The Duffy antigen receptor for chemokines regulates asthma pathophysiology.

BACKGROUND: The Duffy antigen receptor for chemokines (DARC) is an atypical receptor that regulates pro-inflammatory cytokines. However, the role of DARC in asthma pathophysiology is unknown. OBJECTIVE: To determine the role of DARC in allergic airways disease in mice, and the association between DARC single nucleotide polymorphisms (SNPs) and clinical outcomes in patients with asthma. METHODS: Mice with targeted disruption of the Darc gene (Darc∆E2 ) or WT mice were challenged over 3 weeks with house dust mite (HDM) antigen. Allergic airways disease was assessed 24 hours and 7 days following the final challenge. Additionally, associations between DARC SNPs and clinical outcomes were analysed in a cohort of poorly controlled asthmatics. RESULTS: Total airway inflammation following HDM did not differ between Darc∆E2 and WT mice. At 24 hours, Darc∆E2 mice had increased airway hyperresponsiveness; however, at 7 days airway hyperresponsiveness had completely resolved in Darc∆E2 but persisted in WT mice. In poorly controlled asthmatics, DARC SNPs were associated with worse asthma control at randomization and subsequent increased risk of healthcare utilization (odds ratio 3.13(1.37-7.27), P=.0062). CONCLUSIONS AND CLINICAL RELEVANCE: Our animal model and human patient data suggest a novel role for DARC in the temporal regulation in asthma pathophysiology and symptoms.

Biophysical properties and microfilament assembly in neutrophils: modulation by cyclic AMP.

The microfilament lattice, composed primarily of filamentous (F)-actin, determines in large part the mechanical (deformability) properties of neutrophils, and thus may regulate the ability of neutrophils to transit a microvascular bed. Circulating factors may stimulate the neutrophil to become rigid and therefore be retained in the capillaries. We hypothesized that cell stiffening might be attenuated by an increase in intracellular cAMP. A combination of cell filtration and cell poking (mechanical indentation) was used to measure cell deformability. Neutrophils pretreated with dibutyryl cAMP (db-cAMP) or the combination of prostaglandin E2 (PGE2, a stimulator of adenylate cyclase) and isobutylmethylxanthine (IBMX, an inhibitor of phosphodiesterase) demonstrated significant inhibition of the n-formyl-methionyl-leucyl-phenylalanine (fMLP)-inducing stiffening. The inhibition of cell stiffening was associated with an increase in intracellular cAMP as measured by enzyme-linked immunoassay (EIA) and an increase in the activity of the cAMP-dependent kinase (A-kinase). Treatment with PGE2 and IBMX also resulted in a decrease in the F-actin content of stimulated neutrophils as assayed by NBD-phallacidin staining and flow cytometry or by changes in right angle light scattering. Direct addition of cAMP to electropermeabilized neutrophils resulted in attenuation of fMLP-induced actin assembly. Neutrophils stimulated with fMLP demonstrated a rapid redistribution of F-actin from a diffuse cortical location to a peripheral ring as assessed by conventional and scanning confocal fluorescence microscopy. Pretreatment of neutrophils with the combination of IBMX and PGE2 resulted in incomplete development and fragmentation of the cortical ring. We conclude that assembly and redistribution of F-actin may be responsible for cell stiffening after exposure to stimulants and that this response was attenuated by agents that increase intracellular cAMP, by altering the amount and spatial organization of the microfilament component of the cytoskeleton.

Mechanics of stimulated neutrophils: cell stiffening induces retention in capillaries.

The effect of peptide chemoattractants on neutrophil mechanical properties was studied to test the hypothesis that stimulated neutrophils (diameter, 8 micrometers) are retained in pulmonary capillaries (5.5 micrometers) as a result of a decreased ability of the cell to deform within the capillary in response to the hydrodynamic forces of the bloodstream. Increased neutrophil stiffness, actin assembly, and retention in both 5-micrometer pores and the pulmonary vasculature were seen in response to N-formyl-methionyl-leucyl-phenylalanine. These changes were abolished in cells that had been incubated with 2 micromolar cytochalasin D, an agent that disrupts cellular actin organization. A monoclonal antibody directed at the CD11-CD18 adhesive glycoprotein complex did not inhibit the increase in stiffness or retention in pores. These data suggest that neutrophil stiffening may be both necessary and sufficient for the retention that is observed. Hence, neutrophil sequestration in lung and other capillaries in the acute inflammatory process may be the result of increased stiffness stimulated by chemoattractants.

FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants.

Neutrophil adherence induced by lipopolysaccharide in vitro. Role of plasma component interaction with lipopolysaccharide.

Endotoxemia results in neutrophil localization within a number of microcirculatory beds, reflecting in part an adhesive interaction between neutrophils and the vascular endothelial cell. In previous studies, endotoxin or lipopolysaccharide (LPS) treatment of rabbits resulted in neutrophil sequestration at LPS concentrations well below those effective at increasing neutrophil adherence in vitro. We hypothesized that LPS-induced neutrophil adherence involved a plasma component. In the absence of plasma, high concentrations of LPS (10 micrograms/ml) were required to increase human neutrophil adherence to endothelial cells in vitro. With the inclusion of as little as 1% plasma or serum, however, the LPS dose-response curve was markedly shifted, resulting in increments in adherence at 10 ng/ml, and the time course of enhanced adherence was accelerated. Pretreatment studies suggested that the effect of LPS was on the neutrophil rather than the endothelial cell. Immunoprecipitation of 0111:B4 LPS paralleled the loss of functional activity, suggesting that LPS was an integral part of the active complex, rather than altering a plasma component to make it active. The incubation of plasma with LPS decreased the apparent molecular mass of LPS from 500-1,000 kD to approximately 100 kD. The disaggregated 0111:B4 LPS eluted in the range of albumin and was able to increase adherence in the absence of additional plasma. Plasma depleted of lipoproteins or heat treated retained activity, suggesting that the interaction of LPS with HDL or complement did not account for the observed findings. An LPS-binding protein isolated from rabbit serum enhanced the adherence-inducing effects of both 0111:B4 and Re595 LPS. Furthermore, the activity of rabbit serum was abolished after incubation with an antibody directed against this LPS-binding protein (LBP). An antibody directed against CD14, the putative receptor of the LPS-LBP complex, prevented the adhesive response to LPS. These data suggest that LPS is disaggregated by an LBP in serum and plasma to form an active LPS-plasma component complex. This putative complex then interacts with CD14 on the neutrophil so as to induce an adhesive state.

Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP.

Stimulation of human neutrophils with chemoattractants FMLP or platelet activating factor (PAF) results in different but overlapping functional responses. We questioned whether these differences might reflect patterns of intracellular signal transduction. Stimulation with either PAF or FMLP resulted in equivalent phosphorylation and activation of the mitogen-activated protein kinase (MAPk) homologue 38-kD murine MAP kinase homologous to HOG-1 (p38) MAPk. Neither FMLP nor PAF activated c-jun NH2-terminal MAPk (JNKs). Under identical conditions, FMLP but not PAF, resulted in significant p42/44 (ERK) MAPk activation. Both FMLP and PAF activated MAP kinase kinase-3 (MKK3), a known activator of p38 MAPk. Both MAP ERK kinase kinase-1 (MEKK1) and Raf are activated strongly by FMLP, but minimally by PAF. Pertussis toxin blocked FMLP-induced activation of the p42/44 (ERK) MAPk cascade, but not that of p38 MAPk. A specific p38 MAPk inhibitor (SK&F 86002) blocked superoxide anion production in response to FMLP and reduced adhesion and chemotaxis in response to PAF or FMLP. These results demonstrate distinct patterns of intracellular signaling for two chemoattractants and suggest that selective activation of intracellular signaling cascades may underlie different patterns of functional responses.

Additive effect of intravascular complement activation and brief episodes of hypoxia in producing increased permeability in the rabbit lung.

Systemic complement activation with intravascularly administered cobra venom factor (CVF) or infusion of either zymosan-activated rabbit plasma or a fifth component of complement fragment with anaphylatoxin activity in the rabbit have not caused significant increases in bronchoalveolar lavage albumin in rabbits (Webster, R. O., G. L. Larsen, B. C. Mitchell, A. J. Goins, and P. M. Henson. 1982. Am. Rev. Respir. Dis. 125:335-340). To assess if another stimulus (hypoxia) acting in concert with complement activation can produce significant lung injury, rabbits were challenged with CVF alone, 10 min of 12% oxygen alone, or CVF followed by a 10-min exposure to 12% oxygen. Either stimulus alone caused no significant changes in arterial oxygen, pulmonary resistance, or dynamic compliance during the 240 min of observation after either stimulus, and neither stimulus alone caused increased albumin accumulation in bronchoalveolar lavage over a 30-min period at the end of the experiment. However, the combination of insults significantly altered arterial oxygen, pulmonary resistance, and dynamic compliance while also increasing albumin and neutrophils recovered by lavage. The increase in lavage albumin did not appear to be due to hemodynamic events in that the pulmonary artery pressure increased acutely after CVF infusion and again during the hypoxic exposure, but was normal when albumin accumulation in the lung was measured. Neutrophil depletion with nitrogen mustard abolished all of these changes induced by CVF plus hypoxia. In addition, meclofenamate pretreatment and infusion during the 4-h study abolished the increases in lavage albumin and neutrophils as well as the increase in pulmonary artery pressure after CVF. Meclofenamate pretreatment did not, however, block accumulation of albumin in the lung (interstitium). We conclude that complement activation, as an isolated event, will not cause a significant increase in lavage albumin in this model. However, combining complement activation with an episode of hypoxia will lead to an increase in lavage albumin that is dependent on the presence of neutrophils for its expression. Meclofenamate treatment will prevent increases in lavage albumin and neutrophils while not preventing albumin accumulation in the lung (interstitium), suggesting a product of the cyclooxygenase pathway of arachidonic acid metabolism is needed to produce movement of albumin and/or neutrophils across the alveolar epithelium in this model.

Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase.

The neutrophil has been implicated as an important mediator of vascular injury, especially after endotoxemia. This study examines neutrophil-mediated injury to human microvascular endothelial cells in vitro. We found that neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP), the complement fragment C5a, or lipopolysaccharide (LPS) (1-1,000 ng/ml) alone produced minimal endothelial injury over a 4-h assay. In contrast, neutrophils incubated with endothelial cells in the presence of low concentrations of LPS (1-10 ng/ml) could then be stimulated by FMLP or C5a to produce marked endothelial injury. Injury was maximal at concentrations of 100 ng/ml LPS and 10(-7) M FMLP. Pretreatment of neutrophils with LPS resulted in a similar degree of injury, suggesting that LPS effects were largely on the neutrophil. Endothelial cell injury produced by LPS-exposed, FMLP-stimulated neutrophils had a time course similar to that induced by the addition of purified human neutrophil elastase, and different from that induced by hydrogen peroxide (H2O2). Further, neutrophil-mediated injury was not inhibited by scavengers of a variety of oxygen radical species, and occurred with neutrophils from a patient with chronic granulomatous disease, which produced no H2O2. In contrast, the specific serine elastase inhibitor methoxy-succinyl-alanyl-alanyl-prolyl-valyl-chloromethyl ketone inhibited 63% of the neutrophil-mediated injury and 64% of the neutrophil elastase-induced injury. However, neutrophil-mediated injury was not inhibited significantly by 50% serum, 50% plasma, or purified alpha 1 proteinase inhibitor. These results suggest that, in this system, chemotactic factor-stimulated human neutrophil injury of microvascular endothelial cells is enhanced by small amounts of LPS and may be mediated in large part by the action of neutrophil elastase.

Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils.

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.

Critical role of CXCL4 in the lung pathogenesis of influenza (H1N1) respiratory infection.

Annual epidemics and unexpected pandemics of influenza are threats to human health. Lung immune and inflammatory responses, such as those induced by respiratory infection influenza virus, determine the outcome of pulmonary pathogenesis. Platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) has an immunoregulatory role in inflammatory diseases. Here we show that CXCL4 is associated with pulmonary influenza infection and has a critical role in protecting mice from fatal H1N1 virus respiratory infection. CXCL4 knockout resulted in diminished viral clearance from the lung and decreased lung inflammation during early infection but more severe lung pathology relative to wild-type mice during late infection. Additionally, CXCL4 deficiency decreased leukocyte accumulation in the infected lung with markedly decreased neutrophil infiltration into the lung during early infection and extensive leukocyte, especially lymphocyte accumulation at the late infection stage. Loss of CXCL4 did not affect the activation of adaptive immune T and B lymphocytes during the late stage of lung infection. Further study revealed that CXCL4 deficiency inhibited neutrophil recruitment to the infected mouse lung. Thus the above results identify CXCL4 as a vital immunoregulatory chemokine essential for protecting mice against influenza A virus infection, especially as it affects the development of lung injury and neutrophil mobilization to the inflamed lung.

Biosynthesis of platelet activating factor and 1-O-acyl analogues by endothelial cells.

Mass spectrometric procedures have been used to measure 1-O-alkyl-2-O-acetyl-glycero-3-phosphocholine (PAF) and a structural analogue, 1-O-acyl-2-O-acetyl-glycero-3-phosphocholine, biosynthesis in stimulated human umbilical vein endothelial cells (HUVEC). The primary species of acetylated glycerophosphocholine detected were of the 1-O-acyl type, rather than PAF. The amounts of PAF synthesized were much less than reported in earlier studies. In addition, mass spectrometric procedures were used to profile the molecular species of glycerophosphocholine in HUVEC and to determine those species which have arachidonate in the sn-2 position. The pattern of the occurrence of arachidonate in the sn-2 position is very similar to the pattern of synthesis of the acetylated glycerophosphocholines. These results suggest that 1-O-acyl-2-O-acetyl-glycero-3-phosphocholines may have significant but unappreciated biological activities. These results also support the view that glycerophosphocholine molecular species which have arachidonate esterified at the sn-2 position are the immediate precursors for the hydrolysis-acetylation steps which result in the synthesis of sn-2 acetylated glycerophosphocholines, including PAF.

Bacterial lipopolysaccharide enhances chemoattractant-induced elastase secretion by human neutrophils.

Bacterial lipopolysaccharide (LPS) has previously been shown to enhance a number of chemoattractant-induced responses by human neutrophils. The possible role of elastase, a neutral protease with broad substrate specificity, in neutrophil-mediated vascular injury of a variety of diseases prompted us to examine a) whether or not LPS enhances the direct chemoattractant-induced secretion of elastase, b) the quantitative requirements of LPS and chemotactic factors, and c) some structural requirements of LPS for this effect. Our results show that LPS at 10 ng/ml and above, enhanced formyl-methionyl-leucyl-phenylalanine-induced neutrophil secretion of elastase, as well as secretion of myeloperoxidase and vitamin B12-binding protein. This effect was independent of cytochalasins or surface stimulation, and thus may occur during chemotactic factor stimulation in vivo. LPS also enhanced neutrophil secretory responses to the complement fragments C5a, C5a des arg, and, to a lesser degree, to leukotriene B4 and platelet-activating factor. This enhancement effect appeared to require the presence of the lipid A moiety and/or parts of the core polysaccharide but not the O-antigen portion of the LPS molecule. Our findings identify a possible LPS-dependent mechanism of neutrophil elastase-mediated tissue injury in Gram-negative infections.

Selective coupling of the human anaphylatoxin C5a receptor and alpha 16 in human kidney 293 cells.

The peptide C5a which is generated during the complement cascade is an important chemotactic factor involved in the inflammatory response. The C5a receptor (C5aR) primary sequence suggests that it has a serpentine structure of seven transmembrane domains which is typical of classical G-protein-coupled receptors. To investigate the signal transduction mechanism of C5a we transiently expressed the C5aR in combination with different G-protein alpha subunits in human kidney 293 cells and measured the PLC activity induced upon C5a stimulation. Cotransfection of C5aR and alpha 16 stimulated PLC while cotransfection of C5aR with either alpha q or alpha i2 did not.

Neutrophil retention in model capillaries: deformability, geometry, and hydrodynamic forces.

The initial retention of neutrophils within the pulmonary microvascular bed occurs in both physiological and pathological states, yet the factors responsible for this retention are poorly understood. Because the diameter of the neutrophil is approximately 7.03 micron and the mean pulmonary capillary diameter is 5.5 micron, we postulated that geometric constraints imposed by the microvascular bed, the deformability of the neutrophil, and the hydrodynamic characteristics of blood were important determinants of neutrophil retention. We used a filtration system wherein 111In-labeled human neutrophils (111In-N) suspended in a serum-containing buffer were passed through Nuclepore filters of known pore size. Compared with 99mTc-labeled erythrocytes (99mTc-RBC), the passage of 111In-N was delayed and a higher percentage was retained within the filter. Because the neutrophil and RBC are approximately equal in diameter, the deformability of the neutrophil must be less than that of RBC. As the flow rate increased, retention in the filters decreased logarithmically from 72 +/- 5% (flow rate 0.5 ml/min) to 15 +/- 4% (10.0 ml/min). As the number of RBC in the buffer increased, neutrophil retention in 5-micron filters decreased in a linear fashion from 65 +/- 6% at hematocrit of 0 to 33 +/- 2% at hematocrit of 10. The perfusion pressure and shear stress were of critical importance, and there was a logarithmic relationship between retention and perfusion pressure or shear stress (tau), whether the increase in pressure or tau was generated by increasing flow or by increasing the hematocrit of the perfusate. As the pore size of the filter increased, the retention of neutrophils decreased in a logarithmic fashion: from 75 +/- 5% in the 3-micron filter to 4 +/- 1.3% in the 12-micron filter.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of local blood flow in pulmonary inflammation: role for neutrophils, PAF, and thromboxane.

The intrapulmonary instillation of C5a results in a local inflammatory response that, in this site, is accompanied by a decrease in local blood flow. Reversal of this decrease by vasodilators or the thromboxane synthesis inhibitor dazmegral has been shown to result in enhanced lung inflammation. In the present study the mechanisms underlying the decrease in flow in pulmonary inflammation were investigated in the rabbit in vivo and in the isolated blood-perfused rabbit lung. In vivo, the decrease in local blood flow was shown to be dependent on circulating neutrophils. In the isolated blood-perfused lung, inflammation induced by airway instillation of C5a was similar histologically to that seen in vivo and was also accompanied by a decrease in local blood flow. The decrease in blood flow appeared to require circulating neutrophils and was prevented by dazmegral and the platelet-activating factor (PAF) antagonists WEB 2086 and L-659,989. Furthermore, no decrease occurred in aspirin-treated lungs perfused with normal blood, suggesting that the source of thromboxane was lung rather than circulating cells. The decrease in blood flow in inflammation did not appear to be a consequence of hypoxic vasoconstriction. Inflammation in the guinea pig lung was also accompanied by a decrease in local blood flow and was also prevented by dazmegral and PAF antagonists. We conclude that local inflammation in the lung is accompanied by a decrease in blood flow that involves neutrophils and the lipid mediators PAF and thromboxane. We suggest that this form of negative feedback by the neutrophil serves to control the inflammatory response.

Fetal pulmonary vasodilation after exogenous platelet-activating factor.

To determine the fetal pulmonary vascular response to platelet-activating factor (PAF), we studied the hemodynamic effects of the infusion of PAF directly into the left pulmonary artery in 21 chronically catheterized fetal lambs. Left pulmonary arterial blood flow (Q) was measured with electromagnetic flow transducers. Ten-minute infusions of low-dose PAF (10-100 ng/min) produced increases in Q from a baseline of 71 +/- 5 to 207 +/- 20 ml/min (P less than 0.001) without changes in pulmonary arterial pressure. Pulmonary vasodilation with PAF was further confirmed through increases in Q with brief (15-s) infusions and increases in the slope of the pressure-flow relationship as assessed by rapid incremental compressions of the ductus arteriosus during PAF infusion. Infusion of Lyso-PAF had no effect on Q or pulmonary arterial pressure. Treatment with CV-3988, a selective PAF receptor antagonist, but not with meclofenamate, atropine, or diphenhydramine and cimetidine blocked the response to PAF infusion and did not affect baseline tone. Systemic infusion of high-dose PAF (300 ng/min) through the fetal inferior vena cava increased pulmonary arterial pressure (46.5 +/- 1.0 to 54.8 +/- 1.9 mmHg, P less than 0.01) and aorta pressure (44.3 +/- 1.0 to 52.7 +/- 2.2 mmHg, P less than 0.01) while also increasing Q. Neither PAF nor CV-3988 changed the gradient between pulmonary arterial and aorta pressures, suggesting that PAF does not affect ductal tone. We conclude that PAF is a potent fetal pulmonary vasodilator and that the effects are not mediated through cyclooxygenase products or by cholinergic or histaminergic effects.

Effects of neutrophil depletion and repletion on PAF-induced hyperresponsiveness of canine trachea.

Platelet-activating factor (PAF) has been implicated as a mediator of airway hyperresponsiveness. PAF, infused intra-arterially into the canine cervical trachea, causes adherence of neutrophils to vascular endothelium, increases vascular permeability, and increases the responsiveness of tracheal muscle to parasympathetic stimulation. We hypothesized that the increase in airway responsiveness induced by PAF in this model depends on the presence of neutrophils. To test this hypothesis, we perfused a cervical tracheal segment with autologous blood depleted of leukocytes or with similar leukocyte-depleted blood that had been repleted with its neutrophils. Fifteen minutes after the onset of perfusion with either leukocyte-depleted or neutrophil-repleted blood, PAF infusion was begun into the tracheal arterial vasculature. The contractile response of the tracheal muscle to parasympathetic stimulation was measured before and 15 and 30 min after the onset of PAF infusion. PAF did not significantly change the response of tracheal muscle during perfusion with neutrophil-depleted blood but increased the response of tracheal muscle during perfusion with neutrophil-repleted blood. We conclude that the increase in canine tracheal muscle response induced by intra-arterial PAF depends on neutrophils.

Opposing hemodynamic effects of substance P on pulmonary vasculature in rabbits.

Substance P is a peptide implicated in the control of a variety of physiological processes. Although substance P-containing neurons impinge on the pulmonary vasculature, the effects of substance P on the pulmonary circulation have not been systematically investigated. Rabbits were anesthetized with methohexital sodium and paralyzed with pancuronium bromide. Injection of substance P (0.002-0.10 microgram/kg) in the vena cava produced dose-dependent pulmonary vasoconstriction and systemic vasodilation. Pulmonary arterial pressure reached a peak within 15-20 s and declined toward base line over 10 min. Aortic pressure fell rapidly, reaching minimum at 5-10 s. At higher doses cardiac output fell transiently, resulting in a 65% fall in pulmonary vascular conductance. If repeat substance P dosages were administered 15 min apart, there was no tachyphylaxis. Pulmonary vasoconstriction was inhibited by the cyclooxygenase blocker meclofenamate (10 mg/kg) and the thromboxane synthase inhibitor Dazmegrel (UK-38,485) (2 mg/kg). In contrast, vasoconstriction was enhanced by atropine (2 mg/kg). In Dazmegrel-treated animals in whom pulmonary vasoconstriction was established by epinephrine infusion, low doses of substance P produced vasodilation. Our findings indicate that substance P produces pulmonary vasoconstriction via prostaglandin (particularly thromboxane) generation and pulmonary vasodilation via activation of cholinergic pathways.

Endotoxin-pretreated neutrophils increase pulmonary vascular permeability in dogs.

Endotoxin causes pulmonary vascular neutrophil sequestration and injures the lung. Whether this is primarily due to a direct effect of endotoxin on the endothelium or is mediated by an action on the neutrophil is unclear. Canine neutrophils, isolated on plasma-Percoll gradients in vitro, were incubated with Salmonella enteriditis endotoxin, washed, and injected via wedged pulmonary arterial catheters into discrete lung zones of anesthetized dogs, whereas untreated neutrophils were administered into contralateral control lung zones. 113mIn-transferrin was administered intravenously 2 h before the animals were killed. Morphometry and extravascular protein accumulation were assessed at 4 h. Endotoxin treatment of neutrophils ex vivo induced a two- to three-fold increase in neutrophils in these lung zones (0.096 +/- 0.012 vs. 0.05 +/- 0.002 neutrophils/alveolar septal intercept, P less than 0.05). Extravascular-to-intravascular protein ratios in zones receiving endotoxin-treated neutrophils were significantly increased compared with control zones (0.146 +/- 0.02 vs. 0.079 +/- 0.02, P less than 0.05). Because complement fragments increase injury to endothelium in vitro, exogenous C5 fragments were administered to other dogs before administration of neutrophils but failed to significantly increase the extravascular protein signal (0.154 +/- 0.03 vs. 0.124 +/- 0.04). In summary, endotoxin treatment of neutrophils leads to neutrophil sequestration and increased pulmonary extravascular protein accumulation. C5 fragments failed to further enhance the protein accumulation. These data are consistent with an effect of endotoxin on the neutrophil to initiate neutrophil-endothelial interaction and subsequent lung injury.

Effects of shear stress on adhesive interaction between neutrophils and cultured endothelial cells.

The effect of hydrodynamic shear stress on the adhesive interaction between neutrophils and endothelial cells in vitro was investigated using an apparatus similar to a cone-in-plate viscometer. Isolated bovine neutrophils were labeled with 111In and incubated with monolayers of cultured bovine pulmonary arterial endothelial cells in the presence of different degrees of shear stress. Physiologically relevant shear (less than 2 dyn/cm2) was associated with marked decrease in neutrophil adherence. Stimulation with 10% bovine zymosan-activated plasma increased adherence under static conditions but failed to increase adherence conducted during the application of shear stress. Inhibition of endothelial cell prostacyclin production by meclofenamate or aspirin failed to alter the response to shear. Incubation of neutrophils under static conditions for 10, but not 5, min however, markedly enhanced subsequent resistance to shear, suggesting that a time-dependent reaction between neutrophil and endothelial cell was required to induce an increase in the strength of adherence. Analysis of neutrophil migration underneath the monolayer indicated that such migration in no way accounted for resistance to shear, particularly since shear resistance was enhanced on serum-coated plastic as well as endothelial cells. We conclude that hemodynamic factors may play an important role in modulating neutrophil adherence to endothelium in both normal and inflammatory states.