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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.

Recent reports in the literature suggest that iron plays an important role in free radical-mediated injury in biological systems. To assess the role of iron-catalyzed oxidant production in ischemia-reperfusion injury, we examined the influence of deferoxamine (an iron chelator) and apotransferrin (iron transporting protein) on the increased intestinal vascular permeability produced by 1 h of ischemia and reperfusion. Both agents were administered intravascularly as a constant infusion, beginning 5 min before reperfusion. Capillary osmotic reflection coefficients were derived from the relationship between lymph-to-plasma protein concentration ratio and lymph flow in the feline small bowel. Vascular permeability in control intestinal preparations was 0.08 +/- 0.005, however it increased significantly to 0.40 +/- 0.03 in preparations subjected to 1 h of ischemia and 30 min of reperfusion. Vascular permeability in the deferoxamine-(0.15 +/- 0.009) and apotransferrin- (0.17 +/- 0.002) treated animals were significantly lower (P less than 0.01) than in the untreated group. Treatment with iron-loaded deferoxamine or transferrin did not offer any protection against ischemic injury. These findings support the hypothesis that iron plays an important role in the formation of hydroxyl radicals after reperfusion of the ischemic bowel.

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-methionylleucyl-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 51 Cr-ethylenediamineacetate. Luminal FMLP increased 51 Cr-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 HOCI. 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.

Isolated human neutrophilic leukocytes were stimulated to produce hydrogen peroxide (H2O2) and to secrete cytoplasmic granule components including myeloperoxidase into the medium. Myeloperoxidase catalyzed the oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), which reacted with endogenous nitrogen compounds to yield derivatives containing nitrogen-chlorine (N-Cl) bonds. Compounds available for reaction with HOCl were ammonia (NH+4), taurine, alpha-amino acids, and granule proteins and peptides that were released into the medium. A portion of the N-Cl derivatives formed under these conditions accumulated in the extracellular medium. These long lived oxidizing agents were characterized as hydrophilic, low molecular weight, mono-N-chloramine (RNHCl) derivatives based on their absorption spectrum, ability to oxidize 5-thio-2-nitrobenzoic acid and to chlorinate ammonia (NH+4), and behavior upon ultrafiltration, gel chromatography, and extraction with organic solvents. The RNHCl derivatives were of low toxicity, but reacted with NH+4 to yield the lipophilic oxidizing agent monochloramine (NH2Cl). Therefore, the addition of NH+4 conferred bactericidal, cytotoxic, and cytolytic activities on the RNHCl derivatives. The results indicate that taurine and other neutrophil amines protect neutrophils and other cells against oxidative attack by acting as a trap for HOCl and by competing with endogenous NH+4 for reaction with HOCl. However, the RNHCl derivatives act as a reserve of oxidizing equivalents that is converted to a toxic form when an increase in NH+4 concentration favors formation of NH2Cl.

Production of hydrogen peroxide and secretion of myeloperoxidase by stimulated neutrophils resulted in myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl), the reaction of HOCl with taurine to yield taurine monochloramine (TauNHCl), and accumulation of TauNHCl in the extracellular medium. When erythrocytes were present, the yield of TauNHCl was lower as the result of uptake of TauNHCl into erythrocytes. The zwitterion taurine was not taken up, but the anion TauNHCl and other anionic oxidants including taurine dichloramine (TauNCl2) and L-alanine chloramines were transported into erythrocytes by the anion-transport system. Oxidation of intracellular components such as glutathione (GSH) by taurine chloramines resulted in reduction of the chloramines and trapping of taurine within erythrocytes. At high oxidant:erythrocyte ratios, TauNHCl also oxidized hemoglobin (Hb) and depleted ATP, but caused little lysis. TauNCl2 was much more effective as a lytic agent. At low oxidant:erythrocyte ratios, the chloramines caused net loss of GSH when no glucose was provided, but Hb was not oxidized and GSH content returned to normal when glucose was added. Therefore, anionic chloramines may mediate oxidative toxicity when the neutrophil:erythrocyte ratio is high. Under more physiologic conditions, chlorination of taurine by neutrophils and the uptake and reduction of TauNHCl by erythrocytes prevents accumulation of oxidants and may protect blood cells, plasma components, and tissues against oxidative toxicity.

Allopurinol has been shown to provide significant protection against ischemia/reperfusion-induced microvascular and parenchymal cell injury. It has been hypothesized that the protection seen with allopurinol after ischemia/reperfusion (I/R) is caused by inhibition of xanthine oxidase. However, recent reports suggest that the beneficial effects of allopurinol in I/R may be caused by direct free radical scavenging. The objective of this study was to determine whether the regimen of allopurinol administration used in most I/R studies leads to a significant modification of the free radical scavenging properties of extracellular fluid (ECF), i.e., plasma and lymph. Plasma and intestinal lymph samples obtained from both control and allopurinol-treated cats were used to assess the following: 1) allopurinol and oxypurinol concentrations, 2) xanthine oxidase inhibition, 3) myoglobin-catalyzed linolenic acid peroxidation, 4) hypochlorous acid scavenging, and 5) protein and nonprotein sulfhydryl content. ECF from allopurinol-treated animals contained approximately 10 microM each of allopurinol and oxypurinol. Ten percent ECF resulted in 80% inhibition of xanthine oxidase activity. Comparable volumes of control ECF did not inhibit xanthine oxidase. Furthermore, allopurinol treatment did not enhance the antioxidant properties of ECF. The results of this study do not support the contention that the beneficial effects of allopurinol in I/R injury are caused by the scavenging of oxidants produced in ECF by activated granulocytes.

The mechanism that leads to an inhibition of enzyme activity in the presence of high concentrations of substrate was investigated with the two malate dehydrogenase isoenzymes obtained from pig heart. The inhibition is promoted by an abortive binary complex formed by the enzymes and the enol form of of oxalacelate. Neither the oxidized coenzyme nor the reduced coenzyme appears to be involved in the formation of this complex. These results suggest that the mechanism of substrate inhibition that occurs with the pig heart malate dehydrogenases is different from that observed with the lactate dehydrogenases from chicken hearts. The inhibition constants for oxalacetate are 2.0 mM with the mitochondrial enzyme and 4.5 mM with the cytoplasmic enzyme. Since the in vivo concentration of oxalacetate is reported to be about 10 micrometer, these data suggest that the substrate inhibition that is exhibited by the malate dehydrogenases may not be of any significance in vivo.

Ischemia and reperfusion (I/R) of the small intestine initiates a series of events that result in neutrophil-mediated microvascular injury. Recent reports suggest that adenosine possesses anti-inflammatory properties by virtue of its ability to inhibit neutrophil (PMN) superoxide (O2-.) and hydrogen peroxide (H2O2) production and to interfere with PMN adherence to cultured endothelium. In an attempt to further characterize the anti-inflammatory properties of adenosine in vivo we assessed the influence of exogenous adenosine on 1) I/R-induced PMN-mediated microvascular injury in the feline small intestine, 2) feline PMN superoxide production, and 3) I/R-induced PMN adherence to feline mesenteric venular endothelium. We found that intra-arterial administration of adenosine (2 microM) significantly attenuated the I/R-induced increases in intestinal capillary permeability. This protective effect of adenosine could not be explained entirely on its ability to inhibit PMN O2-. (or H2O2) production, since adenosine was effective in inhibiting feline PMN O2-. production by only 20%. Using intravital microscopic techniques in cat mesentery, we found that adenosine did not alter the responses of venular blood flow, shear rate, leukocyte rolling velocity, and leukocyte adherence to I/R when compared with control animals. However, the number of extravasated leukocytes during the ischemic period was significantly reduced by adenosine. Adenosine reduced the number of adherent leukocytes by 25% at 10 and 60 min of reperfusion while leukocyte extravasation was reduced by 65-70% during the same period. Our data indicate that the adenosine-induced suppression of leukocyte extravasation cannot be explained solely by an attenuation in leukocyte adherence to venular endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to determine whether ATP-MgCl2 or isoproterenol pretreatment would attenuate the increase in canine gracilis muscle vascular resistance and permeability associated with 4 h of occlusive ischemia followed by 1 h of reperfusion. To this end, the osmotic reflection coefficient for total plasma proteins (omega), isogravimetric capillary pressure (Pci), precapillary resistance (Ra), postcapillary resistance (Rv), and total vascular resistance (Rt) were determined for the following conditions: control, ischemia, and ischemia plus pretreatment with ATP-MgCl2 or isoproterenol. Reperfusion, after ischemia, significantly reduced omega from 0.94 +/- 0.02 to 0.64 +/- 0.02, whereas Pci was decreased by 50 +/- 4%, indicating a dramatic increase in vascular permeability. Ischemia-reperfusion was also associated with an increase in Rt of 230 +/- 22%. Similar results were obtained in muscles pretreated with isoproterenol. However, in muscles pretreated with ATP-MgCl2, omega averaged 0.98 +/- 0.09, Pci was reduced by only 15 +/- 8%, and Rt was increased by just 25 +/- 12%. The effect of ATP-MgCl2 on neutrophilic oxidative metabolism was evaluated by measuring superoxide production by activated neutrophils in the presence and absence of ATP-MgCl2. Superoxide production by activated neutrophils was significantly attenuated by ATP-MgCl2. The results of these studies indicate that pretreatment with ATP-MgCl2, but not isoproterenol, is remarkably effective in attenuating the increase in skeletal muscle vascular resistance and permeability induced by ischemia-reperfusion. The protective effect of ATP-MgCl2 may be related in part to its ability to inhibit neutrophilic superoxide production.

The aim of the present study was to determine whether the split products of sulfasalazine, sulfapyridine, and 5-aminosalicylic acid can ameliorate ischemia/reperfusion-induced injury to the gastric mucosa. Gastric mucosal damage was assessed by measuring (a) 51 Cr-labeled red blood cell leakage into the gastric lumen, (b) the area of gross mucosal lesions, and (c) the extent of histologically demonstrable mucosal damage. In rats treated with 5-aminosalicylic acid, but not in those treated with sulfapyridine, the leakage of 51 Cr-labeled red blood cells and the area of gross mucosal lesions after ischemia/reperfusion were significantly reduced as compared with untreated (control) rats. Inasmuch as 5-aminosalicylic acid (the therapeutic moiety of sulfasalazine) has been reported to be a hydroxyl radical scavenger, we also assessed the effects of dimethylsulfoxide (another hydroxyl radical scavenger) on ischemia/reperfusion-induced gastric mucosal injury. In rats treated with dimethylsulfoxide, leakage of 51 Cr-labeled red blood cells and the area of gross mucosal lesions after ischemia/reperfusion were significantly reduced as compared with control rats. The results of this study support the contention that ischemia/reperfusion-induced gastric bleeding involves the hydroxyl radical and indicate that 5-aminosalicylic acid significantly attentuates this vascular injury.

The objective of this study was to determine whether superoxide mediates the leukocyte-endothelial cell interactions elicited by reperfusion (reoxygenation) of ischemic (hypoxic) tissues. Mesenteric and intestinal blood flows were reduced to 20% of control for 1 h, followed by 1 h of reperfusion. Sixty minutes after reperfusion, red blood cell velocity (Vr), leukocyte rolling velocity (Vw), and the number of adherent leukocytes were measured in mesenteric venules. Then, either human superoxide dismutase (hSOD), hydrogen peroxide-inactivated hSOD, or MoAb IB4 (a monoclonal antibody against the leukocyte adhesion molecule CD18) was injected intravenously. Ten minutes later, repeat measurements were obtained and compared with pretreatment values. hSOD attenuated reperfusion-induced neutrophil adherence and increased Vw/Vr, an index of the fracture stress between leukocytes and endothelium. Peroxide-inactivated hSOD did not alter any parameter. MoAb IB4 attenuated reperfusion-induced adherence but did not alter Vw/Vr. In a correlate study, cultured bovine microvascular endothelium was exposed to 30 min of anoxia, followed by 60 min of reoxygenation. Cat neutrophils were added during reoxygenation. Reoxygenation-induced leukocyte adherence was attenuated by either hSOD or MoAb IB4 but not by inactivated hSOD. Adherence of phorbol 12-myristate 13-acetate-activated cat neutrophils to plastic was unaffected by hSOD or inactive hSOD, yet MoAb IB4 virtually abolished the response. These results indicate that superoxide mediates reperfusion-induced leukocyte adherence and that endothelial cells are required for this superoxide-mediated adherence.

Xanthine oxidase-derived oxidants and leukocytes have been implicated in the microvascular injury associated with reperfusion of ischemic intestine. The objective of this study was to determine whether xanthine oxidase-derived oxidants play a role in the leukocyte-microvascular interactions initiated by ischemia-reperfusion. Adherence and extravasation of leukocytes were monitored in cat mesenteric venules subjected to 1 h of ischemia (blood flow reduced to 20% of control) and reperfusion. Leukocyte rolling velocity, vessel diameter, and red cell velocity were also measured in control (untreated) animals and in animals pretreated with either allopurinol or superoxide dismutase. The responses of venular blood flow, wall shear rate, and leukocyte rolling velocity to ischemia and reperfusion did not differ between the three experimental groups. In control animals, 1 h of ischemia was associated with significant adherence and extravasation of leukocytes with reperfusion greatly enhancing these responses. Allopurinol treatment did not alter the responses to ischemia per se, yet it largely prevented the further increment in adherence and extravasation associated with reperfusion. Superoxide dismutase treatment attenuated the leukocyte responses elicited by both ischemia and reperfusion. Our observations that both allopurinol and superoxide dismutase attenuate reperfusion-induced leukocyte adherence and extravasation are consistent with the hypothesis that xanthine oxidase-derived oxidants initiate the leukocyte infiltration induced by reperfusion of ischemic intestine.

These studies determine the levels of malate dehydrogenase isoenzymes in cardiac muscle by a steady state kinetic method which depends on the differential inhibition of these isoenzyme forms by high concentrations of oxaloacetate. This inhibition is similar to that exhibited by lactate dehydrogenase in the presence of high concentrations of pyruvate. The results obtained by this method are comparable in resolution to those obtained by CM-Sephadex fractionation and by differential centrifugation for the analyses of mitochondrial malate dehydrogenase and cytoplasmic malate dehydrogenase in tissues. The use of standard curves of percent inhibition of malate dehydrogenase activity plotted against the ratio of mitochondrial MDH activity to the total of mMDH and cMDH activities [ m (m + c) malate dehydrogenase ratio] (percent m -type) is introduced for studies of comparative mitochondrial function in heart muscle of different species or in different tissues of the same species.

Recent studies examining the effect of allopurinol on bacterial killing by leukocytes [Tubaro et al., Biochem. Pharmac. 29, 3018 (1980); Tritsch and Neiswander, Life Sci. 32, 1359 (1983)] have been interpreted by those authors as proof that xanthine oxidase is the major superoxide producing enzyme in activated leukocytes. To test the assertion that xanthine oxidase is involved in the production of superoxide by activated human neutrophils, the xanthine oxidase content of neutrophils was measured, and the effect of allopurinol on neutrophil functions, including superoxide production, was studied. Neutrophils were found to contain a level of xanthine oxidase insufficient to account for the flux of superoxide associated with neutrophil activation. Allopurinol did not inhibit superoxide production induced by opsonized zymosan, phorbol myristic acetate, or formylmethionylleucylphenylalanine. Furthermore, neither chemotaxis nor degranulation was affected by allopurinol. Allopurinol was also found ineffective in blocking superoxide-mediated carrageenan-induced foot edema in the rat. These studies are interpreted as evidence that xanthine oxidase is not a major superoxide-generating system in activated neutrophils as has been suggested by others.

Several different methods are currently used by numerous laboratories for the isolation of human neutrophils. These methods include partial purification by dextran sedimentation followed by water lysis, and more complete purification procedures utilizing discontinuous density gradients coupled with dextran sedimentation and in some cases hypotonic lysis. Some investigators refrain from using certain purification schemes because certain steps or reagents used in a particular method might adversely affect the functional parameter of the neutrophil they wish to measure. In spite of these concerns there has been no systematic comparison of the functional status of neutrophils prepared by the various methodologies. In this study we have compared 4 commonly used methods of isolation with neutrophil function. The results of this study indicate that while neutrophil yield and purity were determined by isolation procedure, all cells were equivalent with regard to chemotactic performance, ability to degranulate, and ability to produce superoxide and hypochlorous acid.

The objective of this study was to determine whether dimethylsulfoxide (DMSO) influences chemoattractant-induced leukocyte adherence in the intestinal microcirculation. The distal colon of Sprague-Dawley rats was prepared for intravital microscopic observation of 25-35 microns diameter venules located in the muscularis externa. The number and average velocity of rolling leukocytes and the number of leukocytes adherent to the venular endothelial surface were determined from recorded video images. After a control period of observation, either N-formyl-methionyl-leucyl-phenylalanine (FMLP) or leukotriene B4 (LTB4) was added to the serosal superfusion solution. Both FMLP (1 microM) and LTB4 (1 microM) consistently caused leukocytes to adhere to venular endothelium. DMSO, at concentrations ranging between 2.5 and 140 mM, inhibited FMLP-induced leukocyte adherence in a dose-related manner. LTB4-induced leukocyte adherence was also significantly attenuated by DMSO. Leukocyte rolling velocity and leukocyte flux were not affected by DMSO. These results indicate that DMSO significantly inhibits leukocyte adherence at concentrations commonly used for its hydroxyl radical scavenging properties.

Myeloperoxidase-catalyzed oxidation of chloride (Cl-) to hypochlorous acid (HOCl) resulted in formation of mono- and dichloramine derivatives (RNHCl and RNCl2) of primary amines. The RNCl2 derivatives could undergo a reaction that resulted in incorporation of the R moiety into proteins. The probable mechanism was attack of RNCl2 or an intermediate formed in the decomposition of RNCl2 on histidine, tyrosine, and cystine residues and on lysine residues at high pH. Incorporation of radioactivity from labeled amines into stable, high molecular weight derivatives of proteins was measured by acid or acetone precipitation and by gel chromatography and electrophoresis. Whereas formation of RNCl2 was favored at low pH, the subsequent incorporation reaction was favored at high pH. Up to several hours were required for the maximum amount of incorporation, which was less than 10% of the label in RNCl2. For the amines tested, incorporation was in the order histamine greater than 1,2-diaminoethane greater than putrescine greater than taurine greater than lysine greater than glucosamine greater than leucine greater than methylamine. Initiation of the reaction required HOCl, and oxidized forms of bromide, iodide, or thiocyanate did not substitute. Inhibitors of incorporation fell into three classes. First, ammonia or amines competed with the labeled amine for reaction with HOCl, so that larger amounts of HOCl were required. Second, readily oxidized substances such as sulfhydryl or diketo compounds or thioethers (methionine) reduced RNCl2. Third, certain compounds competed with protein as the acceptor for the incorporation reaction. The amount required to block incorporation into protein depended on protein concentration. Among these inhibitors were imidazole compounds (histidine), phenols (tyrosine), and disulfides (glutathione disulfide, GSSG). Low yields of derivatives of histidine, tyrosine, and GSSG were detected by thin-layer chromatography. Acid-precipitable derivatives were obtained by reacting RNCl2 with polyhistidine or polytyrosine, and to a lesser extent with polylysine at high pH, but not with other poly(amino acids). Precipitable derivatives were also obtained by incubating MPO-containing extracts from leukocyte granules with hydrogen peroxide, Cl-, and labeled amines. The extracts were found to have a high content of substances with primary amino groups, which competed for incorporation. The results account for oxidative incorporation of amines into proteins in leukocytes and provide evidence that HOCl and nitrogen-chlorine (N-Cl) derivatives are formed in these cells. The characteristics of the incorporation reaction suggest that it would not contribute significantly to the antimicrobial activity of myeloperoxidase (MPO). Nevertheless, the reaction may provide a sensitive method for studying MPO action in vivo.

Previously, we documented that endotoxin induces bacterial translocation from the gut and that inhibition or inactivation of xanthine oxidase activity reduces endotoxin-induced bacterial translocation. Consequently, experiments were performed to correlate endotoxin-induced bacterial translocation with changes in intestinal mucosal structure and xanthine dehydrogenase and oxidase activity. Segments of the jejunum, ileum, cecum, proximal colon, distal colon, and liver were harvested from ICR mice 24 hr after IP administration of E. coli 0111:B4 endotoxin (0.1 mg). Xanthine dehydrogenase and oxidase activities were measured in these samples and correlated with intestinal morphology. Bacteria translocated from the intestines to extraintestinal organs in 70% of the mice receiving endotoxin, while the organs of control mice were sterile (p less than 0.01). Endotoxin injured primarily the ileal and cecal mucosa and increased ileal and hepatic xanthine dehydrogenase and cecal oxidase activities (p less than 0.05). These results suggest that xanthine oxidase-induced mucosal damage plays a role in endotoxin-induced bacterial translocation.

To determine whether leukocytes play an important role in the pathogenesis of the vascular injury (increased vascular permeability and resistance) associated with ischemia-reperfusion, isolated canine gracilis muscles were perfused with autologous whole blood or with whole blood that had been depleted of leukocytes (primarily granulocytes) using Leukopak filters. The osmotic reflection coefficient for total plasma proteins, isogravimetric capillary pressure, and total vascular resistance was determined for the following conditions: control, ischemia (4 h inflow occlusion) plus reperfusion with whole blood, and ischemia plus reperfusion with granulocyte-depleted whole blood. Reperfusion with whole blood was associated with a reduction in the osmotic reflection coefficient from 0.96 to 0.61, whereas isogravimetric capillary pressure was reduced by 40%, indicating a dramatic increase in vascular permeability. Total vascular resistance was increased approximately twofold. Reperfusion with leukocyte-depleted blood largely prevented the increases in vascular permeability and resistance. These data suggest that leukocytes play a major role in the pathogenesis of ischemia-reperfusion injury in skeletal muscle.

The major objective of the present study was to characterize the sequence of events leading to endothelial cytotoxicity induced by oxidants generated extracellularly by xanthine oxidase. 51Cr-labeled monolayers of calf pulmonary artery endothelial cells were exposed to a reaction mixture containing hypoxanthine, xanthine oxidase, and chelated iron (HX/XO) and endothelial cell injury was quantitated as 51Cr release into the media. Catalase, but not mannitol or superoxide dismutase, prevented endothelial cell injury induced by HX/XO, indicating that H2O2 was the mediator of the cytotoxicity. Pretreatment of the cells with free deferoxamine (an iron chelator), but not with deferoxamine bound to dextran (mol wt 40,000), prevented endothelial cell injury induced by HX/XO or H2O2. Of the membrane-permeant hydroxyl radical scavengers dimethylsulfoxide and dimethylthiourea, only dimethylthiourea prevented 1) HX/XO or H2O2-induced endothelial cytotoxicity and 2) deoxyribose degradation by hydroxyl radicals (.OH) generated by an iron-catalyzed reaction on the sugar (site-specific reaction). The concentration of ferritin required to produce significant quantities of .OH was much greater than that present in endothelial cells, and ferritin-catalyzed .OH formation was not affected by deferoxamine, indicating that ferritin-bound iron is most likely not the physiologically active catalyst. We conclude that extracellularly generated H2O2 can enter the cell and interact with nonferritin iron to produce the cytotoxic .OH via a site-specific reaction.

We have previously shown that phorbol myristate acetate (PMA) produces acute lung injury in blood-perfused lungs but not in plasma-dextran-perfused lungs. This is compatible with the concept that its major mechanism of injury is the stimulation of O2 radicals by neutrophils, which in turn increase permeability by damaging the endothelial cells. In this study we measured vascular permeability and resistance before and 1 h after PMA in five groups of blood-perfused dog lungs: PMA alone in one group and pretreatment with catalase, superoxide dismutase, deferoxamine, and adenosine each in four other groups. By the use of two indexes of permeability, the filtration coefficient and the isogravimetric capillary pressure, we found that, compared with PMA alone, catalase, deferoxamine, and adenosine provided significant protection, whereas the results with superoxide dismutase were variable. These four drugs also significantly attenuated the marked increased resistance seen with PMA alone. Although the effects seen with the first three can be explained by their scavenging of O2 radicals, adenosine appears to provide protection through a separate mechanism.

Both experimental colitis and human inflammatory bowel disease are characterized by an increased colonic blood flow. The objective of this study was to define the role of neutrophils in the colonic hyperemia associated with acetic acid-induced colitis in rats. One, two, and five days after the acetic acid enema, the colon was separated into five segments. Regional blood flow to each segment was measured using the radioactive microsphere technique. Tissue-associated myeloperoxidase activity was used as an index of neutrophil infiltration. Rectal blood flow and myeloperoxidase activity increased progressively after the acetic acid enema. At 5 days there were 3.9- and 4.6-fold increases in myeloperoxidase activity and blood flow, respectively. Comparable changes were noted in all bowel segments. The results suggest a temporal relationship between colonic blood flow and the extent of neutrophil infiltration. To assess directly the role of circulating and infiltrated neutrophils as mediators of the colitis-induced hyperemia, animals were rendered neutropenic approximately 8 h before the enema and neutropenia was maintained for another 24 h. Neutropenia did not modify the colitis-induced intestinal hyperemia normally observed at 24 h. We conclude from these findings that vasoactive agents derived from neutrophils do not mediate the increased colonic blood flow in this model of ulcerative colitis.

Stimulation of the oxygen (O2) metabolism of isolated human neutrophilic leukocytes resulted in oxidation of hemoglobin of autologous erythrocytes without erythrocyte lysis. Hb oxidation could be accounted for by reduction of O2 to superoxide (O-2) by the neutrophils, dismutation of O-2 to yield hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), the reaction of HOCl with endogenous ammonia (NH+4) to yield monochloramine ( NH2Cl ), and the oxidative attack of NH2Cl on erythrocytes. NH2Cl was detected when HOCl reacted with the NH+4 and other substances released into the medium by neutrophils. The amount of NH+4 released was sufficient to form the amount of NH2Cl required for the observed Hb oxidation. Oxidation was increased by adding myeloperoxidase or NH+4 to increase NH2Cl formation. Due to the volatility of NH2Cl , Hb was oxidized when neutrophils and erythrocytes were incubated separately in a closed container. Oxidation was decreased by adding catalase to eliminate H2O2, dithiothreitol to reduce HOCl and NH2Cl , or taurine to react with HOCl or NH2Cl to yield taurine monochloramine . NH2Cl was up to 50 times more effective than H2O2, HOCl, or taurine monochloramine as an oxidant for erythrocyte Hb, whereas HOCl was up to 10 times more effective than NH2Cl as a lytic agent. NH2Cl contributes to oxidation of erythrocyte components by stimulated neutrophils and may contribute to other forms of neutrophil oxidative cytotoxicity.

Gastric mucosal clearance of 51Cr-labeled red blood cells (51Cr-RBC) was measured in rats during a 30-min control period, a 30-min ischemic period (hemorrhage to 27 mmHg arterial pressure), and a 60-min reperfusion period (reinfusion of shed blood). In untreated (control) rats, a dramatic rise in the leakage of 51Cr-labeled red blood cells into the gastric lumen was observed during the reperfusion period. Treatment with neutrophil antiserum attenuated 51Cr-labeled red blood cell flux into the gastric lumen. Using the radioactive microsphere technique, neutrophil-depleted animals were shown to have higher blood flows in the ischemic period than the untreated rats. Bleeding of untreated rats to a mean arterial pressure of 40 mmHg resulted in blood flows that were not different from those in antiserum-treated rats bled to 27 mmHg and leakage of 51Cr-labeled red blood cells similar to that measured in antiserum-treated rats. The results of this study indicate that neutrophils play an important role in hemorrhagic shock-induced gastric bleeding.

Recent studies indicate that polymorphonuclear neutrophils (PMNs) infiltrate the intestinal mucosa during ischemia and after reperfusion. To determine whether PMNs mediate the increased microvascular permeability produced by ischemia-reperfusion (I/R) we treated cats with either saline, antineutrophil serum (ANS), or a monoclonal antibody specific for the beta-chain of the CD18 complex (MoAb 60.3) that prevents neutrophil adherence and extravasation. Intestinal microvascular permeability to plasma proteins was measured in control preparations (0.08 +/- 0.007), in preparations subjected to 1 h of ischemia then reperfusion (I/R, 0.32 +/- 0.02), I/R preparations treated with ANS (0.13 +/- 0.01), and I/R preparations treated with MoAb (0.12 +/- 0.003). Our results indicate that both PMN depletion (to less than 10% control) and prevention of PMN adherence significantly attenuate the increased microvascular permeability induced by I/R. These findings, coupled to previous results obtained from this model, support the hypothesis that neutrophils, which accumulate in the mucosa in response to xanthine oxidase activation, mediate the oxyradical-dependent injury produced by reperfusion of the ischemic bowel.

Recent studies have implicated oxygen free radicals in ischemia-reperf usion injury to the gastric mucosa. The aims of the present study were to (a) test the hypothesis that the enzyme xanthine oxidase is the source of the oxygen radicals in the ischemic stomach and (b) determine the importance of the ironcatalyzed Haber—Weiss reaction in,generating the cytotoxic oxygen radicals. Gastric mucosal clearance of 5 'Cr-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 reperf usion period (reinf usion of shed blood). In untreated (control) rats, a dramatic rise (100-fold) in the leakage of 51 Cr-labeled red blood cells into the gastric lumen was observed only during the reperf usion period. A f ter the reperf usion 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 de f eroxamine (an iron chelating agent) or superoxide dismutase (a superoxide scavenger). All three interventions substantially reduced 51 Cr-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 ironcatalyzed Haber—Weiss reaction is largely responsible for hydroxyl radical generation in this model.

The lung is especially sensitive to a variety of vastly different agents and conditions including hyperoxia, certain drugs and xenobiotics, particulate debris, and ischemia/reperfusion. There is a growing body of experimental data to suggest that most, if not all, of these agents or conditions mediate pulmonary injury by forming reactive O2 metabolites such as O2-., H2O2.OH, HOCl, and RNHCl. The presence mechanisms by which these different agents converge to produce free radical-mediated pulmonary injury is not entirely clear. The lung does contain several metabolic pathways that will produce large amounts of reactive O2 metabolites. For example, hyperoxia-induced pulmonary injury may be mediated by oxidants produced by both mitochondrial and microsomal electron transport. Certain drugs and xenobiotics may be metabolized by nonspecific flavoproteins found in the mitochondrial electron transport chain and associated with microsomal mixed function oxidase system to yield a variety of free radicals and oxidants. Inhalation of particulate debris will activate resident phagocytic leukocytes to produce large quantities of cytotoxic oxidants. Ischemia and reperfusion appear to produce substantial amounts of xanthine oxidase-derived oxy-radicals that recruit and activate inflammatory phagocytes to produce cytotoxic HOCl and N-chlorinated oxidants. Finally, inappropriate metabolism of arachidonate by prostaglandin synthetase in the presence of NADH (NADPH) produces a burst of O2-. The fact that the lung contains so many different metabolic avenues for oxidant and free radical production suggests that this particular organ may be the most sensitive to oxidative insult.

The gastrointestinal epithelium is continuously exposed to reactive oxygen metabolites that are generated within the lumen. In spite of this exposure, the healthy epithelium appears unaffected, suggesting efficient mechanisms for protection against these potentially cytotoxic oxidants. The objective of this study is to characterize the interaction between purified gastric mucin and hydroxyl radicals generated from the interaction between ferric iron and ascorbic acid. We found that both native and pronase-treated mucin effectively scavenged hydroxyl radical and that the scavenging properties were not significantly different. The effective concentration of mucin required for a 50% reduction in malondialdehyde production was approximately 10 mg/ml for both native and pronase-treated mucin. In addition, the iron-ascorbic system produced a dramatic decrease (greater than 50%) in the specific viscosity of mucin that was inhibited by catalase, deferoxamine, and mannitol. Superoxide dismutase had no effect. These data suggest that hydroxyl radicals derived from the iron-catalyzed decomposition of hydrogen peroxide are responsible for the depolymerization of native mucin. We propose that mucin may provide protection to the surface epithelium of the gastrointestinal tract by scavenging oxidants produced within the lumen; however, it does so at the expense of its viscoelastic properties.

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 51 Cr-ethylenediaminetetraacetate 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 ethylene diaminetetraacetate 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.