Functional alpha4-integrin: a newly identified pathway of neutrophil recruitment in critically ill septic patients.

Using a novel flow chamber assay system and whole blood, we show that leukocytes from septic individuals have a four-fold elevation of adhesion, but not rolling, on a P-selectin/beta2-integrin substrate. Most leukocytes from septic patients (but not healthy controls) that bound vascular cell adhesion molecule 1 (VCAM-1) were neutrophils. All adhesion was inhibited with an antibody specific for the VCAM-1 ligand alpha4-integrin. The alpha4-integrin was present on neutrophils from septic patients but not on neutrophils from patients with localized bacterial infections. The plasma milieu of septic patients was sufficient to induce neutrophils from healthy subjects to bind VCAM-1 under flow conditions. This is the first description of alpha4-integrin/VCAM-1 pathway of neutrophil recruitment in human disease. This pathway may provide a new therapeutic target to reduce inappropriate neutrophil adhesion without altering the normal yet critical beta2-integrin-mediated adhesive function of neutrophils.

Anaphylaxis-induced mesenteric vascular permeability, granulocyte adhesion, and platelet aggregates in rat.

This study investigates the response of small venules to IgE-dependent, antigen-mediated mast cell activation. Intravital microscopy was utilized to visualize 25- to 40-micron mesenteric venules, mast cell degranulation (on-line detection), vascular permeability changes (albumin leakage), leukocyte adhesion, and the formation of platelet aggregates in rats sensitized with 10 microg of intraperitoneal egg albumin (EA) in saline- or sham-sensitized (saline alone) rats. Sensitized rats challenged with EA (1 mg/ml superfusing mesentery), but not sensitized rats challenged with BSA or sham-sensitized rats challenged with EA, exhibited mast cell degranulation with significant time-dependent increases in vascular permeability (inhibited by diphenhydramine, salbutamol, and indomethacin), leukocyte adhesion (inhibited by Web-2086), and the formation of cellular aggregates (platelet), which were associated with intermittent obstruction of venular flow. Anti-platelet antibody, but not anti-neutrophil antibody or fucoidin (selectin antagonist), prevented platelet aggregate formation. Compound 48/80-induced mast cell degranulation caused similar changes in permeability (via different mediators) and leukocyte adhesion but did not induce platelet aggregation. EA-induced platelet aggregation was not inhibited by any of the mediators tested, and platelets isolated from sensitized rats failed to aggregate in response to direct EA challenge, suggesting release of an unidentified inflammatory mediator as the factor initiating platelet aggregation.

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.

Role of platelet-activating factor in ischemia/reperfusion-induced leukocyte adherence.

The objective of this study was to determine whether platelet-activating factor (PAF) mediates the leukocyte-endothelial cell interactions elicited by ischemia/reperfusion. The rates of adherence and extravasation of leukocytes were monitored in cat mesenteric venules subjected to 60 min of ischemia (blood flow reduced to 20% of control) followed by 60 min of reperfusion. Leukocyte rolling velocity, red blood cell velocity, and vessel diameter were also measured. The experiments were performed in control (untreated) animals and in animals pretreated with one of two PAF receptor antagonists, i.e., BN 52021 or WEB 2086. The responses of venular blood flow, wall shear rate, and vessel diameter did not differ between the three groups. In the control group, 1 h of ischemia was associated with significant adherence and extravasation of leukocytes, with reperfusion greatly enhancing these responses. The rates of leukocyte adherence and extravasation during reperfusion were greatly attenuated by both PAF antagonists. Furthermore, the proportion of adherent leukocytes that ultimately extravasate during reperfusion was markedly reduced by WEB 2086. These results suggest that PAF plays an important role in mediating the adhesive interaction between circulating leukocytes and microvascular endothelium induced by ischemia/reperfusion and that the phospholipid promotes the leukocyte extravasation associated with ischemia/reperfusion.

Endotoxin-induced ascites formation in the rat: partial mediation by platelet-activating factor.

Systemic endotoxemia has been observed in patients with acute and chronic liver failure, and bacterial endotoxin is known to increase vascular permeability. We investigated in the normal rat the effects of intraportal endotoxin administration and the possible mediation of these effects by platelet-activating factor. Injection of endotoxin lipopolysaccharide (10 and 25 mg per kg) in the rat resulted in rapid ascites formation, as well as systemic hypotension, hemoconcentration and acute erosions of the gastrointestinal mucosa. These effects were significantly attenuated by pretreatment with L652,731 and CF-3988, specific platelet-activating factor antagonists. Administration of 25 mg per kg endotoxin also resulted in significant elevations of platelet-activating factor biosynthesis in vitro by samples of duodenum, liver and lung. The effects of endotoxin were mimicked by intraportal infusion of platelet-activating factor (50 ng per kg per min), which induced ascites and gastrointestinal lesions. Platelet-activating factor reduced circulating plasma volume and increased peritoneal permeability to albumin as assessed by the ascites to plasma ratio of labeled albumin. These results, therefore, support a role for platelet-activating factor in mediating endotoxin-induced ascites and gastrointestinal erosions.

Inhibitory effects of dexamethasone in endotoxic shock and its relation to PAF-acether synthesis in the gastrointestinal tract and lung.

Endotoxic shock is accompanied by significant increases in PAF-acether synthesis, particularly by the lung. The onset of tissue damage and increases in vascular permeability in the gastrointestinal tract correlate temporally with the changes in PAF-acether synthesis and have previously been shown to be inhibited by PAF-acether antagonists. In the present study, the effects of pretreatment with dexamethasone on endotoxin-induced hemoconcentration and hypotension were examined in the rat. Furthermore, the effects of dexamethasone on PAF-acether synthesis and gastrointestinal vascular permeability following administration of endotoxin were also studied. Pretreatment with dexamethasone resulted in a significant attenuation of endotoxin-induced hemoconcentration, hypotension and damage in the duodenum and stomach. Dexamethasone also significantly reduced PAF-acether synthesis by the lung. However, dexamethasone pretreatment had no significant effect on endotoxin-induced increases in PAF-acether release and vascular permeability in the gastrointestinal tissues. The mechanism of the protective actions of dexamethasone may be related to inhibition of the release of PAF-acether from the lung. PAF released from gastrointestinal tissues likely contributes little to the systemic disturbances in endotoxic shock.

Beneficial effects of prostaglandin E2 in endotoxic shock are unrelated to effects on PAF-acether synthesis.

The effects of endotoxic shock on the synthesis of PAF-acether by the stomach, duodenum and lung were examined in the rat. Furthermore, the effect of pretreatment with prostaglandin E2 on endotoxin induced PAF-acether synthesis and changes in vascular permeability were examined. Administration of endotoxin resulted in significant increases in PAF-acether synthesis in all tissues studied. Such increases were apparent within 5-15 minutes of the administration of endotoxin, corresponding to the time when significant hypotension, hemoconcentration and increases in gastrointestinal vascular permeability were first observed. Pretreatment with prostaglandin E2 resulted in a significant reduction of endotoxin-induced hypotension, hemoconcentration and changes in vascular permeability in the gastrointestinal tract. However, prostaglandin pretreatment did not significantly alter endotoxin-induced PAF-acether release from the gastrointestinal tissues studied. These results demonstrate that prostaglandin E2 can significantly attenuate several of the systemic and gastrointestinal manifestations of endotoxic shock. The mechanism responsible for these beneficial actions appears to be unrelated to effects of prostaglandin E2 on PAF-acether synthesis.

Assessment of the role of platelet-activating factor in an animal model of inflammatory bowel disease.

Using a rat model of chronic colitis, the role of PAF-acether as a mediator of intestinal inflammation was assessed. Rats were treated with a specific PAF-acether antagonist, BN52021, during the first 4 days after induction of colitis, or during the period of 4-7 days after induction of colitis. The effects of treatment with BN52021 were compared to those of treatment with 5-aminosalicylic acid. BN52021 and 5-aminosalicyclic acid were without significant effect on colonic damage score when administered intracolonically on days 1-4 after induction of colitis. However, when given on days 4-7 after induction of colitis, both drugs significantly accelerated the healing of ulcers and reduced the incidence of adhesions and diarrhea. Intraperitoneal administration of BN52021 also resulted in a significant reduction of colonic damage scores, while administration of 5-aminosalicyclic acid via this route was without significant effect. Using an in vitro superfusion system, the effects of PAF-acether on contractility of segments of ascending colon were assessed. Tissue segments from normal rats contracted to doses of PAF-acether as low as 0.5 pg. However, non-inflamed segments of ascending colon from rats in which colitis was induced 2 weeks earlier were relatively insensitive to PAF-acether. The results of the present study demonstrate that PAF-acether is unlikely to play an important role in the acute inflammatory response in this model, but may be important in the prolongation of inflammation and ulceration in this model. The studies on contractility of ascending colon suggest that changes in tissue sensitivity to PAF-acether may occur as a consequence of inflammation.

The fate of homologous 125I-labelled immunoglobulin G within rat visceral yolk sacs incubated in vitro.

When 125I-labelled rat IgG (immunoglobulin G) is incubated in vitro with visceral yolk sacs from 17.5-day-pregnant rats, the protein is readily degraded. The major radioactive digestion product that accumulates in the medium is [125I]iodo-L-tyrosine. When rotenone (10 microM) is also present in the incubation medium, the rate of digestion of IgG is inhibited to the same extent as the rate of pinocytosis of 125I-labelled polyvinylpyrrolidone. Proteolysis is likewise inhibited when either NH4Cl (30 mM) or leupeptin (30 micrograms/ml) is present in the medium. The above findings strongly suggest that the observed proteolysis occurs within lysosomes. Normally, yolk sacs that have been exposed in vitro to radiolabelled substrates release radioactivity slowly when the tissue is re-incubated, unless the substrate can be degraded within lysosomes and released in the form of low-molecular-weight hydrolysis products. However, in such experiments 125I-labelled rat IgG shows quite exceptional behaviour in being rapidly released in an apparently intact form (as well as being degraded). If an agent that inhibits pinocytosis (e.g. rotenone or 2,4-dinitrophenol) is present in the incubation medium during exposure of the tissue to 125I-labelled rat IgG, it abolishes release of macromolecular radioactivity on re-incubation of the tissue. Enhanced tissue accumulation of 125I-labelled rat IgG, induced by the presence of leupeptin in the medium during the uptake phase, resulted in no concomitant increase in the amount of 125I-labelled IgG released in macromolecular form on re-incubation of the tissue. These findings indicate that the observed rapid release of 125I-labelled IgG is unlikely to represent release from lysosomes and is more compatible with release from a separate class of vesicle that does not fuse with lysosomes.

Rate of pinocytic capture of macromolecular substrates by rat yolk sac incubated in serum-free culture medium.

Pinocytic activity was quantified for rat yolk sacs incubated in a medium that was either serum-free or contained 10% (v/v) of calf serum. Absence of serum from the medium caused a small increase in the rate of pinosome formation, as determined by the rates of capture of both 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose. In contrast, the rates of uptake of substrates ingested by adsorptive pinocytosis were greatly enhanced when serum proteins, which compete for the same binding sites on the plasma membrane as used by adsorbing substrates, were absent. Elimination of such competition greatly simplifies the quantitative analysis of the binding process, and permitted a detailed study of the binding to the plasma membrane of formaldehyde-denatured bovine serum albumin, a protein that is rapidly digested within the lysosomal system after its pinocytic capture. Binding obeyed Michaelis-Menten kinetics and showed a dissociation constant of approx. 1 micron, indicating the high affinity of this protein for binding sites on the surface of actively pinocytosing yolk-sac cells.