Induce Neutrophil Activation Research Articles

The anti-FcγRIII MAb 3G8 induces neutrophil activation via a cooperative action of FcγRIIIb and FcγRIIa

Human neutrophils express two types of Fcγ receptors, the transmembrane FcγRIIa and the glycan-phosphatidylinositol-anchored FcγRIIIb, that show synergism in provoking a cellular response. To analyse further the requirements for this synergism to occur we used the monoclonal antibody 3G8, directed against FcγRIII This antibody is able to induce neutrophil activation, as measured by an increase in the intracellular free Ca 2+ concentration and homotypic neutrophil aggregation, but only when the Fe part of the antibody is able to interact with FcγRIIa. We observed that binding of the Fab parts of 3G8 mAb to two FcγRIIIb molecules and binding of the Fc part to one FcγRIIa molecule is required, because a bispecific antibody, 2B1, in which only one 3G8 Fab is present, did not induce neutrophil activation. Moreover, engagement of one FcγRIIa molecule and two FcγRIIIb molecules on the same cell is instrumental to achieve activation of the mAb 3G8. The activation of neutrophils by the 3G8 antibody represents a further example of synergistic activation of neutrophils via Fcγ receptors.

In vitro modulation of induced neutrophil activation by different surfactant preparations.

Endotracheal surfactant administration has gained an important role in the treatment of respiratory failure. Polymorphonuclear neutrophil granulocyte (PMN) activation mediated by chemoattractants, such as interleukin-8 (IL-8), neutrophil-activating peptide-2 (NAP-2) and formylated bacterial oligopeptides, has been found to be involved in the pathophysiology of acute respiratory failure. We investigated potential modulating effects of commercial surfactant preparations (Exosurf, Alveofact, Curosurf and Survanta) on spontaneous and chemoattractant-induced PMN function. Isolated cytochalasin B (CytB)-treated PMNs from healthy adults were incubated with increasing concentrations of surfactant. The response of the cells was measured in terms of elastase release from the lysosomes within 30 min. The PMNs showed no direct activation by any of the surfactants tested. However, when cells were stimulated with suboptimal dosages of chemokines, such as IL-8 (2 nM) or NAP-2 (100 nM), or formyl-methionyl-leucyl-phenylalanine (fMLP) (50 nM), and co-incubated with increasing concentrations of surfactant (0.05-8 mg.mL-1) the release of elastase was markedly modulated depending on the surfactant preparation used. Whilst Exosurf and Alveofact showed only modest effects on the elastase release induced by all three mediators, Curosurf and Survanta markedly inhibited the cellular response in a dose-dependent manner. At concentrations above 1 mg.mL-1, Curosurf and Survanta decreased the IL-8-, NAP-2- and fMLP-induced elastase release by 83, 67 and 90%, and by 82, 75 and 80%, respectively. In conclusion, exogenous surfactant may modulate the inflammatory response of the airways by affecting the chemoattractant-induced polymorphonuclear neutrophil activation. Surfactant preparations with inhibiting properties on neutrophil activation may participate in the prevention of neutrophil-induced lung damage.

A fusion protein of IL-8 and a Fab antibody fragments binds to IL-8 receptors and induces neutrophil activation.

A fusion protein was generated by genetic engineering which combined a Fab fragment of a monoclonal antibody directed to the human epidermal growth factor receptor with the biologically active N-terminally truncated 2-72 amino acid form of the human chemokine IL-8. The Fab IL-8 fusion protein was expressed in E. coli and antibody binding and IL-8 activity were determined. Our data indicate that the N-terminus of IL-8 remains functional for receptor interaction. The fusion protein showed specific binding to IL-8 receptors, induced IL-8 mediated chemotactic activity, and the release of MPO activity. However, N-terminal fusion of IL-8 to the carboxyl terminus of the Fab fragment resulted in reduced binding to IL-8 receptors and consequently to reduced biologic activity of IL-8. The affinity of the antibody arm for EGF-R was improved when compared to a monovalent Fab. Fusion proteins as described herein may represent improved therapeutics for cancer therapy based on their potential to selectively increase and prolong cytokine concentration in the tumour. Since chemokines such as IL-8 recruit effector cells and stimulate effector cell function in situ, a lymphocyte-independent anti-tumour activity followed by tumour-specific immunity could be proposed.

Correlated plasma elastase and sera cytotoxicity in eclampsia A possible role of endothelin-1 induced neutrophil activation in preeclampsia-eclampsia

The activation of neutrophils was studied in preeclampsia (n = 10) and eclampsia (n = 20) compared to normotensive controls (n = 10) and nonpregnant essential hypertensives (n = 10). Plasma elastase levels were raised in preeclampsia (0.53 +/- 0.32 microgram/mL, P < .002) and eclampsia (1.26 +/- 0.8 microgram/mL, P < .001) respectively compared to normal pregnancies (0.032 +/- 0.009 microgram/mL). The plasma elastases were more elevated in eclamptic cases compared to essential hypertensive (0.53 +/- 0.27 microgram/mL; P = .01) patients. We analyzed the correlation among elastase values, systolic (SBP), mean blood pressures (MBP), endothelin-1 (ET-1) levels and sera cytotoxicity (as measured by fura-2 release from human umbilical venous endothelial cell culture) in eclamptic cases. SBP and MBP were significantly correlated with plasma elastase levels in preeclampsia (r = 0.67, 0.63, respectively; P < .03) and eclampsia (r = 0.49, 0.49, respectively; P < .02). ET-1 levels were correlated with SBP (P = .003) and MBP (P = .001) and corresponding elastase levels (r = 0.606, P < .003) in eclamptic patients. Doses of 10, 25, and 50 pmol/mL of ET-1 increased elastase release in human neutrophil cultures dose and time dependently. Cytotoxicity of eclamptic sera correlated (P < .001) to the corresponding plasma elastase values. Therefore, this study suggests that neutrophil activation and ET-1 induced neutrophil activation occurs in this disease.

慢性副鼻腔炎病態における好中球浸出に対するＩＬ‐８の役割

It is well known that persistent purulent nasal discharge with numerous neutrophils occurs in patients with chronic sinusitis (CS) . In recent years, IL-8 has been reported to be one of the major mediators which induce neutrophil activation and chemotaxis. The present study was performed in an attempt to investigate the role of IL-8 in neutrophil chemotaxis in CS. Immunohistochemical examinations revealed that IL-8 was expressed in polymorphonuclear cells in the nasal discharge and in epithelial and nasal gland cells of the ethmoidal mucosa. IL-8 concentration in the nasal discharge of CS determined by enzyme immunoassay was 28.3±7.3 nM (mean±SEM, n=10), which was significantly higher than that of nasal allergy. These results suggest that chemotactic factors in paranasal sinus effusion including IL-8 derived from epithelial cells and nasal gland cells attract neutrophils from the mucosa, and the neutrophils in the effusion secrete IL-8, which causes further neutrophil accumulation in the paranasal sinus effusion in CS.

Neutrophil activation after percutaneous transluminal coronary angioplasty

We investigated whether percutaneous transluminal coronary angioplasty (PTCA) would induce neutrophil activation in patients with coronary artery disease. Blood samples were taken from the coronary sinus in 14 patients who underwent PTCA and in 9 control subjects who underwent coronary arteriography (CAG). Flow cytometry was used to measure membrane surface expression of β 2 integrin (CD11b) and the generation of hydrogen peroxide in neutrophils after ex vivo phorbol myristate acetate stimulation by 2′,7′-dichlorofluorescein. Neutrophil elastase was measured by an immunoenzymatic method. Surface expression of CD11b increased significantly, approximately twofold, after PTCA but not after CAG. Mean fluorescence intensity of 2′,7′-dichlorofluorescein in stimulated neutrophils decreased significantly after PTCA, suggesting a previous in vivo activation, but not after CAG. Neutrophil elastase increased significantly after PTCA but not after CAG. These data indicate that PTCA induces neutrophil activation and suggest that neutrophils may contribute to the ischemic injury.

Neutrophil Activation by Helicobacter pylori Lipopolysaccharides

Helicobacter pylori-induced release of toxic substances by neutrophils could be of potential importance in the pathogenesis of gastroduodenal inflammatory diseases. Lipopolysaccharide (LPS) has the ability to induce neutrophil activation at very low concentrations. Neutrophil oxidative metabolism and enzyme release were assessed after stimulation of neutrophils with various preparations of LPS from H. pylori and compared with that obtained with Salmonella typhimurium LPS. No direct activation of neutrophils by LPS was observed. Preincubation with LPS showed a significant priming for increased activity on subsequent stimulation, particularly with rough-form LPS. The potency of H. pylori LPS was 10-fold lower than that of S. typhimurium LPS, probably reflecting the unique biochemical structure of H. pylori LPS. Chronic low-grade stimulation by H. pylori LPS in the gastric mucosa may render neutrophils primed for the excessive release of detrimental substances into the tissue on stimulation by other mediators.

α1-Antitrypsin Pittsburgh (Met358 → Arg) Inhibits the Contact Pathway of Intrinsic Coagulation and Alters the Release of Human Neutrophil Elastase during Simulated Extracorporeal Circulation

Cardiopulmonary bypass prolongs bleeding time, increases postoperative blood loss, and triggers activation of plasma proteolytic enzyme systems and blood cells referred to as the "whole body inflammatory response". Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, contact and complement systems. Contact and complement pathway proteins both induce neutrophil activation. alpha 1-antitrypsin Pittsburgh (Met358-->Arg), a mutant of alpha 1-antitrypsin, is a potent inhibitor of plasma kallikrein and thrombin. We investigated whether this recombinant mutant protein inhibited platelet activation, as well as contact and/or complement-induced neutrophil activation during simulated extracorporeal circulation. Arg358 alpha 1-antitrypsin did not prevent the 34% drop in platelet count at 5 min of recirculation, did not block the 50% decrease in ADP-induced platelet aggregation at 120 min of recirculation, nor inhibit the release of 6.06 +/- 1.07 micrograms/ml beta-thromboglobulin at 120 min of recirculation suggesting that the inhibitor had little effect on platelet activation. However, Arg358 alpha 1-antitrypsin totally blocked kallikrein-C1-inhibitor complex formation but not C1-C1-inhibitor complex formation. Most importantly, Arg358 alpha 1-antitrypsin decreased the release of 1.11 +/- 0.16 micrograms/ml human neutrophil elastase by 43%. The attenuation of neutrophil activation in the absence of an effect on complement activation via the classical pathway, supports the concept that kallikrein is a major mediator of neutrophil degranulation during cardiopulmonary bypass.

A histamine derivative increases intracellular calcium mobilization and oxidative metabolism in HL-60 cells

Past work in our laboratory has shown that a derivative of histamine, histamine-trifluoromethyl-toluidide (HTMT), has surprising tissue specificity on lymphocytes and can produce remarkable immunosuppression. This study focuses on the effects of HTMT on Ca2+ mobilization and oxidative metabolism in undifferentiated and DMSO-differentiated HL-60 cells. HTMT caused two phases of increases in intracellular calcium concentrations ([Ca2+]i) in HL-60 cells. The responses were dose dependent, with similar EC50 values (1.7×10−5M for undifferentiated and 1.5×10−5M for differentiated cells). The increase in [Ca2+]i in differentiated cells was much greater than in undifferentiated cells. The maximum responses were observed after the undifferentiated cells were incubated with DMSO for 7 days. The increase in [Ca2+]i induced by HTMT in both types of cells was competitively antagonized by high concentrations of histamine but not by classic histamine receptor antagonists (H1, H2, or H3). The inhibitory effects of histamine on [Ca2+]i accumulation in differentiated cells were partially reversed by histamine H2 receptor antagonist ranitidine, whereas in undifferentiated cells, the effects of histamine on Ca2+ mobilization were not affected by ranitidine. Other cAMP elevating agents did not inhibit increases in [Ca2+]i in undifferentiated cells but did effect [Ca2+]i in differentiated cells. The enhanced response in [Ca2+]i mobilization after differentiation of HL-60 cells appeared to be the result of an increase in the expression/function of receptors for HTMT. One interesting feature of this regulation was the fact that cAMP per se did not regulate HTMT induced Ca2+ mobilization in undifferentiated cells but inhibited the mobilization in differentiated cells. HTMT caused the generation of reactive oxygen species in both undiffrentiated and differentiated HL-60 cells as measured by chemoluminescence and the levels of generation correlated with the mobilization of [Ca2+]i. In addition, the EC50s for the HTMT induced calcium mobilization and the generation of reactive oxygen species were similar, as was the case for histamine induced inhibition (Ki) in both cell types. The data imply a second messenger role for Ca2+ in HTMT induced neutrophil activation.

Reduction of exercise induced neutrophil activation after sucessful coronary ballon angioplasty

Reduction of exercise induced neutrophil activation after sucessful coronary ballon angioplasty

Quantification of Neutrophil Adhesion to Skeletal Muscle Venules Following Ischemia-Reperfusion

Ischemia-reperfusion is encountered in a wide variety of surgical situations. The damage resulting from ischemia-reperfusion may be due, in part, to the infiltration and activation of neutrophils into the reperfused tissue. The purpose of the study was to examine ischemia-reperfusion induced neutrophil activation in skeletal muscle. In control muscles, ischemia in the hamster right cremaster muscle was produced for 5 min after an initial 2 hr 55 min perfusion period. In ischemic muscles, ischemia was produced for 3 hr prior to reperfusion. After the clamps were removed, a video recording of the cremaster microvasculature was made using intravital fluorescence microscopy. Acridine orange was infused intravenously 10 min prior to video recording in order to selectively label and enhance the contrast of neutrophils. The number of neutrophils rolling along the endothelium of 40 to 60 microns-diameter venules in a 1-min period increased from 9.0 in control animal cremaster venules to 24.1 following ischemia-reperfusion (p less than .05; n = 11). The ischemia-reperfusion model developed in this study allows for the direct quantification of neutrophil adhesion in skeletal muscle and can be further used to assess pharmacologic minimization of neutrophil-mediated damage in skeletal muscle.

Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) are in the circulation of most patients with pauci-immune necrotizing vasculitis and pauci-immune crescentic glomerulonephritis. The current study demonstrates an effect of these autoantibodies on neutrophil function in vitro. ANCA cause normal human neutrophils to undergo an oxidative burst and degranulate. Both ANCA phenotypes (i.e., cytoplasmic-pattern ANCA and myeloperoxidase-specific ANCA) induce neutrophil activation. ANCA sera and purified immunoglobulins significantly increase the release of reactive oxygen species when compared with controls. ANCA, in a dose-dependent manner, induce the release of primary granule contents. These effects are markedly enhanced by priming neutrophils with tumor necrosis factor. Flow cytometry studies demonstrate the presence of myeloperoxidase on the surface of neutrophils after cytokine priming, indicating that primed neutrophils have ANCA antigens at their surfaces to interact with ANCA. These observations suggest an in vivo pathogenetic role for ANCA. We propose that, in patients with necrotizing vasculitis, ANCA-induced release of toxic oxygen radicals and noxious granule enzymes from cytokine-primed neutrophils could be mediating vascular inflammation.

Neutrophil activation during cell separation procedures

Leucocytes labelled with 111In or 99Tcm are used as diagnostic agents for detecting sites of infection by scintigraphy. Before radiolabelling is performed, leucocytes are isolated from whole blood. The effect of isolation procedures on neutrophil activation has been studied by measuring the neutrophil elastase produced during incubation of whole blood with three erythrocyte sedimentation agents (dextran 70, hydroxyethyl starch and methylcellulose) and two density gradient media (Percoll and Mono-Poly Resolving Medium). Neutrophil elastase was measured using a standard radioimmunoassay. At 21 degrees C, dextran caused no elastase release while hydroxyethyl starch and methylcellulose induced significant release (p = 0.01 and p less than 0.01 resp.). All three agents caused significant elastase release at 37 degrees C. When whole blood was incubated with Percoll and Mono-Poly Resolving Medium, no release of neutrophil elastase was observed. These results show that neither density gradient medium induces neutrophil activation but that certain erythrocyte sedimentation agents do. Of the three sedimentation agents investigated, dextran is the agent of choice if neutrophil activation is to be minimized.

Leukotriene release enhancing factor. Purification, specific allergen induction, and further biologic properties.

Human PBMC cultured with PHA produced a factor which enhanced neutrophil LTB4 production. After progressive purification by gel filtration and chromatofocusing, leukotriene release enhancing factor (LREF) gave a single band on SDS-PAGE corresponding to a Mr of 60 kDa and a isoelectric point of 5.0 to 5.2. Human T lymphocytes were identified as a source of LREF because a long term human CD4+ T cell line, specific for house dust mite (Dermatophagoides farinae) generated leukotriene release enhancing activity (LREA) when stimulated with the inducing Ag together with irradiated accessory cells. Cultures of accessory cell and Ag alone did not induce neutrophil activation. Similarly, control cultures using an irrelevant allergen (grass pollen) failed to produce this activity. Stimulation of PBMC with anti-CD3 mAb also generated LREA. LREA had other neutrophil-associated biologic activities. These included the enhancement of neutrophil adherence to nylon wool, increased cytotoxicity of opsonised schistosomula of Schistosoma mansoni and upregulation of C receptors (CR1 and CR3). LREA had minimal neutrophil chemotactic activity. Therefore, LREF has potential as a mediator of inflammation and provides a link between lipid mediators, neutrophils, and cell-mediated immunity in the context of allergic diseases.

Lipoxygenase products induce neutrophil activation and increase endothelial permeability after thrombin-induced pulmonary microembolism.

We examined the mechanism of the neutrophil (PMN)-dependent increase in pulmonary vascular permeability to protein after thrombin-induced pulmonary microembolism. Humoral factors that activate PMNs after thrombin-induced pulmonary microembolism were characterized in pulmonary lymph obtained from unanesthetized sheep challenged with intravenous infusion of alpha-thrombin. Time-dependent increases in PMN migration, aggregation, and superoxide anion (O2-) generation were induced by the pulmonary lymph obtained within 20 minutes after thrombin infusion. The pulmonary lymph neutrophil activating factors present in ether extracts of lymph had retention times of leukotriene B4 (LTB4) and monohydroxyeicosatetraenoic acids (HETEs) by high-performance liquid chromatography. The postthrombin lymph samples containing the LTB4 and HETEs increased PMN O2- generation and endothelial monolayer permeability to 125I-albumin in the presence of PMNs layered on the endothelial monolayers. Control lymph samples replete with LTB4, 5-HETE, and 15-HETE induced increases in PMN O2- generation and endothelial monolayer permeability to 125I-albumin in the presence of PMNs layered on the endothelial monolayers. Maximal increases in PMN O2- production and endothelial permeability occurred when LTB4, 5-HETE, and 15-HETE were coincubated with PMNs, indicating a synergistic action of these mediators in inducing PMN activation. Endothelial monolayer permeability to 125I-albumin did not increase with postthrombin lymph samples obtained after pretreatment with the 5-lipoxygenase inhibitor, L-651,392. The results indicate that lipoxygenase products generated in the lungs after thrombin-induced microembolism contribute to increased endothelial permeability secondary to PMN activation.

Oxygen distribution and utilization after phorbol myristate acetate-induced lung injury.

There is considerable evidence suggesting that substrate (e.g., oxygen) distribution and utilization are abnormal in the setting of acute respiratory failure from the adult respiratory distress syndrome (ARDS). The mechanisms that may explain this anomalous relationship include: redistribution of cardiac output resulting in the nonuniform delivery of substrate to regional vascular beds; increases in flow through anatomic precapillary arteriovenous channels; increased organ metabolic demand; and direct injury to the regional microvasculature. To test these hypotheses, we used radiolabeled 15-mu microspheres to measure cardiac output, regional blood flow, total systemic shunt flow, oxygen uptake (VO2), and oxygen delivery (QO2) in dogs after phorbol myristate acetate (PMA: 30 micrograms/kg; n = 5)-induced neutrophil activation and acute lung injury. These studies demonstrated that neutrophil activation and acute lung injury was accompanied by an increase in the level of QO2 (e.g., 19.1 ml/min.kg) necessary to maintain a constant level of VO2. Although PMA-induced acute lung injury did not result in an increase in the fraction of cardiac output comprising total systemic shunt flow or in an increase in whole body metabolic demand compared to control animals (e.g., PMA = 0 micrograms/kg; n = 10), it did result in a significant decrease in cardiac output. In addition, there were significant reductions in blood flow to most organs after PMA-induced acute lung injury that were not reversible with intravascular volume expansion (80 ml isotonic saline/kg; n = 5). The exclusion of reversible volume depletion, anatomic shunts, and increased metabolic demand suggest a role for microvascular injury in the development of the regional blood flow abnormalities and the supply dependence of oxygen uptake observed in this study. In the context of the mechanisms of lung injury known to be operative in this model, these microcirculatory abnormalities may be mediated through the intravascular activation of circulating neutrophils.

Leukotriene B4 synthesis and neutrophil chemotaxis in chronic granulocytic leukaemia.

A sensitive gas chromatography-mass spectrometric method was used to measure the generation in whole blood of leukotriene B4 (LTB4), a potent stimulator of neutrophil chemotaxis, in eight patients with chronic granulocytic leukaemia (CGL) and 12 healthy controls. LTB4 was detectable in unstimulated samples from all the patients (mean 194 (70 SEM) pg/ml), and the capacity for LTB4 production after stimulation with calcium ionophore (A23187) was similar in patients (32.1 (11) ng/10(6) leucocytes) and controls (38.1 (4) ng/10(6) leucocytes). In response to stimuli which induce neutrophil activation, LTB4 production was significantly greater in the patients than in controls: 35.6 (13) v 13.0 (3) ng/ml, p less than 0.05 (f-met-leu-phe); and 42.4 (16) v 14.7 (4) ng/ml, p less than 0.02 (opsonised zymosan). Anti-IgE stimulated considerably more LTB4 production in patients with CGL than in controls (3.86 (1.6) v 0.83 (0.43) ng/ml; p less than 0.005) and this correlated significantly (p less than 0.05) with the basophil count. Neutrophil chemotaxis to LTB4, however, was significantly impaired in the patients with CGL even at the highest concentration of LTB4 (10(-5) M). Chemotaxis to f-met-leu-phe, phagocytosis, and bacterial killing were normal. Thus although LTB4 synthesis is normal or even enhanced in patients with CGL, specific defects in LTB4-mediated responses may contribute to neutrophil dysfunction in this disease.

Thrombin enhances opsonized zymosan-induced chemiluminescence of neutrophils

We examined the effects of α-thrombin (the native enzyme) on neutrophil activation as assessed by the measurement of chemiluminescence. α-Thrombin in physiological concentrations (10 −9 − 10 −8 M) did not induce neutrophil chemiluminescenee. However, when neutrophils were coincubated with opsonized zymosan and α-thrombin, the chemiluminescence response to opsonized zymosan was enhanced in a concentration-dependent manner. The neutrophil chemiluminescence responses to opsonized zymosan and to opsonized zymosan plus α-thrombin were dependent on the generation of oxygen-derived free radicals since the chemiluminescence was inhibited by superoxide dismutase. The results indicate that thrombin per se does not induce neutrophil chemiluminescence. However, thrombin enhances the chemiluminescence response to opsonized zymosan suggesting an interaction between thrombin and complement receptors in inducing neutrophil activation. The chemiluminescence response to thrombin and opsonized zymosan is the result of oxygen-derived free radicals.