Neutrophilic proteases: mediators of formyl-methionyl-leucyl-phenylalanine-induced ileitis in rats

N-formyl-methionyl-leucyl-phenylalanine (FMLP), a tripeptide of bacterial origin that activates and attracts neutrophils, increases mucosal permeability when placed in the lumen of rat ileum. Although studies using neutropenic animals demonstrate the essential role of neutrophils in FMLP-induced mucosal injury, the neutrophil-derived chemical mediator of this injury process remains undefined. The objective of this study was to determine whether neutrophilic proteases mediate FMLP-induced increases in mucosal permeability. The blood-to-lumen clearance of 51Cr-ethylenediaminetetraacetate was used to monitor mucosal permeability in the terminal ileum of Sprague-Dawley rats. In control (untreated) animals luminal perfusion with 10(-5) M FMLP resulted in twofold and fourfold increases in 51Cr-ethylenediaminetetraacetate clearance after 1 and 2 h of FMLP exposure, respectively. Pretreatment with the nonspecific serine protease inhibitor, soybean trypsin inhibitor (15 mg/kg), significantly attenuated the clearance responses normally observed during luminal perfusion with FMLP. The specific elastase inhibitors MeOSuc-Ala-Ala-Pro-Val-CH2Cl (10 mg/kg) and Eglin c (8 mg/kg) significantly attenuated the FMLP-induced increases in ethylenediaminetetraacetate clearance observed after both 1 and 2 h of exposure. The results of this study indicate that neutrophilic proteases mediate at least part of the increased mucosal permeability induced by luminal exposure to FMLP.     

Sulfasalazine metabolites and dapsone attenuate formyl-methionyl-leucyl-phenylalanine-induced mucosal injury in rat ileum

The effects of 5-aminosalicylic acid (5-ASA), 4-ASA, N-acetyl-5-ASA, and sulfapyridine on mucosal permeability were determined in an experimental model of acute ileitis. In addition, the antiinflammatory drug dapsone was tested. The distal 10 cm of rat ileum was perfused with formyl-methionyl-leucyl-phenylalanine (FMLP) (10(-5) M), a bacterial peptide that activates and attracts neutrophils. Changes in mucosal permeability were assessed using the blood-to-lumen clearance of 51Cr-ethylene-diamineacetate. Luminal FMLP increased 51Cr-labeled ethylenediamineacetate clearance twofold and fourfold in the first and second hour, respectively. Addition of 5-ASA (10 mM), 4-ASA (10 mM), or dapsone (4 mM) to the luminal perfusate after 60 min of FMLP perfusion greatly attenuated the increased mucosal permeability observed after 120 min of FMLP perfusion. Neither N-acetyl-5-ASA (10 mM) nor sulfapyridine (5 mM) had an effect on the FMLP-induced increase in mucosal permeability. We characterized the inhibitory effect of these drugs on the catalytic activity of myeloperoxidase and tested their ability to scavenge hypochlorous acid in vitro. 5-Aminosalicylic acid, 4-ASA, and dapsone demonstrated a powerful inhibitory effect on the catalytic activity of myeloperoxidase, whereas all drugs were equally effective in scavenging HOCl. In additional in vitro experiments we were unable to demonstrate an inhibitory effect of either of the drugs on the catalytic activity of neutrophilic elastase. Our results indicate that inhibition of neutrophilic myeloperoxidase may be an important mechanism by which 5-ASA, 4-ASA, and dapsone attenuate FMLP-induced mucosal injury.     

Intestinal microvascular exchange in the rat during luminal perfusion with formyl-methionyl-leucyl-phenylalanine

Formyl-methionyl-leucyl-phenylalanine (FMLP), a peptide released from bacteria in the gut lumen, is known to both attract and activate neutrophils. The aim of this study was to determine whether luminal perfusion with 1 microM FMLP alters microvascular permeability, blood flow, and neutrophil migration in the small intestine of control rats and rats treated with antineutrophil serum. Microvascular permeability to total plasma proteins was determined from an analysis of lymphatic protein fluxes. Myeloperoxidase activity was used as an index of tissue neutrophil count. Intestinal blood flow was measured using radiolabeled microspheres and the reference blood sample method. In control rats, luminal perfusion with FMLP caused significant increases in blood flow, lymph flow, lymph protein clearance, and microvascular permeability, but it did not alter tissue myeloperoxidase activity. In rats treated with antineutrophil serum, tissue myeloperoxidase levels were reduced by approximately 55%, and the FMLP-induced changes in lymph flow, lymph protein clearance, and microvascular permeability were significantly attenuated. In vitro experiments with isolated rat neutrophils revealed that 1 microM FMLP elicits significant chemotaxis and degranulation yet minimally enhances superoxide production. The results of this study indicate that peptides produced by microorganisms in the gut lumen can increase intestinal microvascular permeability. The FMLP-induced alterations in microvascular exchange appear to be mediated by activated neutrophils.     

The chemotactic peptide N-formyl methionyl-leucyl-phenylalanine increases mucosal permeability in the distal ileum of the rat

Activation of granulocytes within the lamina propria by luminally derived bacterial products may represent an important mechanism in the pathogenesis of inflammatory bowel disease. The objective of this study was to determine the effects of luminal perfusion with N-formyl methionyl-leucyl-phenylalanine (FMLP), a bacterial product that attracts and activates granulocytes, on mucosal permeability in different regions of the rat small intestine and colon. Mucosal permeability was measured using blood-to-lumen clearance of 51Cr-ethylenediamine-tetraacetate during luminal perfusion with FMLP (10(-3) to 10(-8) M) dissolved in Tyrode's solution. Of the bowel segments studied, mucosal permeability was significantly increased by FMLP only in the distal 10 cm of ileum. The minimal FMLP concentration required to increase mucosal permeability was 10(-6) M. The increased mucosal permeability induced by FMLP could be prevented by depletion of circulating granulocytes with antineutrophil serum. The greater sensitivity of the distal ileum to FMLP did not correlate with a higher tissue myeloperoxidase activity, but it was associated with a higher basal ethylenediaminetetraacetate clearance. These observations indicate that a high basal mucosal permeability to solutes the size of FMLP (5-6 A radius), rather than a greater number of resident granulocytes in the lamina propria, predisposes the terminal ileum to the inflammatory actions of FMLP.     

Role of oxygen radicals in ischemia-induced lesions in the cat stomach

Ischemia in a stomach that contains acid may produce severe gastric mucosal injury. The extent to which oxygen-derived free radicals are involved in the pathogenesis of this injury was investigated in the present study. Local gastric ischemia was achieved by reducing celiac artery pressure to 30 mmHg for 1 h. Ischemic injury was assessed by recording the loss of 125I-albumin and 51Cr-red cells across the gastric mucosa. Cats were treated with a xanthine oxidase inhibitor (allopurinol), a superoxide radical scavenging enzyme (superoxide dismutase), and a scavenger of hydroxyl radicals (dimethyl sulfoxide). The damage associated with ischemia only occurred during reperfusion of the stomach and was worst in the antrum. The level of xanthine oxidase in the antrum was twice that of the corpus. Treatment with allopurinol, superoxide dismutase, and dimethyl sulfoxide reduced 51Cr-red cell loss to 15%, 25%, and 21% of control (untreated) animals, respectively. The data indicate that oxygen-derived free radicals play a role in ischemic injury to the stomach and that the hydroxyl radical, a secondary radical produced from the superoxide anion, appears to be the major oxygen radical contributing to ischemic damage.     

Sequence of gastric mucosal injury following ischemia and reperfusion

The mechanisms of gastric mucosal injury following a period of ischemia remain unclear. The aim of this study was to determine the relative contributions of ischemia, reperfusion, and reactive oxygen metabolites to mucosal injury induced by temporary occlusion of the celiac artery. Rats were subjected to 30 min of gastric ischemia in the presence of 100 mM HCl. Reperfusion periods ranged from 1 min to 24 hr. Drug treatments included allopurinol (100 mg/kg) or a combination of superoxide dismutase (15,000 units/kg), catalase (90,000 units/kg), and desferrioxamine (50 mg/kg). Mucosal injury was assessed by quantitative histology and the extent of macroscopic hemorrhage. Approximately one third of the total injury to the volume of the mucosa (11.8 +/- 9.1%) was due to ischemia alone. Another third was blocked by allopurinol or superoxide dismutase, catalase, and desferrioxamine (22.1 +/- 6.9%, P less than 0.001; and 25.9 +/- 4.6%, P less than 0.01), respectively, compared with control (32.5 +/- 5.1%). In contrast, extensive surface mucosal injury (62.2 +/- 27.6%) occurred primarily during ischemia and was not affected by antioxidants. Macroscopic hemorrhage was halved by treatment with allopurinol (17.5 +/- 12.6%, P less than 0.01) or superoxide dismutase, catalase, and desferrioxamine (15.9 +/- 14.5%, P less than 0.01). We conclude that temporary celiac occlusion results in gastric mucosal damage that consists of both ischemic and reperfusion components. The majority of surface mucosal injury occurred during ischemia, whereas injury to the volume of the mucosa and the vasculature occurred equally during reperfusion and was associated with reactive oxygen metabolites.     

Effects of recombinant human granulocyte colony-stimulating factor on neutrophil function in normal rats.

We studied the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on neutrophil functions in vitro using neutrophils isolated from the venous blood of normal rats. FMLP-induced superoxide anion (O2-) release, phagocytosis, and FMLP-induced chemotaxis were evaluated. These functions were significantly enhanced by rG-CSF treatment. In addition to performing neutrophil function assays, we evaluated FMLP binding to rat neutrophils after rG-CSF treatment. FMLP specific binding was not changed by rG-CSF treatment. In addition, we intravenously injected rG-CSF (10 micrograms/kg) or control vehicle into rats for 7 consecutive days, and evaluated the functions of neutrophils isolated from venous blood at 6 h after the final injection. The neutrophil count in the peripheral blood of rG-CSF-treated rats was increased significantly compared with that in control rats. FMLP-induced O2- release, phagocytosis, FMLP-induced chemotaxis and spontaneous migration of rG-CSF-treated neutrophils were significantly enhanced in comparison with those in control rats. These findings demonstrate that rG-CSF not only increases neutrophil counts in peripheral blood, but that it also enhances neutrophil functions, both in vitro and in vivo.     

The effect of anticholinergic and beta-agonist pretreatment on bronchoconstriction induced by N-formyl-methionyl-leucyl-phenylalanine

N-Formyl-methionyl-leucyl-phenylalanine (FMLP), an acylated tripeptide bacterial neutrophil chemotactic factor, has been shown to be a bronchoconstrictor by inhalation in man in vivo. It thus has a putative role in the generation of bronchoconstriction associated with bacterial bronchial infection. We have investigated the effect of pretreatment with the anticholinergic agent, ipratropium bromide (IB), and the beta 2-agonist, fenoterol (F), on FMLP-induced bronchoconstriction. Ten non-asthmatic subjects aged 21-28 yrs performed dose-response curves to nebulized FMLP on 3 study days after pretreatment with saline, F or IB. Amongst the 8 subjects who bronchoconstricted by 20% to FMLP there was a significant increase in the provocative concentration causing a 20% fall in forced expiratory volume in one second (FEV1) (PC20FMLP) after both IB and F. F was significantly better than IB. When comparison was made using absolute fall in FEV1 and including all 10 subjects the same results were found. Partial inhibition of FMLP-induced bronchoconstriction by IB suggests that part of the effect of FMLP is vagally mediated. We suggest that F is acting via modulation of FMLP-induced rises in intracellular free calcium.     

Effect of reactive oxygen metabolites on endothelial permeability: role of nitric oxide and iron.

OBJECTIVE: We evaluated the effects of the xanthine oxidase (XO)-derived reactive oxygen metabolites on the permeability of bovine pulmonary artery-endothelial monolayers and examined how iron and nitric oxide (NO) participate in these changes in permeability. METHODS: Permeability was measured using a cell-column chromatographic method in which monolayers were exposed to combinations of agents. RESULTS: Exposure of monolayers to a superoxide/peroxide generator, xanthine (X, 0.1 mM)/XO (25 mU/mL), increased solute permeability after 10 minutes, but the same dose of either X or XO alone did not. Exposure of monolayers to peroxide (0.1 mM) also increased permeability, but only after 70 minutes. This X/XO permeability was attenuated by either catalase, superoxide dismutase, methionine (1 mM), an oxy-radical scavenger, or desferrioxamine (0.1 mM), an iron chelator. Spermine NONOate (SNO), an NO donor, attenuated X/XO permeability at 0.1 mM, but this protection was not significant at 0.01 or 1 mM. Spermine NONOate (0.1 mM) did not alter the permeability produced by 0.1 mM peroxide. L-N5-(1-iminoethyl)-ornithine (10 microM), an NO synthase inhibitor, completely blocked peroxide-, and partially attenuated X/XO-mediated permeability. However, 3-morphosynodiomine (SIN-1, 1 mM) plus catalase (1,000 U/mL), a peroxynitrite generator, did not alter permeability. CONCLUSIONS: Xanthine/Xanthine Oxidase permeability involves peroxide, superoxide, oxy-radicals, and iron. Endogenous NO may regulate peroxide-, but not superoxide-mediated permeability. The protective effects of exogenous NO on the X/XO permeability may represent interactions between superoxide, peroxide, and cell surface-bound iron.     

Effects of ethanol on the chemotactic peptide-induced second messenger generation and superoxide production in polymorphonuclear leukocytes.

The generation of oxygen radicals by polymorphonuclear leukocytes (PMNL) plays a pivotal role for host defense. Since ethanol reduced FMLP- but not PMA-induced superoxide ion (O2-) formation by PMNL, the effects of ethanol on second messenger systems in PMNL were studied. FMLP induced a biphasic rise in cytosolic calcium concentrations, [Ca2+]i. Ethanol treatment abolished the second phase (believed to reflect Ca2+ influx), an effect also observed in PMNL treated with La3+ or suspended in Ca(2+)-free buffer. The FMLP-induced inositol trisphosphate generation was unaffected by ethanol, whereas diacylglycerol formation was, as expected, markedly reduced. Propranolol, an inhibitor of diacylglycerol formation from phosphatidic acid, caused a prolonged transmembrane influx of Ca2+ and partially reversed the inhibitory effect of ethanol on FMLP-induced O2- production. Thus, the ability of ethanol to inhibit FMLP-induced O2- generation in neutrophils seems to be due to both impaired influx of Ca2+ across the plasma membrane and reduced phospholipase D-mediated generation of phosphatidic acid.     

Gastric mucosal injury in the rat. Role of iron and xanthine oxidase

Recent studies have implicated oxygen free radicals in ischemia-reperfusion injury to the gastric mucosa. The aims of the present study were to test the hypothesis that the enzyme xanthine oxidase is the source of the oxygen radicals in the ischemic stomach and determine the importance of the iron-catalyzed Haber-Weiss reaction in generating the cytotoxic oxygen radicals. Gastric mucosal clearance of 51Cr-labeled red blood cells was measured during a 30-min control period, a 30-min ischemic period (hemorrhage to 25 mmHg arterial pressure), and a 60-80-min reperfusion period (reinfusion of shed blood). In untreated (control) rats, a dramatic rise (100-fold) in the leakage of 51Cr-labeled red blood cells into the gastric lumen was observed only during the reperfusion period. After the reperfusion period, gastric mucosal damage was further assessed using gross lesion area and histology. Rats were placed on a sodium tungstate diet (to inactivate xanthine oxidase), or treated with either deferoxamine (an iron chelating agent) or superoxide dismutase (a superoxide scavenger). All three interventions substantially reduced 51Cr-labeled red blood cell clearance and gross lesion area relative to untreated rats. However, tissue injury assessed histologically was similar in both treated and untreated animals. The results of this study support the hypothesis that oxygen free radicals mediate the hemorrhagic shock-induced extravasation of red blood cells. The data also indicate that xanthine oxidase is the source of the oxy-radicals and that the iron-catalyzed Haber-Weiss reaction is largely responsible for hydroxyl radical generation in this model.     

Platelet-dependent modulation of polymorphonuclear leukocyte chemiluminescence

Human blood platelets decreased luminol-enhanced chemiluminescence of human polymorphonuclear leukocytes (PMNL) stimulated with FMLP or Ca2+-ionophore A23187 by 56 or 47%, respectively. Horseradish peroxidase potentiated the decreasing effect of platelets on A23187-stimulated PMNL (92% inhibition) or reversed inhibition of FMLP-induced chemiluminescence to 94% potentiation, indicating dependence of platelet activity on availability of extracellular peroxidase. Moreover, platelet activity may depend also on the extent of platelet activation, as non-activated platelets (in the presence of FMLP) were found to potentiate PMNL-generated chemiluminescence, while platelets activated with A23187 displayed the opposite effect. Interference of platelets with formation and liberation of superoxide anion was indicated by platelet-modified isoluminol chemiluminescence. Superoxide dismutase with catalase and sodium azide were used, respectively, to differentiate the intracellular and the extracellular part of the chemiluminescence signal. Platelets were found to be capable of modifying both components of chemiluminescence, i.e., oxygen metabolites produced on the plasma membrane as well as on membranes of intracellular granules.     

The role of oxygen-derived free radicals in ischemia-induced increases in canine skeletal muscle vascular permeability

Previous studies indicate that vascular permeability is increased in skeletal muscle subjected to 4 hours of inflow occlusion. However, the mechanism(s) underlying the increase in permeability are unknown. The aim of this study was to assess the role of oxygen-derived free radicals and histamine as putative mediators of the increased permeability in skeletal muscle subjected to 4 hours of inflow occlusion. The osmotic reflection coefficient for total plasma proteins and isogravimetric capillary pressure were estimated in canine gracilis muscle for the following conditions: control, ischemia, and ischemia plus pretreatment with allopurinol (a xanthine oxidase inhibitor), catalase (a peroxidase that reduces hydrogen peroxide to water and molecular oxygen), superoxide dismutase (a superoxide anion scavenger), dimethyl sulfoxide (a hydroxyl radical scavenger), diphenhydramine (a histamine H1-receptor blocker), or cimetidine (a histamine H2-receptor blocker). Ischemia, followed by reperfusion, significantly reduced the reflection coefficient from 0.94 +/- 0.02 to 0.64 +/- 0.02 and isogravimetric capillary pressure from 13.8 +/- 1.0 mm Hg to 6.9 +/- 0.4 mmHg, indicating a dramatic increase in microvascular permeability. Prior treatment with diphenhydramine or cimetidine did not significantly alter the permeability increase induced by ischemia. However, pretreatment with allopurinol, catalase, superoxide dismutase, or dimethylsulfoxide did significantly attenuate the increase in vascular permeability. The results of this study indicate that oxygen radicals are primarily responsible for the increased vascular permeability produced by ischemia-reperfusion, that the hydroxyl radical may represent the primary damaging radical, and that xanthine oxidase may represent the primary source of oxygen-derived free radicals in ischemic skeletal muscle.     

Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.

Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.     

Platelet-dependent modulation of polymorphonuclear leukocyte chemiluminescence

Human blood platelets decreased luminol-enhanced chemiluminescence of human polymorphonuclear leukocytes (PMNL) stimulated with FMLP or Ca2+-ionophore A23187 by 56 or 47%, respectively. Horseradish peroxidase potentiated the decreasing effect of platelets on A23187-stimulated PMNL (92% inhibition) or reversed inhibition of FMLP-induced chemiluminescence to 94% potentiation, indicating dependence of platelet activity on availability of extracellular peroxidase. Moreover, platelet activity may depend also on the extent of platelet activation, as non-activated platelets (in the presence of FMLP) were found to potentiate PMNL-generated chemiluminescence, while platelets activated with A23187 displayed the opposite effect. Interference of platelets with formation and liberation of superoxide anion was indicated by platelet-modified isoluminol chemiluminescence. Superoxide dismutase with catalase and sodium azide were used, respectively, to differentiate the intracellular and the extracellular part of the chemiluminescence signal. Platelets were found to be capable of modifying both components of chemiluminescence, i.e., oxygen metabolites produced on the plasma membrane as well as on membranes of intracellular granules.     

Matrix metalloproteinase-9 release from human leukocytes.

Although proteinases are thought to contribute to the pathogenesis of bronchial asthma and COPD, the mechanism of proteinase release from inflammatory cells has not been thoroughly clarified. We examined matrix metalloproteinase (MMP-9) release from human leukocytes using soluble agonists such as C5a, FMLP, and PAF. Mononuclear cells, neutrophils, and eosinophils isolated from human leukocytes were incubated with C5a, FMLP, or PAF for 20 min. MMP-9 in supernatants was measured by ELISA. Among mononuclear cells, neutrophils, and eosinophils, MMP-9 was released mainly from neutrophils. FMLP was the most effective stimulus of MMP-9 release from neutrophils among three agonists: C5a, FMLP, and PAF. GM-CSF clearly enhanced FMLP-induced MMP-9 release. Pretreatment of neutrophils with pertussis toxin (PTX) resulted in the inhibition of FMLP-induced MMP-9 release, indicating the contribution of PTX-sensitive G-proteins to intracellular signal transduction in FMLP-induced MMP-9 release. These results suggest that neutrophils release large amounts of MMP-9 in response to FMLP, which is a bacterial product analogue. It cannot be excluded that MMP-9 released from neutrophils may be involved in the pathogenesis of bronchial asthma and COPD.     

Reperfusion-induced arrhythmias and oxygen-derived free radicals. Studies with "anti-free radical" interventions and a free radical-generating system in the isolated perfused rat heart

We have assessed, whether six agents, that either inhibit free radical formation or scavenge free radicals once they are produced, can reduce the incidence of reperfusion-induced arrhythmias, whether a free radical-generating system (FeCl3 X adenosine diphosphate) can increase the incidence of reperfusion-induced arrhythmias, and whether "anti-free radical" interventions can reduce reperfusion rhythm disturbances caused by the addition of FeCl3 X adenosine diphosphate. With the isolated, perfused rat heart (n = 15 in each group), inclusion of L-methionine (1 and 10 mM), superoxide dismutase (2.5 X 10(4) and 1 X 10(5) U/liter), catalase (5 X 10(4), 5 X 10(5), and 1 X 10(6) U/liter), mannitol (50 mM), glutathione (10 microM), or desferrioxamine (150 microM) significantly reduced the incidence of reperfusion-induced ventricular fibrillation and, in many cases, the incidence of reperfusion-induced ventricular tachycardia. The mean duration of sinus rhythm during reperfusion was also increased significantly. Perfusion of hearts with boiled superoxide dismutase (1 X 10(5) U/liter) or boiled catalase (1 X 10(6) U/liter) did not decrease arrhythmias. Conversely, under conditions where, in the control group, the incidence of reperfusion arrhythmias was lowered by increasing perfusate potassium to 6.5 mM, the addition of the free radical-generating system FeCl3 X adenosine diphosphate (0.1 microM X 1 microM) to the perfusion fluid increased dramatically the incidence of reperfusion-induced ventricular fibrillation and tachycardia. Simultaneous perfusion with FeCl3 X adenosine diphosphate and superoxide dismutase (1 X 10(5) U/liter), catalase (1 X 10(6) U/liter), mannitol (50 mM), methionine (10 mM), or desferrioxamine (150 microM) again reduced the incidence of reperfusion-induced arrhythmias and increased the duration of normal sinus rhythm during the reperfusion phase. Thus, addition of six "anti-free radical" interventions reduced the incidence of reperfusion-induced arrhythmias, addition of a free radical-generating system increased the incidence of reperfusion-induced arrhythmias, and simultaneous perfusion of the hearts with FeCl3 X adenosine diphosphate and "anti-free radical" interventions again reduced reperfusion rhythm disturbances. These results are further evidence supporting the hypothesis that oxygen-derived free radicals play an important role in the genesis of reperfusion-induced arrhythmias.     

Role of xanthine oxidase-derived oxidants and leukocytes in ethanol-induced jejunal mucosal injury

Previous reports indicate that intestinal intraluminal ethanol increases mucosal permeability (an index of mucosal injury) and histamine release by mast cells, and that the released histamine plays a role in mediating the increased permeability. In the present study, we investigated whether reactive oxygen metabolites and their major sources (xanthine oxidase and leukocytes) were involved in these ethanol effects. In rabbits, segments of the jejunum were perfused with a control solution or with 6% ethanol. In these segments, mucosal permeability was assessed by determining jejunal clearance of i.v. administered⁵¹Cr-ethylenediaminetetraacetate (⁵¹Cr-EDTA) and¹²⁵I-bovine serum albumin (¹²⁵I-BSA), and mast cell histamine release was estimated from the histamine concentration of the gut effluent. Ethanol increased⁵¹Cr-EDTA clearance,¹²⁵I-BSA clearance, and histamine release. These ethanol effects decreased when the animals were given superoxide dismutase plus catalase (scavenger of O^2– and H₂O₂, respectively), allopurinol, or oxypurinol (xanthine oxidase inhibitors). Administration of a monoclonal antibody (R15.7) against leukocyte adhesion molecule, CD18, inhibited completely the ethanol-induced increased⁵¹Cr-EDTA and¹²⁵I-BSA clearances and histamine release. These and supplementary data suggest that (a) ethanol-induced mucosal injury and mast cell histamine release are mediated primarily by leukocytes, and (b) oxy radicals, especially those generated by xanthine oxidase, mediate these ethanol effects mainly by promoting leukocyte infiltration.     

Neutrophil-mediated injury to gastric mucosal surface cells

Neutrophils (PMNs) have been implicated in the pathogenesis of gastritis. This study evaluates the magnitude and mode of PMN-mediated damage to gastric mucosal surface cells (GSC) in a system independent of vascular and neural factors. Rabbit GSC were freshly isolated and preloaded with⁵¹Cr. GSC were then incubated for 1 hr or 4 hr with freshly isolated human PMNs at varying effector-to-target cell ratios. Injury to GSC was assessed as percent specific⁵¹Cr released and by electron microscopy. We found minimal GSC injury using nonactivated PMNs. Incubation with PMNs activated with formylmethionyl-leucyl-phenalalanine (FMLP), however, resulted in significant GSC injury at the 20:1 PMN/GSC ratio, 33.2±1.8%⁵¹Cr release (P<0.001 compared to nonactivated PMNs). Electron microscopy revealed well-preserved gastric surface cells after exposure to nonstimulated PMNs. GSC exposed to activated PMNs (20:1 PMN/GSC ratio) were severely injured. Proteinase inhibitors and dimethylsulfoxide failed to diminish PMN-mediated GSC injury. Conversely, superoxide dismutase (SOD) inhibited GSC injury by more than 50% (P<0.001). In addition, glutathione peroxidase inhibited injury by 84% (P<0.001). These data suggest that neutrophil-mediated injury to gastric surface cellsin vitro involves superoxide anion and hypochlorous acid and not neutral trypsinlike proteinases or hydroxyl radicals.     

Density heterogeneity of neutrophilic polymorphonuclear leukocytes: gradient fractionation and relationship to chemotactic stimulation

When elicited murine peritoneal exudate cells were subjected to Percoll density gradient centrifugation, polymorphonuclear neutrophils (PMN) were found to distribute over a broad spectrum of buoyant densities (1.10-1.06 g/ml). PMN isolated between approximately 1.10 and 1.085 g/ml were referred to as high density PMN (HD-PMN), and those isolated at approximately 1.085-1.06 g/ml were designated intermediate density PMN (ID-PMN). Cells were characterized on the basis of morphology and specific markers: PMN by lactoferrin immunocytofluorescence and macrophages by nicotinamide adenine dinucleotide glycohydrase activity. Macrophages banded near the top of the gradient with a peak at 1.04 g/ml. At increasing times following elicitation, the ratio of HD to ID- PMN decreased. Decreased density of either murine HD-PMN or human peripheral blood PMN could be induced in vitro by exposure of the cells to endotoxin-activated serum. A decrease in buoyant density of human PMN was also demonstrated in vitro using the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The response was time dependent, related to dose, and appeared to be mediated by the cell membrane receptor for FMLP. A competitive antagonist of FMLP binding, carbobenzoxy-phenylalanyl-methionine, inhibited the density change with a calculated Kd similar to that reported for inhibition of FMLP-induced aggregation, degranulation, locomotion, and superoxide production. The FMLP-induced decrease in PMN density was shown to be directly correlated with increases in relative mean cell volume. The density response is a new measurement of PMN interaction with specific chemotactic factors, which may be important in the generation of PMN heterogeneity observed in elicited peritoneal exudate cells. In addition, this approach offers a means of physically separating "activated" from "resting" PMN and of studying resultant biochemical differences between these cell populations using both in vivo and in vitro systems.