulating factor enhances EBV-induced synthesis of chemotactic factors in human neutrophils.

We have recently demonstrated that EBV binds to human neutrophils and stimulates a wide range of activities, including homeotypic aggregation, total RNA synthesis, and expression of the chemokines IL-8 and macrophage inflammatory protein-1alpha (MIP-1alpha). Neutrophil function is also known to be modulated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have therefore investigated the modulation of EBV-induced activation of human neutrophils by GM-CSF. Treatment of neutrophils with GM-CSF before EBV activation enhanced the production of both MIP-1alpha and IL-8. The IL-8 produced under these conditions was biologically active as determined in the calcium mobilization assay. GM-CSF was also found to increase the ability of EBV to prime neutrophils for increased leukotriene B4 (LTB4) synthesis. Prior treatment of GM-CSF with neutralizing Abs inhibited these effects. GM-CSF also increased the specific binding of FITC-EBV to the neutrophil surface, as evaluated by fluorocytometry. Local production of GM-CSF in tissues invaded by EBV could therefore serve to potentiate a host defense mechanism directed toward the destruction of the infectious virus via increased production of chemotactic factors. Since both IL-8 and MIP-1alpha are reported to be chemoattractants in vitro for T cells and T and B cells, respectively, the ability of EBV to induce their production by neutrophils may enhance its ability to infect B and T lymphocytes via increased recruitment to sites of infection.