Enhancement of human T cell responses to allogeneic stimuli by factor VIII concentrates

The effect of factor VIII concentrate, from commercial and National Health Services manufacturers, on in vitro lymphocyte proliferative response to allogeneic stimulator cells was investigated. Factor VIII preparations 'purified' by ion exchange or by monoclonal antibody affinity had no effect in this assay but lymphocyte proliferation in response to allogeneic cells was markedly and consistently enhanced by some intermediate purity factor VIII concentrates and, to a lesser extent, by a factor IX preparation. These preparations did not stimulate lymphocyte proliferation in the absence of other mitogens. The co-mitogenic factor(s) present in intermediate purity factor VIII was not identified. However, enhanced proliferation was not due to factor VIII itself, nor to albumin or fibronectin. The clinical relevance of the immunomodulatory activity of intermediate purity factor VIII concentrates in vitro is discussed.     

Effect of theophylline on airway responses to inhaled platelet-activating factor in man

It has been suggested that theophylline may possess anti-inflammatory actions which underlie its antiasthma properties. We examined whether theophylline could inhibit the bronchoconstriction and the bronchial hyperresponsiveness induced by inhaled platelet-activating factor (PAF) in eight nonasthmatic subjects in a double-blind, cross-over study. After oral theophylline (6 mg.kg-1), plasma theophylline at 1 h was 10.4 +/- 1.8 mg.ml-1 (mean +/- SEM) compared to 0.39 +/- 0.19 mg.ml-1 on the placebo day (p less than 0.005). PAF, inhaled in five successive doses every 15 min, caused a 56 +/- 11% fall in Vp30 (flow at 30% of vital capacity from a partial expiratory manoeuvre) after the first dose at 5 min, and diminishing responses with successive doses. Theophylline had no significant effect on PAF-induced bronchoconstriction. PAF caused a significant decrease in PC40 (the concentration of methacholine needed to cause 40% fall in baseline Vp30) from a baseline of 12.8 mg.ml-1 (geometric standard error of mean (GSEM) 1.98) to 7.9 (1.79) mg.ml-1 on day 3 and 6.9 (1.74) on day 7 (p less than 0.02). There was no significant difference when mean PC40 values on corresponding days after PAF were compared between placebo and theophylline treatment periods. Our results suggest that theophylline has negligible influence on the airway effects of PAF.     

Inhaled platelet-activating factor causes pulmonary neutrophil sequestration in normal humans.

Inhaled platelet-activating factor (PAF) causes bronchoconstriction and transient peripheral neutropenia in humans. We studied eight normal subjects to investigate whether inhaled PAF caused pulmonary neutrophil sequestration. All subjects received autologous 99mTc-red cells as a blood pool marker, seven received 111In-neutrophils, and one received 111In-platelets. Six subjects inhaled 48 micrograms of PAF. There was immediate pulmonary sequestration of 111In-neutrophils, maximal (218% baseline) at 6 min (p less than 0.001), returning to normal by 3 h. There was no change in circulating platelet count or pulmonary 111In-platelet transit. Methacholine inhalation caused equivalent bronchoconstriction to PAF, but it had no effect on neutrophil count or pulmonary 111In-neutrophil activity. We have demonstrated pulmonary neutrophil, but not platelet, sequestration after PAF. This supports a role for PAF as an inflammatory mediator in humans. This may be a useful model for exploring pulmonary neutrophil kinetics and preinflammatory processes.     

Metabolic, membrane, and functional responses of human polymorphonuclear leukocytes to platelet-activating factor

The phospholipid mediator of anaphylaxis, platelet-activating factor (PAF) is chemotactic for polymorphonuclear leukocytes (PMN). We have examined this agent's effects on several other PMN functions. Human PMN were prepared from heparinized venous blood by Ficoll gradient. Metabolic burst was examined by measurement of O2 use and O2.- production in the presence or absence of PAF (10(-6)--10(-9) M). Unless cells were treated with cytochalasin-B (5 micrograms/ml), no significant respiratory burst was demonstrated. However, pretreatment with PAF (10(-7) M) enhanced approximately threefold the O2 utilization found when cells were subsequently stimulated with 10(-7) M FMLP. PAF also stimulated arachidonic acid metabolism in 14C-arachidonic acid- labeled PMN. Thin-layer chromatography analysis of chloroform-methanol extracts showed substances that comigrated with authentic 5- hydroxyeicosatetraenoic acid had a marked increase in radioactivity following PAF stimulation at 10(-7) M. PAF failed to stimulate release of granule enzymes, B-glucuronidase, lysozyme, or myeloperoxidase unless cytochalasin-B were added. PAF from 10(-6) M to 10(-10) M affected PMN surface responses. PMN labeled with the fluorescent dye, chlorotetracycline, showed decreased fluorescence upon addition of PAF, suggesting translocation of membrane-bound cations. Further, the rate of migration of PMN in an electric field was decreased following PAF exposure, a change consistent with reduced cell surface charge. PMN self-aggregation and adherence to endothelial cells were both influenced by PAF (10(-6) M--10(-9) M). Aggregation was markedly stimulated by the compound, and the percent PMN adhering to endothelial cell monolayers increased almost twofold in the presence of 10(-8) M PAF. Thus, PAF promotes a variety of PMN responses: enhances respiratory burst, stimulates arachidonic acid turnover, alters cell membrane cation content and surface charge, and promotes PMN self- aggregation as well as adherence to endothelial cells.     

Gastrointestinal damage induced by platelet-activating factor

Intravenous infusion of platelet-activating factor (PAF-acether) induces extensive vasocongestion and damage in the stomach and small intestine of the rat. The effect of pretreatment with the corticoid dexamethasone (2 mg/kg) on such gastrointestinal damage in the rat has now been investigated, using macroscopic observation and the release of acid phosphatase, as an enzyme marker of cell disruption. Administration of PAF-acether (100 ng/kg/min intravenously, for 10 min) induced focal, intense hyperemia and hemorrhage in all the regions of the gastrointestinal tract, with the exception of the distal colon. This damage was associated with an increase in the release of acid phosphatase into the lumen of the stomach, duodenum, jejunum, and ileum following incubation in vitro. Pretreatment with dexamethasone reduced the macroscopically apparent gastrointestinal damage following PAF-acether administration and abolished the intraluminal release of acid phosphatase. Dexamethasone also significantly suppressed the hemoconcentration, as determined by changes in platelet and erythrocyte count and hematocrit, 30 min after PAF-acether infusion. Thus, as in endotoxin shock, this glucocorticoid can reduce the gastrointestinal damage associated with PAF-acether administration.Dr. J.L. Wallace is the recipient of a scholarship from the Medical Research Council of Canada.     

Bacterial responses to neutrophil phagocytosis

PURPOSE OF REVIEW: This review focuses on adaptive bacterial interactions with neutrophils, emphasizing information communicated within the past year about bacterial factors that respond to contact with or phagocytosis by PMN. RECENT FINDINGS: Since the discovery of type III and IV secretion, progress in the analysis of bacterial interactions with host phagocytes has been extensive but largely focused on the macrophage. The remarkable growth of information about bacterial subversion of macrophage metabolism has been summarized in several excellent reviews. The scope of progress on neutrophil-bacteria interactions is more limited and dominated by recent studies of the granulocyte pathogen, Anaplasma phagocytophilum, the agent of granulocytic ehrlichiosis. SUMMARY: For many pathogens, contact with or ingestion by phagocytes elicits a vigorous but varied microbial response. The response repertoire includes activation of type III and type IV secretion systems that inject effector molecules into the host cell. Effectors modify host cell signaling and metabolic pathways to favor survival of the microbe. Whereas microbial secretory structures are few in kind and relatively conserved, effector molecules are numerous and variable. Effectors may promote phagocytosis by nonphagocytic cells or suppress phagocytosis by macrophages and neutrophils. They may suppress assembly or misdirect localization of the phagocyte NADPH oxidase that is responsible for generating toxic oxidants, and they may suppress phagosome-lysosome fusion. Phagocytosed bacteria may also up-regulate the expression of defensive proteins that attenuate the effects of phagocyte-derived antimicrobial toxins. These pathogenic stratagems probably have their origins in the competition among single-celled organisms, eukaryotes versus prokaryotes, that arose early in evolution.     

Modulation by norepinephrine of neural responses to estradiol

Pharmacological studies have suggested that neurotransmitter activity impinging on steroid-concentrating cells can affect the steroid receptor system within those cells, modifying behavioral responses to the hormone. The present experiments revealed that the alpha 1-noradrenergic antagonist prazosin, administered to ovariectomized rats at the time of each of two pulses of estradiol, inhibited the appearance of sexual receptivity. Prazosin also substantially reduced the levels of estrogen receptors within hypothalamic cell nuclei following an injection of estradiol. Manipulation of noradrenergic inputs into the hypothalamus by lesioning brain stem norepinephrine cell groups with 6-hydroxydopamine (6OHDA) also reduced the level of nuclear estrogen receptors following an injection of estradiol. Although this effect of 6OHDA lesions was observed in two separate experiments, in other experiments 6OHDA had no effect on estrogen receptors. In some instances, there was a positive correlation between nuclear estrogen receptor levels in the hypothalamus and the levels of norepinephrine. The results are consistent with the hypothesis that brain stem inputs to the hypothalamus are able to modulate neural responses to steroids and specifically that noradrenergic inputs are able to modulate neural responses to estradiol. However, there are additional undiscovered variables that preclude statements of a simple relationship between norepinephrine levels and estrogen receptor levels.     

Modulation by pentobarbital of neutrophil responses to inhaled E. coli endotoxin in sheep: role of lung epithelium.

Neutrophils (PMNs) are implicated in the pathogenesis of acute respiratory distress syndrome (ARDS). The role of the epithelium in the modulation of PMN migration within the lungs was examined. Epithelial integrity and PMN concentrations in the lung air spaces and lymph were measured in sheep anaesthetized with either halothane (1-2.5%) or intravenous pentobarbital (12+/-4 mg x kg(-1) x h(-1)). Ventilation with an aerosol containing 25 mg Escherichia Coli endotoxin (lipopolysaccharide; LPS) effected neutrophil recruitment to the air spaces. Lymphatic clearance of aerosolized 99mTc-DTPA provided an index of epithelial integrity. Three hours after the deposition of LPS, the lung lining fluid of sheep anaesthetized with halothane (n=7) had 4.9+/-3.2x10(6) PMN.mL(-1), but the lung lymph had almost no PMNs (3+/-8%). Sheep anaesthetized with pentobarbital (n=6) had fewer PMNs in the air spaces (2.4+/-1.2X10(6) mL(-1)) and more PMNs in the lung lymph (30+/-20%). Control sheep (n=5) that received no LPS had almost no PMNs in the airspaces or lung lymph, regardless of the anaesthesia. Three additional sheep that remained awake after receiving LPS also had no PMNs in the lung lymph. The PMN fraction in the lung lymph correlated well with the extra-alveolar epithelial permeability measured by lymphatic clearance of aerosolized diethylenetriamine penta-acetic acid (r=0.81, p<0.001). Aerosolized lipopolysaccharide recruits neutrophils into the lungs of sheep, but they appear to remain in the airspaces unless extra-alveolar permeability is increased by agents such as pentobarbital.     

Measurement of platelet-activating factor by physicochemical technique s

The measurement of PAF by physiochemical methods perhaps in the future will become the normal mode for the measurement of this molecule. This is due in part to the limitations in the bioassay and radiometric assays but also to the fact that the development of facile techniques that are acceptable to the community both in terms of operation, sensitivity, and accuracy are attainable. It is with these latter forms of measurement that significant advances can be made in the understanding of the biosynthesis and metabolism of PAF after specific stimuli of either isolated cells, organs, or the intact organism. Recent studies using mass spectrometry have suggested that 95% of PAF administered via the airway was retained within the lung primarily in only 2 cell types: the alveolar type II cell and the nonciliated bronchiolar epithelial cell (Clara cell). Further characterization of the metabolites in these lungs suggested a biochemical interconnection between PAF and arachidonate metabolism.     

In vitro and in vivo effects of treatment by platelet-activating factor on N-formyl-met-leu-phe-mediated responses of polymorphonuclear leucocytes

Two chemoattractants, the peptide N-formyl-met-leu-phe (FMLP), and the ether phospholipid, platelet activating factor (PAF), each stimulate a variety of in vitro responses in polymorphonuclear leucocytes (PMN). Because often more than one inflammatory mediator is active during inflammation, we determined the effect on PMN of sequential stimulation with these two agents. Before FMLP stimulation, human PMN were exposed to PAF, at concentrations which gave little or no response when administered alone. PAF enhanced FMLP-elicited superoxide release in a dose-dependent fashion. Likewise, release of granular lysozyme from the cells was increased in PAF treated cells. Similar treatment with other phospholipids, including the lyso derivation of PAF, failed to produced these effects. Incubation with nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, had little effect on the enhancement of lysozyme release by PAF. To determine if enhancing effects by PAF might occur also in vivo, we studied rabbits receiving PAF and/or FMLP intravenously. When rabbits received 0.01 micrograms PAF (a dose which does not elicit the sustained neutropenia observed with higher doses of PAF) followed by 0.05 micrograms FMLP the absolute granulocyte count (AGC) dropped at 1 min (46 +/- 11% of original value), and continued to fall (24 +/- 12% at 10 min). Controls, treated with the suspending fluid for PAF, and then 0.05 micrograms FMLP, had a similar 1 min AGC value, but at 10 min AGC returned to 65 +/- 6.1% (P less than 0.001 for comparison of 10 min values). Thus PAF pretreatment enhanced FMLP-elicited granulocytopenia in vivo. Study of in vitro human PMN aggregation revealed that, at certain relative concentrations of PAF and FMLP, aggregation was enhanced. These studies show that both in vitro and in vivo responses of FMLP-stimulated PMN may be exaggerated by pre-exposure to PAF.     

Platelet-activating factor primes neutrophil responses to agonists: role in promoting neutrophil-mediated endothelial damage

During inflammation polymorphonuclear cells (PMNs) are exposed to agonistic stimuli including activated complement, kallikrein, arachidonic acid metabolites, monokines, and platelet-activating factor (PAF). We report that PAF not only directly activates PMNs but in miniscule quantities (10(-12) mol/L) "primes" them as well, that is, permits PMNs to respond to subsequent stimuli that would be otherwise ineffectual. PAF priming of responses including superoxide generation, elastase release, and aggregation is time dependent and is maximal within five minutes. PAF need not be present during the subsequent exhibition of PMN agonists, but priming is inhibited by cold and is also inhibited by the PAF receptor antagonists BN 52021, L-652, and kadsurenone. An intact PAF molecule is required because lyso-PAF and methoxy-PAF do not prime PMN responses. PAF priming is associated with both enhanced expression of the adhesive glycoprotein identified by OKM- 1 antibody and an enhanced rise in intracellular calcium levels in response to the subsequent addition of agonists such as FMLP. PMNs primed with PAF and stimulated with either F-Met-Leu-Phe or phorbol esters are more effective in lysing and detaching cultured human endothelial cells--damage that can also be inhibited by the PAF antagonists. Because PAF is synthesized and exhibited on surfaces of endothelial cells perturbed by coagulation, we suggest that this lipid may potentiate otherwise trivial activators of marginated PMNs so that they become damaging to the PAF-synthesizing endothelium itself. If so, our studies suggest a possible therapeutic role for PAF inhibitors in excessive inflammatory states.     

Cardiovascular effects of platelet-activating factor

Platelet-activating factor (PAF) is a unique biologically active phospholipid that is synthesized by many cells and tissues and has actions far more diverse than just platelet activation. PAF is probably a mediator of the normal inflammatory and thrombotic responses. Inappropriate or excessive production of PAF can lead to vascular injury, particularly via its proinflammatory effects, and marked hemodynamic derangements. There is evidence that PAF may play a role in clinical syndromes of shock, infarction, and reperfusion injury. Antagonists of the PAF receptor have been developed and have therapeutic potential in these common life-threatening disorders.     

The presence of platelet-activating factor associated with eosinophil and/or neutrophil accumulations in the pleural fluids

Platelet-activating factor (PAF) has been reported to play a role in the inflammatory reaction, but the mechanism of PAF in humans is still unclear. We examined the presence of PAF in pleural fluids from 23 patients with pleural effusion and in all cases detected PAF associated with eosinophil and/or neutrophil infiltrations. The amounts of PAF in pleural fluids were, respectively, 340, 50 to 170, and 1,250 to 2,130 fmol/ml for a patient with eosinophilic pneumonia, those with pneumothorax (n = 9), and empyema (n = 3). In contrast, patients with tuberculous pleuritis (n = 2), lung edema (n = 3), or malignant disease (n = 5) had no detectable amounts of pleural fluid PAF (less than or equal to 10 fmol/ml). The amount of PAF showed a close correlation with the numbers of eosinophils and neutrophils in the pleural fluids. Furthermore, PAF was mostly detected in the cellular fractions, and the molecular species of PAF from the patients with empyema were almost consistent with those of PAF generated by human blood neutrophils. These results indicate that neutrophils and, presumably, eosinophils were the cellular source of PAF in the pleural fluids in the pathologic state of inflammation.     

Cardiac myocytes activated by septic plasma promote neutrophil transendothelial migration: role of platelet-activating factor and the chemokines LIX and KC

Cardiac myocytes isolated from rats with peritonitis (cecal ligation and perforation; CLP) promote PMN transendothelial migration. Herein, we assessed (1) the mechanisms involved in cardiac myocyte activation during peritonitis and (2) the means by which these activated myocytes promote PMN transendothelial migration. Plasma obtained from mice subjected to CLP (septic plasma) activated isolated cardiac myocytes as evidenced by (1) increased nuclear levels of nuclear factor-kappaB (NF-kappaB) and (2) their ability to promote PMN migration across endothelial cell monolayers. Pretreatment of septic plasma with an antibody against tumor necrosis factor-alpha (TNF-alpha), but not interleukin-1beta (IL-1beta), blunted the ability of septic plasma to activate the myocytes. However, septic plasma obtained from TNF-alpha-deficient mice could still activate the myocytes; an effect attenuated by an antibody against IL-1beta. If the myocytes were pretreated with a proteasome inhibitor (MG 132) to prevent NF-kappaB activation, the myocyte-induced PMN transendothelial migration was compromised. The activated myocytes released platelet-activating factor (PAF), and myocyte-induced PMN migration was abrogated by a PAF receptor antagonist (WEB 2086). These myocytes also released the CXC chemokines LIX and KC; an event prevented by MG 132. Antibodies against LIX and KC abrogated the myocyte-induced PMN migration. However, LIX and KC, but not PAF, could promote PMN migration when used at concentrations produced by activated myocytes. These observations indicate that TNF-alpha and IL-1beta are, in part, responsible for the ability of septic plasma to activate cardiac myocytes. The activated myocytes promote PMN transendothelial migration, an effect attributable to LIX and KC, and possibly, PAF.     

Chemorepulsion of trichomonads by products of neutrophil oxidative metabolism

To determine whether secreted neutrophil products affect the migration of motile microorganisms such as Trichomonas vaginalis, stimulated human neutrophils and cell-free oxygen metabolites were used as stimuli in a multiwell filter chemotaxis assay using tritiated T. vaginalis. When stimulated neutrophils were present on the opposite side of the filter, migration of T. vaginalis into the filter was significantly diminished, and this reduction varied with the dose of neutrophil stimulus. The reduction of movement was abrogated by the addition of catalase and superoxide dismutase to scavenge oxygen metabolites. Studies with cell-free hydrogen peroxide or hypochlorite preparations indicated that the reduction in trichomonal trapping in the filter was due to chemorepulsion and not to a nonspecific decrease in motility or adherence. These findings suggest that active migration away from neutrophil products might be a means by which trichomonads avoid the microbicidal functions of host phagocytes.