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PubMed1985

The role of neutrophil membrane glycoprotein 150 (Gp-150) in neutrophil-mediated endothelial cell injury in vitro.

In this study we examined the importance of neutrophil adherence in neutrophil-mediated endothelial cell injury. Phorbol myristate acetate (PMA)-activated neutrophils from a patient with a congenital defect in neutrophil adherence (Gp-150 deficiency) and PMA-activated normal neutrophils pretreated with monoclonal antibody (MoAb) 60.3 were used. Both Gp-150-deficient and MoAb 60.3-treated normal neutrophils failed to adhere to cultured human umbilical vein endothelial cell (HEC) monolayers when activated by PMA (adherence less than 10% with patient and MoAb 60.3-treated cells compared with 53 +/- 3% with normal cells). The addition of PMA-activated normal neutrophils to 51Cr-labeled HEC monolayers failed to induce significant 51Cr release but did produce marked HEC detachment (percentage of detachment 50 +/- 3 at 6 hr). In marked contrast, PMA-activated Gp-150-deficient neutrophils failed to induce significant HEC detachment (percentage of detachment zero (0) at 6 hr). Moreover, the addition of MoAb 60.3 to normal neutrophils inhibited neutrophil-mediated HEC detachment in a time- and dose-dependent fashion. Non-lytic HEC detachment was determined to be largely oxygen radical independent, because PMA-activated chronic granulomatous disease neutrophils and PMA-activated normal neutrophils produced similar disruption of HEC monolayers. Soybean trypsin inhibitor, a chloromethylketone elastase inhibitor, and autologous serum all failed to inhibit neutrophil-mediated HEC detachment. From these studies there is no evidence that nonlytic HEC detachment by PMA-activated neutrophils is mediated by the neutrophil-derived proteases, elastase and cathepsin G. Neutrophil-mediated HEC detachment also required intact neutrophils, because postsecretory medium from PMA-activated normal neutrophils and a suspension of frozen-thawed PMA-activated normal neutrophils were without effect. These in vitro studies indicate that the neutrophil cell surface glycoprotein Gp-150 is required for nonlytic HEC detachment by intact PMA-activated neutrophils.

Animals↗

PubMed1996

Neutral endopeptidase 24.11 in neutrophils modulates protective effects of natriuretic peptides against neutrophils-induced endothelial cytotoxity.

This study was performed to determine effects of atrial and brain natriuretic peptides (ANP, BNP) on neutrophils-induced endothelial injury which is known to play a role in the pathophysiology of ischemia/reperfusion myocardial injury and to examine whether the effects of ANP and BNP on neutrophils are modulated by neutral endopeptidase 24.11 (NEP) in neutrophils themselves. The incubation of human neutrophils with ANP and BNP inhibited the neutrophils-induced detachment of cultured human endothelial cells (HEC). The inhibitory effect of ANP and BNP was associated with the suppressions of the neutrophils adhesiveness to HEC, CD18 expression on the neutrophils and elastase release from the neutrophils. Coincubation with UK73967 or phosphoramidon, inhibitors of NEP, potentiated all of the effects of ANP and BNP on the neutrophil functions, and the NEP inhibitors protected degradation of ANP and BNP by the neutrophils. NEP enzymatic activity in the particulate fractions and immunoreactive NEP expression were found to increase in the neutrophils from patients with early phase of acute myocardial infarction (AMI) by 5.2- and by 4.2-fold of the neutrophils from patients with late phase of AMI, respectively. In an in vivo canine model of myocardial ischemia/reperfusion, the intravenous administration of UK73967 suppressed the neutrophil adherence to endothelium and the neutrophil accumulation in the ischemic/reperfused myocardium. The results indicate that ANP and BNP, which are known to increase in AMI, modulate the neutrophil functions and exert protective effects against the neutrophils-induced endothelial cytotoxity. But the effects are suppressed due to their degradation by the neutrophil own NEP. Thus, neutrophil NEP, which also increases in AMI, may play a role in the pathophysiology of neutrophils-mediated ischemia/reperfusion endothelial and myocardial injury.

Animals↗

PubMed1981

Stimulation of human neutrophil chemiluminescence by soluble immune complexes and antibodies to neutrophils.

Activation of the neutrophil surface membrane may occur with a variety of stimuli, leading to enhanced glucose oxidation via the hexose monophosphate shunt and production of toxic oxygen radicals such as superoxide anion. This metabolic stimulation may be accompanied by light emission or chemiluminescence. The objective of these studies was to determine the effects of soluble and insoluble immune complexes, IgG aggregates, and rabbit anti-neutrophil serum on neutrophil activation as measured by chemiluminescence. Also, the uptake of immune complexes by neutrophils was determined and was correlated with the solubility of the complexes and the levels of induced chemiluminescence. The results indicate that stimulation of neutrophil chemiluminescence and uptake of immune complexes by neutrophils were both closely correlated with the percent precipitation of the immune complexes. Insoluble immune complexes that were avidly ingested by neutrophils also stimulated maximum levels of chemiluminescence. Nevertheless, elevated levels of chemiluminescence were induced by soluble immune complexes that were poorly ingested by neutrophils. Similarly, soluble aggregated IgG induced elevated levels of neutrophil chemiluminescence in a dose-dependent manner. Aggregated IgG induced up to sixfold higher levels of neutrophil chemiluminescence than did equal amounts of monomeric IgG. Finally, rabbit anti-neutrophil serum also caused increased levels of neutrophil chemiluminescence. Since previous studies have shown that sera from some patients with rheumatoid arthritis and Felty's syndrome have increased amounts of neutrophil-binding IgG due to the presence of both immune complexes and neutrophil-reactive antibodies, the effect of these sera on neutrophil chemiluminescence was studied. Positive correlations were observed between the level of neutrophil chemiluminescence induced by the sera and the amount of serum IgG neutrophil-binding activity (r = 0.72, p less than 0.001) as well as the level of immune complexes determined by C1q binding (r = 0.76, p less than or equal to 0.001). Such neutrophil activation by immune complexes or antibodies may reflect production of toxic oxygen radicals and could subsequently affect neutrophil function.

Antibodies↗

PubMed1998

Neutrophil cathepsin G modulates platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion.

1. Close contact between platelets and neutrophils modulates their cellular interactions in thrombotic and inflammatory states, with stimulation of P-selectin expression on platelets by agonists such as thrombin and neutrophil-derived cathepsin G being critical in mediating platelet-neutrophil adhesion. This study compared the effects of thrombin and cathepsin G on platelet P-selectin expression and on P-selectin-mediated platelet-neutrophil adhesion. 2. Washed platelets and platelet-neutrophil mixed cell suspensions (platelet/neutrophil ratio, 10:1) were incubated with either the supernatant of activated neutrophils, purified cathepsin G or thrombin. Platelet P-selectin expression and platelet adhesion to neutrophils was quantified by flow fluorocytometric analysis. 3. The supernatant from activated neutrophils stimulated platelet P-selectin expression comparable to that produced by purified cathepsin G or thrombin. P-selectin expression induced by both activated neutrophil supernatant and purified cathepsin G was completely inhibited by alpha 1-antichymotrypsin, a specific inhibitor of cathepsin G. Unlike thrombin, which induced maximum platelet P-selectin expression by 10 min, sustained to 120 min, cathepsin G induced an initial large increase in platelet P-selectin expression, followed by a progressive reduction over 30-60 min to baseline levels. 4. Co-incubation of neutrophils with thrombin-stimulated platelets resulted in a significant increase in P-selectin-mediated platelet-neutrophil adhesion, which was completely inhibited by preincubation of neutrophils with anti-sialyl Lewis(x) monoclonal antibody. Thrombin produced maximum platelet-neutrophil adhesion by 10 min which remained stable over 120 min. In contrast, cathepsin G-stimulated platelets did not adhere to neutrophils over 120 min of co-incubation. Addition of cathepsin G to thrombin-stimulated platelets caused a progressive reduction over 30-60 min to baseline levels of platelet-neutrophil adhesion. 5. Neutrophil-derived cathepsin G is a potent platelet activator, but unlike thrombin it causes a time-dependent loss of platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion. Therefore, cathepsin G may modulate thrombin-mediated platelet-neutrophil adhesive interactions in inflammation and thrombosis.

Adult↗

PubMed1996

Effects of E-selectin and P-selectin blockade on neutrophil sequestration in tissues and neutrophil oxidative burst in burned rats.

OBJECTIVE: Neutrophil deposition in tissues (leukosequestration) after shock may produce local tissue injury from proteases and high-energy oxygen species released from sequestered neutrophils. The initial step in the binding of neutrophils to capillary endothelium is the interaction of adhesion molecule (selectin) receptors between neutrophils and endothelial cells. We quantified leukosequestration in the tissues of burned rats using two methods of analysis: a) measurement of lung myeloperoxidase; and b) measurement of radiolabeled neutrophils and erythrocytes deposited in multiple tissues. We then determined the ability of a selectin receptor blocking agent to affect neutrophil deposition in tissues after burn injury. DESIGN: Prospective, controlled, laboratory study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats (200 to 300 g). INTERVENTIONS: After tracheostomy and venous cannulation, rats received 17% total body surface area full-thickness contact burns and were resuscitated with saline (20 mL i.p.). Experimental animals received 2 mg/kg body weight i.v. administration of a P- and E-selectin blocking monoclonal antibody, CY-1747, immediately after burn. Lung tissue neutrophils were estimated by measuring myeloperoxidase in lung tissue. Neutrophil retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (preburn) and differentially radiolabeling neutrophils (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hrs after burn, and measuring tissue radioactivity 30 mins later. Edema was estimated by measuring extravasated 125 I-labeled albumin in the various tissues. Peripheral blood neutrophils were analyzed for intracellular hydrogen peroxide content, utilizing a fluorescent dye that reacts with hydrogen peroxide, coupled with analysis of cell fluorescence by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Myeloperoxidase concentration was increased in lungs 5 hrs after burn (p < .05), indicating neutrophil deposition. Radioisotope studies demonstrated significant (p < .05) leukosequestration into the lung, gut, kidney, skin, and brain tissues at 5 hrs after burn. Flow cytometry showed increased intracellular hydrogen peroxide content in peripheral blood neutrophils 5 hrs after burn. Tissue edema, manifested by radiolabeled albumin retention, was not seen in any tissues. Postburn neutrophil deposition in lungs and liver was blocked (p < .05) by administration of CY-1747 after burn, but maximal neutrophil hydrogen peroxide content was unaffected. CONCLUSION: Burn injury in rats results in accumulation of neutrophils in multiple tissues. Neutrophil deposition in the lungs and liver is blocked by administration of the E/P-selectin blocking antibody, CY-1747. Since sequestration of metabolically active neutrophils may induce tissue injury, therapies that block postburn leukosequestration may improve clinical outcomes by limiting remote tissue injury.

Animals↗

PubMed1998

Neutrophil-platelet adhesion: relative roles of platelet P-selectin and neutrophil beta2 (DC18) integrins.

Neutrophils and platelets interact both physically and metabolically during inflammation and thrombosis, but the mechanisms responsible for their adhesion remain incompletely understood. Neutrophil-platelet adhesion was measured after specific stimulation of neutrophils, platelets, or both and quantified by flow cytometry. Specific stimulation of either the neutrophil or the platelet led to a marked increase in the percentage of neutrophils that bound platelets, although platelet stimulation led to a large increase and neutrophil stimulation to only a small increase in the number of platelets per neutrophil. Stimulation of both cells further increased the number of neutrophil-platelet adhesive events and led to large numbers of platelets binding to each neutrophil. Confirming previous observations, blocking antibodies to platelet P-selectin (CD62P) partially inhibited adhesion. However, blockade of the neutrophil beta2 integrin CD11b/CD18 also inhibited the percentage of neutrophils that bound platelets. Combining P-selectin and CD11b/18 blockade further inhibited the stimulated increase in the percentage of neutrophils binding platelets and the increased number of platelets per neutrophil. Both cell adhesion molecules were active even when only a single cell type was primarily activated, supporting physiologically important transcellular activation. These data suggest that: (1) neutrophil-platelet adhesion can be initiated by specific activation of either the neutrophil or the platelet and that specific activation of either cell type leads to distinct patterns of adhesion, and (2) neutrophil-platelet adhesion uses both platelet P-selectin and the neutrophil beta2 integrin CD11b/CD18 when the cells are primarily or secondarily activated.

Antibodies↗

PubMed1984

Neutrophil-endothelial cell interactions. Modulation of neutrophil adhesiveness induced by complement fragments C5a and C5a des arg and formyl-methionyl-leucyl-phenylalanine in vitro.

Neutrophil adherence to vascular endothelial cells is the initial event in the emigration of neutrophils through blood vessel walls to tissue sites of inflammation; this process is attributed to the generation of extravascular chemotactic factors. To investigate the effect of chemotactic factors on neutrophil adherence to endothelium, we developed a sensitive, reproducible in vitro microtiter adherence assay. Base-line nonstimulated adhesion of human neutrophils to cultured human umbilical vein endothelial cell monolayers was 35.2 +/- 0.9%, which is equivalent to three to four neutrophils per endothelial cell. Addition of either purified complement fragment C5a des arg, or formyl-methionyl-leucyl-phenylalanine (FMLP), in concentrations ranging from 10(-10) to 10(-6) M, increased neutrophil adherence to endothelium in a dose-dependent manner. Purified C5a and C5a des arg were essentially equal in their ability to enhance neutrophil adherence, in contrast to the previously described greater in vitro potency of C5a compared with C5a des arg in stimulating neutrophil chemotaxis and enzyme release. Nonstimulated neutrophils adhered preferentially to human endothelial cells compared with fibroblasts or smooth muscle cells, suggesting that endothelial cells may make a unique contribution to the base-line adhesive interaction. However, chemotactic factors appear to enhance neutrophil adherence to endothelium by exerting an effect primarily on the neutrophil. In the presence of chemotactic factor, neutrophils adhered equally well to different cell types or to protein-coated plastic. Pretreatment of endothelial cells with chemotactic factor for as long as 4 h failed to increase subsequent neutrophil adherence. In contrast, pretreatment of neutrophils with chemotactic factor increased adherence to endothelium. Chemotactic factor-stimulated neutrophil adherence to endothelium occurred rapidly (within 2 min), diminished upon removal of stimulus, but could be rapidly and maximally restimulated upon readdition of the original dose of chemotactic factor. Thus, adherence to endothelium stimulated by chemotactic factor would appear to be a dynamic neutrophil response capable of rapid modulation, possibly important to the ability of neutrophils to adhere to and then migrate through vessel walls to localize at sites of inflammation.

Animals↗

PubMed1995

Platelet-induced neutrophil activation: platelet-expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11C/CD18.

Mutual contacts and platelet-expressed fibrinogen seem to be required for the stimulation of neutrophils by activated platelets. The beta 2-integrins CD11b/CD18 and CD11c/CD18 are potential receptors for fibrinogen on neutrophils. In order to investigate whether binding of fibrinogen to these integrins is involved, monoclonal antibodies (MoAbs) and Gly-Pro-Arg-Pro (GPRP) peptide that inhibits fibrinogen binding to CD11c/CD18 were checked for their effects on the interaction of activated platelets and neutrophils. The luminol-amplified chemiluminescence (CL) as a measure for the oxidative burst of neutrophils was recorded simultaneously to the platelet aggregation in mixed cell suspensions. The adhesion of platelets and neutrophils was determined microscopically. The thromboxane A2 mimetic U46619 was used as a potent platelet agonist but that does not stimulate neutrophils. aggregation and a strong CL of neutrophils. The platelet-induced activation of neutrophils required added fibrinogen which fibronectin or thrombospondin could not substitute for. Cytochalasin D (Cyto D) that blocks actin polymerization totally abrogated the platelet-induced Cl of neutrophils. The MoAb OKM1 against CD11b, which blocks fibrinogen binding to CD11b/CD18 as well as the MoAbs IOT16 and IOT18 directed against CD11a and CD18, respectively, had no effect. In contrast, the MoAb LeuM5 which inhibits the binding of fibrinogen to CD11c/CD18 revealed a strong inhibition. Furthermore, GPRP peptide which CD11c/CD18 recognizes on the A alpha-chain of fibrinogen also strongly inhibited the platelet-induced CL of neutrophils, whereas control peptides such as Gly-His-Arg-Pro (GHRP) or Gly-Pro-Gly-Gly (GPGG) had no effect. In contrast to the platelet-induced CL of neutrophils, Cyto D, MoAb against CD11c and GPRP peptide did not inhibit the CL induced by FMLP and PAF in pure neutrophil suspensions. They also did not affect U46619-induced platelet aggregation. The adhesion of platelets and neutrophils was neither dependent on added fibrinogen nor inhibited by Cyto D, MoAb against CD11c and the GPRP-peptide. Therefore fibrinogen and actin polymerization seem not to be required for the adhesion of neutrophils to platelets. However, the activation of neutrophils depends on the interaction of CD11c/CD18 with the A alpha-chain of platelet-expressed fibrinogen and the contractile system of neutrophils.

Blood Platelets↗

PubMed1996

Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation of P-selectin in vitro.

Leukocytes attach to and roll on inflamed endothelium and on leukocyte monolayers that form on the endothelial cells. Leukocyte-leukocyte interactions occurring under hydrodynamic shear stress are mediated by binding of L-selectin to unknown sialomucin-like glycoproteins. We show that purified neutrophil PSGL-1, a sialomucin glycoprotein that serves as a ligand for both P- and E-selectin, can also support the attachment and rolling of free flowing neutrophils in vitro. Neutrophil rolling on PSGL-1 was abolished by the anti-L-selectin mAb DREG200 and by the anti-PSGL-1 mAb PL1, indicating that L-selectin can interact directly with PSGL-1. Neutrophil rolling on neutrophil monolayers was also blocked by PL1 (60 +/- 9% SEM inhibition); however, DREG200 blocked more efficiently (93 +/- 7% SEM inhibition), suggesting that other L-selectin ligands may exist on the neutrophil surface. These studies demonstrate that PSGL-1 on the neutrophil surface is a major functional ligand for L-selectin. The avidity of this L-selectin-dependent adhesion event was sufficient to allow individual neutrophils rolling on P-selectin to capture free flowing neutrophils, which progressed to form linear strings and discrete foci of rolling neutrophils. Neutrophil accumulation on P-selectin accelerated with time as a result of neutrophil-assisted capture of free flowing neutrophils. When neutrophil-neutrophil interactions were blocked by DREG200, neutrophils accumulated on P-selectin in a random pattern and at a uniform rate. Thus, leukocyte-assisted capture of flowing leukocytes may play an important role in amplifying the rate of initial leukocyte recruitment at sites of inflammation.

Antibodies, Monoclonal↗

PubMed1993

Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels.

The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil-neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

Animals↗

PubMed2006

Neutrophil labeling with [(99m)Tc]-technetium stannous colloid is complement receptor 3-mediated and increases the neutrophil priming response to lipopolysaccharide.

INTRODUCTION: [(99m)Tc]-technetium stannous colloid (TcSnC)-labeled white cells are used to image inflammation. Neutrophil labeling with TcSnC is probably phagocytic, but the phagocytic receptor involved is not known. We hypothesised that complement receptor 3 (CR3) plays a key role. Phagocytic labeling could theoretically result in neutrophil activation or priming, affecting the behaviour of labeled cells. Fluorescence-activated cell sorter (FACS) analysis side scatter measurements can assess neutrophil activation and priming. METHODS: We tested whether TcSnC neutrophil labeling is CR3-mediated by assessing if neutrophil uptake of TcSnC was inhibited by a monoclonal antibody (mAb) directed at the CD11b component of CR3. We tested if TcSnC-labeled neutrophils show altered activation or priming status, comparing FACS side scatter in labeled and unlabeled neutrophils and examining the effect of lipopolysaccharide (LPS), a known priming agent. RESULTS: Anti-CD11b mAb reduced neutrophil uptake of TcSnC in a dose-dependent fashion. Labeled neutrophils did not show significantly increased side scatter compared to controls. LPS significantly increased side scatter in control cells and labeled neutrophils. However, the increase was significantly greater in labeled neutrophils than unlabeled cells. CONCLUSIONS: Neutrophil labeling with TcSnC is related to the function of CR3, a receptor which plays a central role in phagocytosis. TcSnC labeling did not significantly activate or prime neutrophils. However, labeled neutrophils showed a greater priming response to LPS. This could result in labeled neutrophils demonstrating increased adhesion on activated endothelium at sites of infection.

CD11b Antigen↗

PubMed2002

Neutrophil defense in patients undergoing bone marrow transplantation: bactericidal/permeability-increasing protein (BPI) and defensins in graft-derived neutrophils.

BACKGROUND: Even after neutrophil counts return to near normal levels, patients undergoing myeloablative chemotherapy and bone marrow transplantation (BMT) are at risk for invasive bacterial infections, raising the possibility that their neutrophil function might be impaired. To assess potential qualitative defects in neutrophil function in patients undergoing BMT, we measured neutrophil content of the antimicrobial (poly)peptides BPI and defensins. METHODS: Neutrophil extracts were analyzed for content of BPI by Western blotting and ELISA and for defensin peptides by acid-urea polyacrylamide gel electrophoresis (PAGE). Antibacterial activity of neutrophil extracts was measured against Escherichia coli K1/r, a clinical isolate sensitive to synergistic killing by BPI and defensins. RESULTS: Neutrophil extract BPI content on post-BMT days +20, +30, and +100 (169+/-35, 232+/-57, and 160+/-55 ng per 106 neutrophils, respectively) was similar to the neutrophil BPI content of normal controls (163+/-35 ng per 106 neutrophils). Neutrophil defensin content also did not vary during this time-span. Activity of neutrophil extracts against E. coli K1/r did not differ between BMT patients and controls. CONCLUSION: At post-BMT days +20 to +100, neutrophils derived from engrafted marrow contain normal quantities of BPI and defensins. Any deficiencies of neutrophil function during this phase are not due to inadequate expression of these antimicrobial (poly)peptides but could reflect abnormalities in other aspects of neutrophil function.

Adolescent↗

PubMed1992

Neutrophil kinetics shortly after initial administration of recombinant human granulocyte colony-stimulating factor: neutrophil alkaline phosphatase activity as an endogenous marker.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered (1.5 micrograms/kg body weight) subcutaneously once daily for 5 to 9 days to 5 patients with malignant lymphoma. In all patients, initial administration of rhG-CSF induced a rapid fall in the neutrophil count within 30 minutes, followed by a recovery and an increase in the neutrophil count within 150 min. A rapid fall in the neutrophil count was accompanied by increased expression of neutrophil C3bi-receptors, and neutrophils left in the circulation had lower activity of neutrophil alkaline phosphatase (NAP) and phagocytosis. A decrease in the NAP scores observed at 30 min reflected a preferential decrease of neutrophils with high NAP activity. A recovery and an increase in the neutrophil count were accompanied by a further decrease of NAP scores, which was caused by a preferential increase of neutrophils with lower NAP activity. The NAP scores of mature neutrophils from peripheral blood were not affected by in vitro treatment of cells with rhG-CSF for up to 150 min at 37 degrees C. These findings and the previous observations that neutrophils in the circulating and marginal pools have high NAP activity and neutrophils in the bone marrow pool have low NAP activity taken together suggest that, following initial administration of rhG-CSF, functionally active neutrophils leave the bloodstream preferentially, which is primarily followed by an influx of neutrophils from the bone marrow, but not by demargination of sequestered neutrophils.

Adolescent↗

PubMed1998

Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.

BACKGROUND: After activation, platelets adhere to neutrophils via P-selectin and beta2-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. METHODS AND RESULTS: Whole blood was sheared at approximately 100 s(-1). The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (approximately 30%) and neutrophil aggregation (approximately 70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. CONCLUSIONS: The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

Abciximab↗

PubMed1989

Neutrophil elastase augments acute edematous injury in isolated rat lungs perfused with neutrophil cytoplasts.

To assess the contribution of neutrophil elastase to neutrophil-mediated acute edematous lung injury, isolated rat lungs were perfused with human neutrophil cytoplasts that produce O2 metabolites normally but contain little of the neutrophil elastase usually found in neutrophils. We found that addition of neutrophil cytoplasts and phorbol myristate acetate (PMA) caused less edematous injury in isolated lungs than addition of neutrophils and PMA. However, addition of neutrophil elastase along with neutrophil cytoplasts and PMA increased the amounts of lung injury found in lungs perfused with neutrophil cytoplasts and PMA to levels that equaled the amount of lung injury found after addition of normal neutrophils and PMA. By comparison, addition of purified neutrophil elastase, PMA alone, or human neutrophils alone did not cause lung injury. The results indicate that neutrophil elastase is a necessary component for the maximal development of acute edematous injury in isolated lungs perfused with neutrophils and PMA.

Acute Disease↗

PubMed1986

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.

Amino Acid Chloromethyl Ketones↗

PubMed2005

Interleukin-15 increases neutrophil adhesion onto human respiratory epithelial A549 cells and attracts neutrophils in vivo.

Interleukin-15 (IL-15) is a neutrophil agonist that plays a role in inflammatory disorders, including a variety of pulmonary diseases. Adhesion of neutrophils onto pulmonary cells is a major event leading to development of inflammation. Recently, elevated levels of IL-15 have been associated with different pulmonary diseases. There is no clear evidence that IL-15 modulates cell surface expression of adhesion molecules in neutrophils, or that IL-15 is involved in neutrophil adhesion onto pulmonary cells. Also, it is not clear if IL-15 induces a neutrophilic inflammation in vivo. This study was aimed at elucidation of these issues. Neutrophils were treated with IL-15 and cell surface expression of CD11a, CD11b, CD11c and CD18 was monitored by flow cytometry. The human respiratory epithelial A549 cell line was used as a substrate for the neutrophil adhesion assay and cell surface expression of CD50, CD54 and CD106 was monitored in IL-15-induced A549 cells. The murine air pouch model was used for investigating potential neutrophilic inflammation induced by IL-15 in vivo. IL-15 significantly increased neutrophil cell surface expression of CD11b and CD18 and up-regulated A549 cell surface expression of CD54. Moreover, A549 cells were found to express IL-15R components and adhesion of neutrophils onto A549 cells was increased when neutrophils or A549 cells were treated with IL-15. Finally, IL-15 induced neutrophilic inflammation in vivo and concentrations of IL-6 and CXCL2/MIP-2 were increased in IL-15-induced pouches. IL-15 might participate in inflammatory pulmonary diseases by attracting neutrophils, modulating cell surface expression molecules and increasing neutrophil adhesion onto pulmonary cells.

Animals↗

PubMed1998

Reduction in neutrophil cell surface expression of tumor necrosis factor receptors but not Fas after transmigration: implications for the regulation of neutrophil apoptosis.

OBJECTIVES: To test the hypothesis that loss of polymorphonuclear neutrophil tumor necrosis factor alpha (TNF-alpha) receptors during transmigration renders the exudate neutrophil refractory to TNF-alpha-mediated stimulation of apoptosis; and to investigate the surface expression of Fas on both circulating and exudate neutrophils. DESIGN: A prospective cohort study. SETTING: Surgical laboratory of a tertiary care hospital. PARTICIPANTS: Twenty-one healthy human volunteers. INTERVENTIONS: All subjects had circulating neutrophils and exudate neutrophils collected by venipuncture and skin window methods, respectively. MAIN OUTCOME MEASURES: Circulating and exudate neutrophils were incubated in culture medium (1.0x10(6) neutrophils per milliliter) alone or with TNF-alpha (100 ng/mL). Apoptosis was evaluated by flow cytometry (annexin V-fluorescein isothiocyanate and propidium iodide). Tumor necrosis factor alpha-phycoerythrin and anti-human Fas-fluorescein isothiocyanate were used to evaluate neutrophil TNF-alpha receptors and surface expression of Fas. RESULTS: Exudate neutrophils had a significant delay in apoptosis rates when compared with circulating neutrophils. The percentage of neutrophils expressing TNF-alpha receptors was significantly diminished after exudation (80%+/-15% vs 33%+/-9%; P<.001), as was the median channel number of TNF-alpha phycoerythrin fluorescence (8.1+/-1.6 vs 5.2+/-0.5; P=.001). However, the expression of Fas was unchanged after transmigration (percentage positive for Fas: 98.7%+/-0.7% vs 92.8%+/-3.4%, P=.89; Fas antibody-fluorescein isothiocyanate median channel fluorescence: 12.2+/-1.1 vs 13.1+/-1.2; P=.80). Exposure of exudate neutrophils to TNF-alpha failed to increase their rate of apoptosis. CONCLUSIONS: Exudate polymorphonuclear neutrophils are confirmed to have delayed apoptosis. Loss of TNF-alpha receptors during transmigration is necessary for neutrophil survival in the extravascular inflammatory milieu.

Apoptosis↗