Nitric oxide as antiinflammatory agent.
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Animals↗
Biomedical subjects
P Kubes
Publications and source records attributed to P Kubes.
Nitric oxide synthase activity in portal-hypertensive and cirrhotic rats.
BACKGROUND/AIMS: The hyperdynamic circulation of cirrhosis and portal hypertension has been postulated to be due to the vasodilatory effects of nitric oxide. However, studies using pharmacological inhibitors of nitric oxide synthase have yielded conflicting results. We aimed to measure nitric oxide synthase activity in tissues from two different rat models of cirrhosis and portal hypertension. METHODS: Cirrhosis was induced by chronic bile duct ligation, and prehepatic portal hypertension by portal vein stenosis. Controls were sham-operated. A fourth group was treated with lipopolysaccharide endotoxin. Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthase activity was assayed by measuring the conversion rate of 14C-arginine to 14C-citrulline in homogenates of stomach, jejunum, liver, kidney and aorta. RESULTS: Jejunal homogenates from the portal vein-stenosed rats showed a significant 10-fold elevation of Ca(2+)-dependent nitric oxide synthase activity. Cirrhotic rat kidney showed significantly decreased Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthase activity. Endotoxin treatment increased Ca(2+)-independent nitric oxide synthase activity in jejunum and liver. There was no increase in Ca(2+)-independent nitric oxide synthase activity in any tissues from cirrhotic or portal hypertensive rats. CONCLUSIONS: We conclude that the lack of increase in Ca(2+)-independent nitric oxide synthase activity does not support the hypothesis that nitric oxide is the major cause of hyperdynamic circulation in cirrhosis.
Animals↗
Monosodium urate crystals promote neutrophil adhesion via a CD18-independent and selectin-independent mechanism.
The primary objective of this study was to determine whether monosodium urate (MSU) crystals induced neutrophil adhesion to cellular substrata and, if so, then to elucidate the molecular mechanisms involved. Human umbilical vein endothelial cells (HUVEC), as well as various other cellular substrata, were treated with various sized MSU crystals, washed, and then coincubated in the presence of neutrophils for 60 min. HUVEC exposed to MSU crystals but not to silica crystals or uric acid promoted neutrophil adhesion in a dose- and size-dependent manner, an event also observed with monolayers of rabbit synovial cells and rat intestinal epithelial cells. The increased neutrophil adhesion could not be attenuated by anti-CD18, anti-intracellular adhesion molecule-1, or various anti-selectin antibodies, despite the fact that scanning electron microscopy revealed that neutrophils were adhering primarily to the endothelial cells rather than to exposed crystals. CD18-deficient neutrophils adhered to MSU crystal-treated HUVEC as effectively as their CD18-positive counterparts. The neutrophil adhesion was temperature dependent but did not require protein synthesis. Additionally, HUVEC phagocytosis of crystals was necessary for subsequent neutrophil-endothelial cell interactions to transpire. Pretreatment of endothelial cells and neutrophils with colchicine significantly reduced the adhesive interaction. Our data demonstrate that exposure of endothelial and other cells to MSU crystals promotes neutrophil adhesion that occurs by a firm CD18-independent and selectin-independent adhesive mechanism.
Antibodies, Monoclonal↗
A critical role for nitric oxide in intestinal barrier function and dysfunction.
There is growing evidence that endogenous nitric oxide (NO) regulates mucosal barrier integrity under physiological conditions and counters the increase in mucosal permeability associated with acute pathophysiological states. The potential mechanisms of action for the protective effects of NO are discussed. These include maintenance of blood flow, inhibition of platelet and leukocyte adhesion and/or aggregation within the vasculature, modulation of mast cell reactivity, and scavenging of reactive oxygen metabolites such as superoxide. On the basis of the data presented, we conclude that both constitutive nitric oxide synthase (cNOS)-derived endogenous NO and exogenous NO (from NO donors) appear to reduce the sequelae of acute inflammation. The second section of this review summarizes the data germane to prolonged (chronic) inflammatory conditions associated with the overproduction of NO from the inducible form of NOS (iNOS). Some emphasis is placed on the role of NO in sepsis and inflammatory bowel disease (IBD), and data to suggest that NO, or more specifically a NO-derived mediator, is involved in these disorders are summarized. These studies are compared with recent publications suggesting that inhibition of NO synthesis with nonspecific inhibitors of NOS or selective iNOS inhibitors may not protect in models of sepsis or IBD. Overall, the review highlights the potential importance of the type of NOS enzyme involved in the particular inflammatory process being studied.
Animals↗
Endogenous glucocorticoids inhibit neutrophil recruitment to inflammatory sites in cholestatic rats.
Since glucocorticoids have been shown to inhibit leukocyte accumulation to inflammatory sites (6,9) and endogenous glucocorticoid levels are elevated in our cholestatic rat model, we investigated their role during acute inflammation. Sprague-Dawley rats (150-200 g) were either bile duct resected (BDR) or sham resected (sham). Five days later, a 2% carrageenan solution in saline was injected subcutaneously into preformed air pouches on their backs. Animals were killed 5 h later, and inflammatory response was quantitated by measuring the exudate volume, cell count, and myeloperoxidase (mainly in neutrophils) activity. We also pretreated BDR/sham rats with RU-486 (2 mg/kg ip), a glucocorticoid receptor antagonist, 1 h before carrageenan injection. BDR rats exhibited an impaired inflammatory response reflected by 20, 52, and 42% decreases in exudate volume, cell count, and myeloperoxidase activity, respectively (all P < 0.05). RU-486 treatment significantly increased the inflammatory response in BDR rats (to sham levels), but not in sham rats. These results suggest that endogenous glucocorticoid suppresses the inflammatory response in BDR rats.
Animals↗
Hydrogen peroxide induces leukocyte rolling: modulation by endogenous antioxidant mechanisms including NO.
In this study, intravital microscopy was used to examine the mechanisms that regulate H2O2-induced leukocyte rolling within rat mesenteric venules in vivo. H2O2 elicited leukocyte rolling within a narrow response window between 10 and 500 microM H2O2. Continuous superfusion with 100 microM H2O2 induced a large but transient increase in the flux of rolling leukocytes, whereas a short 5-min pulse elicited a sustained increase in rolling flux. Both treatments caused increases in leukocyte adhesion. H2O2-induced increases in leukocyte flux and adhesion could be prevented with an anti-P-selectin antibody. Inhibition of endogenous catalase (aminotriazole), glutathione (diethyl maleate), or nitric oxide (NG-nitro-L-arginine methyl ester) shifted the effective concentration of H2O2; continuous superfusion with 10 microM H2O2 now elicited large and sustained increases in leukocyte rolling flux, whereas 100 microM H2O2 elicited less than optimal responses. Dual antioxidant inhibition further reduced the effective H2O2 concentration to 1 microM H2O2. A nitric oxide donor prevented the increased rolling flux induced by 100 microM H2O2. These findings suggest that endogenous antioxidants are important regulators of H2O2-induced, P-selectin-dependent leukocyte rolling in vivo.
Animals↗
Rapid mast cell activation causes leukocyte-dependent and -independent permeability alterations.
The major objective of this study was to systematically elucidate the mechanisms underlying microvascular permeability in rat mesenteric venules after the activation of perivascular mast cells. Intravital microscopy was used to assess polymorphonuclear leukocyte (PMN) infiltration and microvascular permeability alterations in single 25- to 40-micron diameter venules. Ruthenium red was used to detect mast cell activation on-line. Exposure of mast cells to compound 48/80 (CMP 48/80) caused a rapid mast cell activation and increase in microvascular permeability (within 15 min) that was maintained for the duration of the experiment. CMP 48/80 also increased PMN adhesion to the microvascular endothelium. Anti-PMN serum, as well as various antiadhesion therapies, including CL26 (anti-CD18 antibody) and fucoidan (selectin-immunoneutralizing carbohydrate), revealed that the early microvascular permeability was PMN independent. However, these regimens significantly reduced plasma protein leakage out of venules between 30 and 60 min. Methysergide (serotonin receptor antagonist), but not diphenhydramine (histamine receptor antagonist), inhibited the early PMN-independent microvascular permeability. Finally, a platelet-activating factor (PAF)-receptor antagonist did not affect the early phase of microvascular permeability but reversed the later phase, consistent with PAF's role as a proadhesive molecule for PMN during mast cell activation. These data demonstrate that, within the first hour of mast cell activation, a biphasic PMN-independent and -dependent response in microvascular permeability is observed. The data also raise the possibility that histamine's physiological role in this model may be unrelated to alterations in microvascular permeability.
Animals↗
A balance between nitric oxide and oxidants regulates mast cell-dependent neutrophil-endothelial cell interactions.
Nitric oxide (NO) synthesis inhibition causes neutrophil adhesion to endothelium via a mast cell- and oxidant-dependent mechanism. The objective of this study was to delineate the cascade of events in the mast cell- and oxidant-induced neutrophil-endothelium interactions after NO synthesis inhibition. Mast cells were isolated and purified from the rat peritoneal cavity and coadministered with neutrophils to wells of endothelium. This system was treated with an NO synthesis inhibitor (NG-nitro-L-arginine methyl ester; L-NAME) for 60 minutes. L-NAME did not induce neutrophil-endothelium interactions in the absence of mast cells, but the addition of mast cells in a ratio as low as 1:50 mast cells to neutrophils was sufficient to induce a large increase in neutrophil adhesion to endothelium within 20 to 25 minutes. L-arginine, NO donors, and 8-bromo-cGMP reversed the L-NAME effect, whereas NG-nitro-D-arginine methyl ester alone had no proadhesive effect. The adhesion was inhibited by an anti-CD18 or an anti-intracellular adhesion molecule-1 antibody and a platelet-activating factor-receptor antagonist. Inhibition of NO in isolated endothelial monolayers induced oxidant release (reduction of cytochrome C) into extracellular fluid. The endothelium-derived superoxide contributed to the mast cell-induced adhesion, inasmuch as the extracellular antioxidant superoxide dismutase reduced the neutrophil adhesion response as did disruption of endothelial function. There was some direct activation of mast cells with L-NAME (independent of endothelium) inasmuch as intracellular calcium and oxidative stress increased within mast cells after L-NAME treatment, and this translated into increased neutrophil adhesion to nonendothelial substrata. These data demonstrate that depletion of NO increases oxidative stress within mast cells and endothelium and together these events promote neutrophil adhesion within the vasculature.
Animals↗
Leukocyte-endothelial cell interactions evoked by mast cells.
In this review we have summarized some of the evidence to support the view that mast cells play a critical role in leukocyte recruitment to sites of inflammation. Initially, data using a pharmacological tool, compound 48/80, which directly activates mast cells, is reviewed, demonstrating that this reagent can induce the multi-step recruitment of leukocytes (rolling, adhesion and emigration) to sites of inflammation. The adhesive mechanisms and pro-inflammatory mediators implicated in mast cell-induced leukocyte recruitment are discussed. Additionally, data are presented to implicate mast cells in delayed-type hypersensitivity reactions as they pertain to leukocyte recruitment. There is a growing body of evidence to suggest that mast cells also recruit leukocytes in IgE-independent leukocyte recruitment. Ischemia/reperfusion- and bacterial toxin- (Helicobacter pylori and Clostridium difficile) induced leukocyte recruitment is at least in part mast cell dependent. Future directions including preliminary work highlighting the role of nitric oxide as a modulator of mast cell function and subsequent leukocyte recruitment is also discussed.
Endothelium, Vascular↗
Desmopressin induces endothelial P-selectin expression and leukocyte rolling in postcapillary venules.
Desmopressin, (DDAVP; 1-desamino-8-D-arginine vasopressin) increases the release and activity of von Willebrand factor (vWF); however, its effects on the other major constituent of endothelial Weibel-Palade bodies, P-selectin, has not been investigated. DDAVP-induced P-selectin expression may explain DDAVP's efficacy in bleeding disorders in which vWF levels are normal. Therefore, the objective of this study is to assess the effect of DDAVP on P-selectin expression on endothelial cells of postcapillary venules in vivo and on human umbilical vein endothelium in vitro, and to determine whether DDAVP has direct effects on leukocyte behavior in postcapillary venules. DDAVP (0.1 and 1.0 microgram/mL) induced a significant but transient increase in P-selectin expression on human umbilical vein endothelial cells as well as on rat and human platelets. Immunohistochemical analysis of rat postcapillary venules showed that in contrast to saline, DDAVP injection (1 microgram/kg, intravenous) induced significant endothelial P-selectin expression. DDAVP administration also induced a rapid and significant increase in leukocyte rolling in rat mesenteric venules in vivo. This response was entirely dependent on P-selectin, as an anti-P-selectin antibody rapidly reversed the DDAVP-induced increase in leukocyte rolling. DDAVP induced leukocyte rolling in medium (20 to 40 microns) and large (> 40 microns), but not small (< 20 microns), postcapillary venules. In animals that were treated with DDAVP, there was a steady and significant increase in leukocyte adhesion. This study shows that DDAVP can directly induce P-selectin expression on endothelium in vitro and in vivo and that the latter response is capable of supporting prolonged leukocyte rolling in rat postcapillary venules.
Animals↗
Mechanisms underlying acute mast cell-induced leukocyte rolling and adhesion in vivo.
It has been proposed that a primary detector mechanism for tissue infection or injury may be the mast cell that releases agents that recruit leukocytes to the appropriate site at risk. The objective of this study was to evaluate the early mechanisms involved in mast cell-induced leukocyte recruitment. We used intravital microscopy to visualize leukocyte-rolling flux and adhesion in single 25 to 40 microns venules in mesenteric preparations that were treated with the mast cell-degranulating agent, compound 48/80 (CMP 48/80). Superfusion of the rat mesentery with CMP 48/80 caused a dose-dependent rise in the number of rolling and adherent cells, events significantly reduced by: 1) mast cell stabilizers, ketotifen, or cromolyn, and 2) chronic treatment with CMP 48/80 to deplete mast cell constituents. The increase in leukocyte flux associated with CMP 48/80 was blocked by diphenhydramine (H1-receptor antagonist) and an anti-P-selectin Ab (PB1.3), but not by the 5-lipoxygenase inhibitor, MK 886. The reduction in the flux of rolling leukocytes translated into fewer adherent leukocytes with diphenhydramine or PB1.3. The CMP 48/80-induced rise in leukocyte adhesion, but not leukocyte flux, was reduced by the platelet-activating factor (PAF)-receptor antagonist (WEB 2086) and an anti-CD18 Ab (CL26). MK 886 did not prevent the increased leukocyte adhesion. In vitro data revealed that mast cell-derived PAF induced essentially all of the leukocyte adhesion to endothelium or protein-coated plastic. These data suggest that mast cell degranulation induces P-selectin-dependent leukocyte rolling and CD18-dependent leukocyte adhesion via histamine and PAF, respectively.
Animals↗
Neutrophil adhesion is impaired in rat model of cholestasis.
BACKGROUND & AIMS: A high incidence of septic complications has been documented in patients with obstructive jaundice undergoing surgery. Because neutrophils play a central role in the response to infection, the aim of this study was to investigate neutrophil function in a rat model of acute cholestasis induced by bile duct resection. METHODS: Neutrophil function was assessed using both in vivo and in vitro techniques. RESULTS: The inflammatory response was defective in vivo in bile duct-resected (BDR) rats compared with sham-resected controls. Furthermore, proinflammatory stimulus-induced neutrophil adherence and emigration from mesenteric values, assessed in vivo using intravital microscopy, was attenuated in BDR vs. sham rats. Neutrophils isolated from BDR and sham rats studied in vitro adhered to a rat biological substratum similarly. However, stimulated neutrophil adherence was eliminated in the presence of BDR but not sham plasma. Similar findings were observed using plasma obtained from a patient with obstructive cholestasis. Experiments performed to further characterize this antiadhesive factor in BDR plasma suggest that the factor is probably a heat-stable glycoprotein. CONCLUSIONS: Cholestasis in the rat is associated with a defective acute inflammatory response that appears, at least in part, to be caused by a constituent of cholestatic plasma that is antiadhesive to neutrophils.
Acute Disease↗
Excess nitric oxide does not cause cellular, vascular, or mucosal dysfunction in the cat small intestine.
The overproduction of nitric oxide in the small bowel has been invoked as a cytotoxic event in the vascular, mucosal, and whole organ dysfunction associated with inflammation. We assessed whether exogenous administration of nitric oxide in the form of nitric oxide donors (CAS 754, SIN-1) could cause microvascular and mucosal barrier dysfunction in vivo or epithelial and endothelial cell permeability alterations and cell injury in vitro. Increasing concentrations of CAS 754 or SIN-1 were infused locally into autoperfused segments of cat ileum at 30-min intervals. Baseline epithelial permeability (blood-to-lumen clearance of 51Cr-EDTA) was not affected by CAS 754, whereas vascular protein clearance was reduced. The latter effect could almost entirely be explained by a decrease in intestinal capillary hydrostatic pressure. Therefore, in some experiments venous pressure was elevated and the microvascular reflection coefficient for total proteins was estimated at filtration-independent rates. This direct measurement of microvascular permeability was unaffected by exogenous nitric oxide. CAS 754 did not increase permeability across monolayers of endothelial or epithelial cells and did not cause cell injury. Next, we assessed the possibility that excess nitric oxide may be detrimental, but only in inflamed intestine, by infusing CAS 754 with platelet-activating factor; the latter directly increases microvascular and mucosal permeability. CAS 754 did not exacerbate but rather reduced platelet-activating factor-induced rise in microvascular and mucosal permeability. These results suggest that high concentrations of nitric oxide do not cause breakdown of mucosal or microvascular barrier integrity under normal or inflammatory conditions.
Animals↗
Leukotriene C4/D4 induces P-selectin and sialyl Lewis(x)-dependent alterations in leukocyte kinetics in vivo.
The objective of this study was to assess the effect of leukotriene C4 (LTC4) on the flux of rolling leukocytes, leukocyte rolling velocity, and leukocyte adhesion in postcapillary venules in vivo and to study the underlying molecular mechanisms involved. LTC4 (20 nmol/L) induced a rapid and significant increase in leukocyte rolling flux that was inhibitable by an anti-P-selectin antibody and soluble sialyl Lewis(x) (sLe(x)). LTC4 also induced a significant reduction in leukocyte rolling velocity, an event that was independent of P-selectin but entirely dependent on sLe(x). This LTC4-induced reduction in leukocyte rolling velocity was independent of any hemodynamic alterations. Another P-selectin effector, histamine, did not affect leukocyte rolling velocity even at > 5000 times the concentration of LTC4. Treatment with an anti-L-selectin antibody had no effect on the LTC4-induced increase in leukocyte rolling or reduction in rolling velocity. Inhibition of LTC4 bioconversion to LTD4 by pretreatment with L-serine (100 mumol/L) prevented the LTC4-induced increase in leukocyte rolling flux and the LTC4-induced reduction in leukocyte rolling velocity. A subtle, yet significant, increase in leukocyte adhesion was also observed with LTC4. Pretreatment with a platelet-activating factor receptor antagonist returned the LTC4-induced leukocyte rolling velocity to baseline levels. The addition of a very low concentration of platelet-activating factor (1 nmol/L) induced significant leukocyte adhesion in the presence of LTC4 but not histamine.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Therapeutic potential of inhibiting leukocyte rolling in ischemia/reperfusion.
Leukocyte rolling has been postulated to be mandatory for subsequent leukocyte adhesion and tissue injury observed during ischemia/reperfusion. The objective of this study was to systematically assess this hypothesis at the microvascular level by examining the effects of various concentrations of a selectin-binding carbohydrate (fucoidin) on the increased rolling and adhesion of leukocytes in postischemic venules. The contribution of L-selectin and/or P-selectin to leukocyte rolling were also assessed in this model. Using intravital microscopy we observed that 60 min of ischemia followed by reperfusion caused a profound increase in leukocyte rolling and adhesion. A high dose of fucoidin (25 mg/kg) reduced leukocyte rolling by > 90% and significantly reduced leukocyte adhesion, whereas a lower dose of fucoidin still reduced leukocyte rolling by 60% but had no effect on leukocyte adhesion. Moreover, despite the profound reduction in leukocyte rolling with fucoidin, the remaining rolling cells were able to firmly adhere via a CD18-dependent mechanism, particularly in those postcapillary venules with reduced (30-50%) shear rates. The increased rolling was also reduced 60% by either an anti-P-selectin antibody, an anti-L-selectin antibody, or a combination of the two antibodies, but this reduction in rolling cells did not translate into significantly reduced leukocyte adhesion. Our data suggest that L-selectin, P-selectin, and a fucoidin-sensitive pathway contribute to the significant increase in reperfusion-induced leukocyte rolling. However, targeting leukocyte rolling as a form of therapy requires very significant efficacy (> 90%) to achieve reasonable (approximately 50%) attenuation in leukocyte adhesion in postischemic venules.
Animals↗
Nitric oxide affects microvascular permeability in the intact and inflamed vasculature.
There is a growing body of evidence that nitric oxide modulates the movement of fluid and protein out of the vasculature. However, the results obtained from whole organ, single vessel, and endothelial monolayer studies are often not consistent. In fact, there is much evidence that endogenous nitric oxide increases microvascular permeability and equally compelling evidence to suggest that endogenous nitric oxide decreases microvascular permeability. In this review we summarize and critically evaluate the data published to date regarding the role of nitric oxide as a modulator of microvascular permeability under both normal conditions and following exposure to various stimuli. The last section of the chapter discusses the importance of nitric oxide as a modulator of microvascular permeability in inflammatory conditions. Particular emphasis is placed on the stimuli used, the organ systems studied, and the measurements performed to investigate the effects of nitric oxide on microvascular permeability alterations.
Animals↗
A novel beta 1-dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin B or endothelial transmigration.
It is generally accepted that the beta 2-integrin is restricted to mononuclear leukocytes. The objective of this study was to determine whether neutrophils can also express beta 1-integrin (specifically alpha 4 beta 1) and whether this can support neutrophil adhesion to endothelial cells and to extracellular matrix. We stimulated neutrophils with dihydrocytochalasin B (DHCB) and various chemotactic stimuli and observed that chemotactic stimuli induced neutrophil adhesion via beta 2-integrin (CD18), whereas DHCB and either fMLP, PAF, or IL-8 induced adhesion to endothelium or protein-coated plastic that was not inhibitable by anti-CD18 antibody. beta 2-integrin-deficient cells, which did not respond to chemotactic stimuli alone, also adhered avidly in the presence of chemotactic stimuli and DHCB. The induced neutrophil adhesion was inhibited by antibody to beta 1- or alpha 4-integrin chains, but only if an anti-beta 2-integrin antibody was also present. Flow cytometry revealed increased expression of both beta 1 and alpha 4 in the presence of fMLP plus DHCB. Transendothelial migration of neutrophils induced by chemotactic stimuli alone also increased expression of beta 1 and alpha 4. Transmigration across deendothelialized membranes induced a similar beta 1 expression on neutrophils suggesting that events other than an endothelial signal elicited beta 1-integrin expression. Transmigration-induced beta 1-dependent expression translated into only modest adhesion to protein-coated plastic. These data suggest that both a pharmacological (DHCB) and a physiological (transmigration) stimulus can invoke expression of alpha 4 and beta 1 on human neutrophils to mediate adhesion.
Antigens, CD↗
Nitric oxide is an antiadhesive molecule for leukocytes.
There is growing evidence that nitric oxide (NO.), a biologically active gas continuously produced by endothelium, is a homeostatic regulator of leukocyte adhesion in the microcirculation. Inhibition of NO. production leads to increased leukocyte rolling and adhesion in various vascular beds and two adhesion molecules, P-selectin and CD11/CD18, have been implicated in these processes. The role of mast cells and mast cell-derived mediators as potential contributors to the increased adhesion are discussed in this review. Moreover, oxidants may initiate the leukocyte recruitment after NO. synthesis inhibition. Recent data demonstrating increased oxidative stress in endothelium deprived of NO. are summarized. The role of NO. as an anti-inflammatory and antiadhesive modulator in postischemic venules of various organs is also discussed. The beneficial effect of NO. donors in this inflammatory condition is summarized. Finally, the potential use of NO. donating drugs in concert with available pharmaceutical compounds to reduce inflammation is reviewed.
Animals↗