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PubMed1983

Exposure of human neutrophils to chemotactic factors potentiates activation of the respiratory burst enzyme.

NADPH oxidase activity in particulate fractions from human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan was enhanced by prior exposure of the neutrophils to chemotactic factors. Enhanced activity was seen measuring both NADPH-dependent chemiluminescence and superoxide anion production. Enhancement was observed to be both time and dose dependent with several chemotactic stimuli, including casein, N-formyl-methionyl-leucyl-phenylalanine (f-MLP), and C5a. F-MLP and C5a showed similar patterns, with peak enhancement occurring within 2 to 15 min of preincubation and lasting up to 1 hr. In contrast, enhancement of PMA-stimulated oxidase activity by casein was more gradual and sustained, lasting up to 2 hr. Fractions from cells treated only with chemotactic factors and not stimulated with PMA showed no oxidase activity. Kinetic studies of this enhanced activity show that chemotactic factors induce increases in Vmax values but do not significantly alter Km values for the oxidase. Further experiments using agents that modulate degranulation suggest that enzyme release is not involved in this enhancement. These data suggest that pretreatment with chemotactic factors results in an increase in the amount of activated oxidase in membrane fractions obtained from PMA-stimulated neutrophils. This alteration of NADPH oxidase activity provides a subcellular basis for the enhanced bactericidal activity and increased oxidative metabolism seen in neutrophils treated with chemotactic factors.

Chemotactic Factors↗
PubMed1995

In vivo blood monocyte migration to acute inflammatory reactions, IL-1 alpha, TNF-alpha, IFN-gamma, and C5a utilizes LFA-1, Mac-1, and VLA-4. The relative importance of each integrin.

UNLABELLED: The role of the monocyte integrins, Mac-1, LFA-1, and VLA-4, on the adhesion of rat blood monocytes to rat microvascular endothelial cells in vitro and the importance of these receptors in monocyte migration to inflammation in vivo were evaluated. Monocyte adhesion to cytokine (IL-1, IFN-gamma, and TNF-alpha)-stimulated endothelial cells was mediated by Mac-1, LFA-1, and VLA-4, but Mac-1 appeared to be less important than LFA-1 or VLA-4. After i.v. injection, large numbers of 51Cr-labeled blood monocytes migrated within 2 h to dermal inflammatory sites induced by C5a, IL-1 alpha, IFN-gamma, TNF-alpha, LPS, and poly inosinic:cytidylic acid. Anti-Mac-1 mAb treatment had no effect, whereas anti-LFA-1 inhibited migration to C5a and the cytokines by 20 to 40%. Blocking both Mac-1 and LFA-1 decreased monocyte accumulation by 50 to 70% to all stimuli. Anti-VLA-4 inhibited monocyte migration to IL-1 alpha, IFN-gamma, TNF-alpha, and LPS, but not to C5a. Combining anti-Mac-1 with anti-VLA-4 did not increase this inhibition, whereas blocking VLA-4 and LFA-1 together further suppressed (60-85%) migration. Combined treatment with mAb to all three integrins inhibited > 98% of the monocyte migration to the inflammatory stimuli. IN CONCLUSION: 1) 51Cr blood monocytes can be used to quantify monocyte migration to inflammatory reactions in the rat. 2) Monocytes use Mac-1, LFA-1, and VLA-4 for in vitro adhesion and in vivo migration to cutaneous inflammation, and these integrins are essential for normal migration because blockade of all three virtually abolishes monocyte accumulation. 3) Mac-1 plays a less important role than LFA-1, as LFA-1 appears to substitute for Mac-1, and VLA-4 and LFA-1 can mediate much of the adhesion and migration. 4) The initiating inflammatory stimulus also modifies monocyte integrin usage, supporting the multistep combinatorial model of leukocyte extravasation.

Animals↗
PubMed1998

Neutrophils stimulated with a variety of chemoattractants exhibit rapid activation of p21-activated kinases (Paks): separate signals are required for activation and inactivation of paks.

Activation of the p21-activated protein kinases (Paks) was compared in neutrophils stimulated with a wide variety of agonists that bind to receptors coupled to heterotrimeric G proteins. Neutrophils stimulated with sulfatide, a ligand for the L-selectin receptor, or the chemoattractant fMet-Leu-Phe (fMLP), platelet-activating factor, leukotriene B4, interleukin-8, or the chemokine RANTES exhibited a rapid and transient activation of the 63- and 69-kDa Paks. These kinases exhibited maximal activation with each of these agonists within 15 s followed by significant inactivation at 3 min. In contrast, neutrophils treated with the chemoattractant and anaphylatoxin C5a exhibited a prolonged activation (>15 min) of these Paks even though the receptor for this ligand may activate the same overall population of complex G proteins as the fMLP receptor. Addition of fMLP to neutrophils already stimulated with C5a resulted in the inactivation of the 63- and 69-kDa Paks. Optimal activation of Paks could be observed at concentrations of these agonists that elicited only shape changes and chemotaxis in neutrophils. While all of the agonists listed above triggered quantitatively similar activation of the 63- and 69-kDa Paks, fMLP was far superior to the other stimuli in triggering activation of the c-Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein kinase (MAPK). These data indicate that separate signals are required for activation and inactivation of Paks and that, in contrast to other cell types, activated Pak does not trigger activation of JNK or p38-MAPK in neutrophils. These results are consistent with the recent hypothesis that G-protein-coupled receptors may initiate signals independent of those transmitted by the alpha and betagamma subunits of complex G proteins.

Animals↗
PubMed1995

Human heart mast cells. Isolation, purification, ultrastructure, and immunologic characterization.

We have isolated, partially purified, and characterized the mast cells from human heart tissue. The histamine content of left and right ventricles and septum of hearts obtained from 25 patients undergoing heart transplantation was 5.4 +/- 0.6, 5.3 +/- 0.5, and 5.6 +/- 0.5 micrograms/g of wet tissue, respectively. Ultrastructural study of cardiac mast cells revealed scroll, crystal, and mixed granules, homogeneously dense granules, and lipid bodies in the cytoplasm. A mild collagenase digestion was used to disperse the heart mast cells; the average yield was 3.2 +/- 0.6% (range: 0.8 to 13.6%). The average histamine and tryptase content/heart mast cells was 3.3 +/- 0.2 pg (n = 25) and 24.2 +/- 4.3 micrograms/10(6) cells (n = 11), respectively. Survival of cardiac mast cells after overnight culture was 71.9 +/- 5.4% (n = 23). The purification of human heart mast cells can be brought from less than 0.1 to 12% by a combination of low-speed centrifugation over albumin (2%) solution and Percoll gradient. Viability as shown by trypan blue exclusion was greater than 90%. Heart mast cells released histamine in response to immunologic (anti-IgE, anti-Fc epsilon RI, and C5a) and nonimmunologic stimuli (recombinant human stem cell factor, A23187, and compound 48/80) but did not respond to substance P, FMLP, 12-O-tetradecanoylphorbol-13-acetate, or acetylcholine. There was a linear correlation between the percentage of release caused by anti-IgE and anti-Fc epsilon RI, whereas there was no correlation between the release caused by C5a and anti-IgE-mediated stimuli. Cross-linking with anti-IgE of IgE on heart mast cells induced the release of tryptase (10.1 +/- 2.1 micrograms/10(7) cells; n = 10) and the de novo synthesis of PGD2 (17.3 +/- 4.3 ng/10(6) cells; n = 10) and of leukotriene C4 (19.1 +/- 4.5 ng/10(6) cells; n = 10). There was a linear correlation between the percentage of histamine secretion and tryptase release (r = 0.67; p < 0.001) induced by cross-linking of Fc epsilon RI. similarly, there was a significant correlation between percentage of histamine secretion and PGD2 (r = 0.63; p < 0.001) and LTC4 (r = 0.64; p < 0.001) release. Immunoelectron microscopy demonstrated the presence of chymase in cardiac mast cells. Mast cells isolated from human heart can be a useful model with which to study the role of these cells and their mediators in cardiac anaphylaxis and cardiovascular diseases.

Cell Separation↗
PubMed1996

Chemotactic response of human alveolar macrophages and blood monocytes elicited by exposure to sulfur dioxide.

An experimental study was undertaken to investigate the in vitro effect of sulfur dioxide on the chemotactic activity of alveolar macrophages (AM) and blood monocytes (BM). The cells were placed on a polycarbonate membrane and exposed to SO2 0.5, 1.5 and 2.5 ppm for 15 min. Control experiments were performed with exposure of the cells to synthetic air with 5% CO2. After gas exposure the cells were incubated with the chemotactic active agent C5a in 5% carbon dioxide (CO2) at 37 degrees C for 60 min. The numbers of AM and BM passing actively through the membrane were quantified using light microscopy. Our results show a dose-dependent reduction in the migration rate of cells under SO2 exposure. SO2 0.5 ppm induced a 29% and SO2 2.5 ppm a 53% decrease in migration of AM compared with the control exposure to synthetic air (P < 0.01). Identical experiments with BM resulted in a decrease in migration of up to 57% (P < 0.01). At SO2 concentrations of up to 2.5 ppm no significant cytotoxic effects were observed for AM or BM. The data demonstrate that exposure to SO2 may reduce the chemotactic activity of AM and BM. Our results further suggest that the decrease in cell migration induced by SO2 is due to changes in chemotactic mechanisms and not to cell death.

Aged↗
PubMed1999

Hemofiltrate from patients with severe sepsis and depressed left ventricular contractility contains cardiotoxic compounds.

Myocardial dysfunction due to sepsis is common in patients with multiple organ dysfunction syndrome and is believed to be produced by inflammatory mediators. Some of these mediators may be eliminated by continuous hemofiltration, which is a standard procedure in an ICU for renal replacement therapy. This study was designed to directly compare the effects of ultrafiltrates from patients with sepsis (UFs) with ultrafiltrates from healthy volunteers (UFh) in well-characterized cardiomyocyte culture systems. Isovolemic hemofiltration (filtration rate: 2 L/h, polyamide membrane) was performed during 12 hours in 5 patients with severe sepsis (Elebute Score >20) and simultaneously reduced left ventricular contractility (left ventricular stroke work index [LVSWI] <30 g m/m2) and in 5 healthy volunteers. Inflammatory mediator concentrations (interleukin [IL]-1beta, IL-6, IL-8, tumor necrosis factor [TNF] alpha, C3a, and C5a) were measured in plasma and ultrafiltrate samples taken shortly after the beginning of the hemofiltration procedure. Cell culture experiments were done comparing UFs with UFh by using spontaneously beating or electrically driven neonatal rat cardiomyocyte cultures. UFs contained significantly higher amounts of IL-1, IL-8, and C3a when compared to UFh. Simultaneously, UFs induced a decrease in the contraction frequency of electrically-stimulated cardiomyocytes, whereas UFh had no effect. The cardiotoxic effect could be reversed by the addition of a high concentration (2.4 mM) of Ca++. Hemofiltration did not alter parameters of cardiac performance during 12 hours in patients with sepsis. UFs induced significant cardiotoxic effects in rat cardiomyocytes, whereas UFh showed no cardiotoxicity. Contact of blood with the hemofiltration membrane did not induce activation of cardiotoxic mediators. Significantly higher filtration rates may be required to improve left ventricular contractility in patients with sepsis by hemofiltration.

Adult↗
PubMed1991

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.

Animals↗
PubMed1990

Platelet activating factor stimulates cyclo-oxygenase activity in guinea pig eosinophils. Concerted biosynthesis of thromboxane A2 and E-series prostaglandins.

The effect of platelet activating factor (PAF) on the generation of cyclo-oxygenase-derived arachidonic acid metabolites was examined on purified eosinophils harvested from the peritoneal cavity of male guinea pigs. PAF produced a concentration-dependent increase in the amount of immunoreactive thromboxane B2 (TXB2) and PGE1/E2 released from these inflammatory cells at a relative molar ratio of 30:1. The EC50 of PAF was 20 to 40 nM and maximum stimulation (4.5-fold) of both prostanoids occurred at 1 microM PAF. The ability of PAF to generate TXA2 was rapid (t 1/2 = 9 s), transient (40 s), noncytotoxic, and noncompetitively antagonized by the PAF-receptor blocking drug, WEB 2086. On an equimolar (100 nM) basis, PAF was significantly more effective than C5a, fMLP, and PMA at stimulating TXB2 release but markedly less potent than the calcium ionophore, calcimycin. Pretreatment of eosinophils with the cyclo-oxygenase inhibitor flurbiprofen (8 microM for 5 min) abolished the ability of PAF to promote both TXB2 and PGE1/E2 release. Likewise, dazmegrel (50 microM for 5 min), a selective inhibitor of thromboxane synthetase, abolished PAF-stimulated TXB2 release but markedly augmented the elaboration of PGE1/E2. Inhibition of cyclo-oxygenase with flurbiprofen affected neither the ability of PAF to elevate the intracellular calcium ion concentration (measured by fura-2 fluorescence) nor its appetency to generate superoxide anions at any PAF concentration examined. It is concluded that activation of guinea pig eosinophils by PAF is receptor-mediated and independent of the concomitant generation of cyclo-oxygenase-derived excitatory prostanoids. Inasmuch as TXA2 may contribute to the pathogenesis of bronchial hyperreactivity, then these data implicate the eosinophil as a potential source of this lipid mediator.

Animals↗
PubMed1993

Impairment of chemoattractant-stimulated hexose uptake in neonatal neutrophils.

Neonatal neutrophils (polymorphonuclear leukocytes [PMN]) exhibit a well-documented deficiency in chemotaxis, the nature of which has not been fully elucidated. To determine whether impaired ability of neonatal PMN to increase hexose uptake in response to chemoattractants could contribute to this defect, we compared uptake of 2-deoxy-D-glucose (2-DOG) in stimulated versus resting PMN from neonates (cord blood) and healthy adults. Compared with unstimulated values; N-formyl-methionyl-leucyl-phenylalanine (fMLP) (optimal at 10 nmol/L) caused a threefold to fourfold increase in 2-DOG uptake by adult PMN. Unstimulated 2-DOG uptake by neonatal PMN was slightly higher than that for adult cells, but fMLP caused only a minimal (less than twofold) increase, and optimally stimulated uptake was significantly lower than for adult PMN (P < .01 for adult versus neonatal stimulated uptake; n = 6). Findings were similar when ionomycin or C5a was used as a stimulus. Optimal fMLP stimulation of adult PMN was associated with a marked decrease in the Km for 2-DOG uptake, from 0.74 +/- 0.11 to 0.23 +/- 0.03 mmol/L (delta Km = -0.51 +/- 0.12 mmol/L; n = 6). In contrast, there was relatively little fMLP-induced change in the Km for uptake of 2-DOG by neonatal PMN (from 0.44 +/- 0.04 mmol/L to 0.32 +/- 0.019 mmol/L n = 6); delta Km = -0.12 +/- 0.04 mmol/L; P = .011 for adult versus neonatal delta Km. Stimulation with fMLP was not accompanied by a significant change in the Vmax for 2-DOG uptake with either adult or neonatal PMN, and the respective values for Vmax were similar. We conclude that the chemoattractant-induced increase in hexose uptake by PMN is deficient in neonates compared with adults and that this deficiency involves mechanisms that determine the Km for this process. This impairment may contribute to defective chemotaxis in neonatal PMN.

Adult↗
PubMed2003

Immunization to nicotine with a peptide-based vaccine composed of a conformationally biased agonist of C5a as a molecular adjuvant.

This paper describes the synthesis of a nicotine hapten (Nic) that possesses a carboxyl sidearm functional group allowing for conjugation to a peptide via amide bond formation. Nic was attached to the N-terminal amino group of a 19-residue peptide composed of a conformationally biased agonist of human C5a (YSFKPMPLaR), which is used as a molecular adjuvant and a B cell epitope of human MUC1 glycoprotein (YKQGGFLGL) to yield a peptide-based nicotine vaccine, NicYKQGGFLGLYSFKPMPLaR. Rats immunized with this vaccine were significantly less sensitive to behavioral effects (a Pavlovian discrimination task) induced by their exposure to high concentrations of nicotine (0.4 mg/kg) relative to their non-vaccinated counterparts. The attenuation of these nicotine-induced behavioral effects emanated from the presence of nicotine-specific antibodies (Abs) that were present in the sera of vaccinated rats even after their repeated exposure to high concentrations of nicotine during the time required to perform the behavioral assays. These results suggest that immunization with NicYKQGGFLGLYSFKPMPLaR in the absence of adjuvant is an effective means of inducing a nicotine-specific Ab response, which is capable of attenuating nicotine-induced behavioral/psychoactive effects.

Adjuvants, Immunologic↗
PubMed1994

Dexamethasone inhibits basophil migration.

Glucocorticoids have been shown to inhibit the local accumulation of basophils during the allergen-induced late-phase reaction (LPR). Since migration is an essential step in the recruitment of basophils from the circulation, we examined whether the widely used glucocorticoid, dexamethasone (DEX), directly acts on basophils to inhibit the migration caused by C5a, interleukin (IL)-3, and IL-8. When purified basophils were preincubated with various concentrations of DEX, a dose-dependent inhibition was observed; DEX at concentrations as low as 1 nM reduced the number of migrated basophils by 30-40%; at higher concentrations, it showed a slightly stronger inhibitory effect. There was no significant difference in the effect of DEX on the migration caused by the three chemoattractants. The action of DEX took place rapidly; apparent inhibition was observed even when migration was initiated without preincubation. Although the inhibitory effect of this agent was not reversed when DEX was removed by washing, the inhibition was not mediated by the toxicity as measured by the trypan-blue exclusion test. These results indicate that the in vivo blocking effect of glucocorticoids on basophil accumulation during LPR is mediated in part by direct action to inhibit the migration of basophils.

Basophils↗
PubMed1994

Characterization of two different forms of mitogen-activated protein kinase kinase induced in polymorphonuclear leukocytes following stimulation by N-formylmethionyl-leucyl-phenylalanine or granulocyte-macrophage colony-stimulating factor.

Incubation of polymorphonuclear leukocytes with chemoattractants, granulocyte-macrophage colony-stimulating factor (GM-CSF), or phorbol 12-myristate 13-acetate (PMA) activated both mitogen-activated protein kinase kinase (MAPKK) and mitogen-activated protein kinase (MAPK). Activation by chemoattractants was rapid and transient, being maximal by 1 min and decreasing by 10 min. The order of efficacy was formyl-met-leu-phe > C5a > > LTB4 > interleukin 8 > platelet-activating factor. In contrast, activation by GM-CSF or PMA was slow and sustained being maximal at 5 min and with little decrease by 30 min. Sustained MAPK activation required continuous activation of the MAPKK. The MAPKK induced by N-formylmethionyl-leucyl-phenylalanine, GM-CSF, or PMA was resolved into two forms by anion exchange chromatography (Mono Q). Both corresponded to a 45-kDa MAPKK antigen by Western blotting and were inactivated by serine/threonine protein phosphatase 2A. Rechromatography of both forms after dephosphorylation resulted in the antigen's eluting slightly earlier on the Mono Q gradient than when in the active state. However, the two peaks remained separate, suggesting that they are not merely different phosphoforms of the same enzyme. The MAPK cascade is a signaling pathway common to many polymorphonuclear leukocyte stimulants, which may be activated transiently or in a sustained manner.

Blotting, Western↗
PubMed1991

Differential inhibition of human neutrophil activation by cyclosporins A, D, and H. Cyclosporin H is a potent and effective inhibitor of formyl peptide-induced superoxide formation.

Cyclosporin (Cs)A but not CsH inhibits activation of human lymphocytes. We studied the effects of CsA, CsD, and CsH on human neutrophil activation induced by chemoattractants and by various substances that circumvent receptor stimulation. CsH inhibited superoxide (O2-) formation induced by the chemotactic peptide, FMLP (30 nM), with a half-maximal effect at 40 nM. O2- formation was abolished by CsH at 1 microM. CsH increased the concentration of FMLP causing half-maximal activation of O2- formation from 30 nM to 0.8 microM and substantially reduced the stimulatory effect of FMLP at supra-maximally effective concentrations. The inhibitory effect of CsH on O2- formation was evident immediately after addition to neutrophils. CsH also markedly inhibited the increase in cytosolic Ca2+ ([Ca2+]i), beta-glucuronidase, and lysozyme release and aggregation stimulated by FMLP. CsA and CsD were considerably less effective than CsH to inhibit FMLP-induced O2- formation. CsA and CsD were without effect on exocytosis, rises in [Ca2+]i, and aggregation induced by the chemotactic peptide. Cyclosporines inhibited FMLP-induced O2- formation in an additive manner, indicating that they acted through a mechanism they had in common. Cyclosporines only slightly inhibited O2- formation and lysozyme release induced by C5a. Aggregation and rises in [Ca2+]i stimulated by C5a were not affected by cyclosporines, and they did not inhibit O2- formation and exocytosis induced by platelet-activating factor and leukotriene B4. Cyclosporines partially inhibited O2- formations induced by NaF and gamma-hexachlorocyclohexane. CsA marginally inhibited PMA-induced O2- formation and lysozyme release. CsA, CsD, and CsH did not inhibit arachidonic acid-induced O2- formation and its potentiation by NaF or stable guanine nucleotides in a cell-free system from DMSO-differentiated HL-60 cells. CsH partially inhibited binding of FML [3H]P to formyl peptide receptors in membranes from DMSO- or dibutyryl cAMP-differentiated HL-60 cells. Our data show that: 1) cyclosporines differentially inhibit activation of human neutrophils; and 2) CsH is, indeed, not immunologically inactive but is a potent and effective inhibitor of FMLP-induced O2- formation. 3) CsH interferes with agonist binding to formyl peptide receptors and in addition, cyclosporines may also act at sites distal to chemoattractant receptors.

Bucladesine↗
PubMed2000

Eosinophils maintain their capacity to signal and release eosinophil cationic protein upon repetitive stimulation with the same agonist.

Eosinophils contain in their granules eosinophil cationic protein (ECP) and other basic proteins that have been implicated in immunity to parasites and pathophysiology of chronic allergic responses. In a model of eosinophil degranulation, we show that eosinophils release ECP upon short-term GM-CSF priming and stimulation with either platelet-activating factor (PAF) or the anaphylatoxin C5a, but not eotaxin. Restimulation with the same agonist (PAF or C5a) was unsuccessful as assessed by monitoring intracellular calcium concentration and ECP release. In contrast, upon an intermediate washing step, eosinophils rapidly transduced PAF and C5a signals followed by significant ECP releases. Ligand-binding studies demonstrated that only a proportion of PAF receptors is internalized upon cell stimulation and that washing of the cells removes the agonist from the cell surface. Upon repetitive stimulation, eosinophils with less than 50% of the original ECP content were obtained. Such eosinophils did not increase cellular ECP levels even in the presence of the eosinophil survival factor GM-CSF in overnight cultures. In vivo studies revealed that eosinophils always express detectable amounts of ECP under chronic inflammatory conditions. In conclusion, we have shown that eosinophils maintain their capacity to degranulate upon repetitive stimulation with the same agonist as long as the receptor is not occupied from a previous stimulation. The cellular content of ECP appears to be a no limiting factor in the case of repetitive stimulation, implying that mature eosinophils may not require a significant ECP resynthesis.

Blood Proteins↗
PubMed1987

Hydroxy radical as autotoxin in chemotactically activated neutrophils.

Chemotactic stimulation of neutrophils can result in the production and release of toxic oxygen metabolites which, in turn, may affect neutrophil function. We have investigated the effects of catalase, superoxide dismutase, hydroxyl radical scavengers and a iron chelator on chemotactic responses of rat peritoneal neutrophils in vitro. Neutrophils were quantitatively assessed in micropore filters for chemotactic migration to several different chemotactic peptides including C5a, present in inulin-activated rat serum, the non-oxidizable tripeptide N-formyl-norleucyl-leucyl-phenylalanine, and the oxidizable peptide N-formyl-methionyl-leucyl-phenylalanine. The presence of catalase in the suspension of neutrophils caused a dose-dependent enhancement of chemotactic responses to each of the three chemotactic stimuli, whereas addition of superoxide dismutase had little effect. Hydroxyl radical scavengers (mannitol, dimethyl sulfoxide) or the iron chelator, deferoxamine, also afforded significant, dose-dependent enhancement of neutrophyl chemotaxis, while the iron-saturated chelator was devoid of chemotaxis-enhancing properties. Furthermore, stimulation of rat neutrophils with phorbol myristate acetate resulted in the appearance of products of lipid peroxidation (conjugated Schiff bases), the generation of which could be prevented by the hydroxyl radical scavenger, dimethyl thiourea. These data suggest that catalase, hydroxyl radical scavengers, and iron chelators protect chemotactically stimulated neutrophils from autotoxicity caused by neutrophil-derived hydrogen peroxide and its iron-catalyzed conversion product, hydroxyl radical.

Animals↗
PubMed1992

Heterogeneity in the mobilization of cytoplasmic calcium by human polymorphonuclear leukocytes in response to fMLP, C5a and IL-8/NAP-1.

The visible excitation and emission wave-lengths of the recently developed fluorescent Ca2+ indicator fluo-3 permit analysis of the intracellular Ca2+ concentration, [Ca2+]i, in flow cytometry with a 488-nm argon laser. The role of [Ca2+]i in human polymorphonuclear leukocyte heterogeneity was investigated in response to formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and interleukin 8/neutrophil attractant/activation protein 1 (IL-8/NAP-1) by flow cytometry. The [Ca2+]i changes in different subpopulations within a heterogeneous cell suspension were resolved upon stimulation with fMLP. Using an anti-CD16 phycoerythrin-conjugated antibody and fluo-3 simultaneously, neutrophils affected and nonaffected in Ca2+ mobilization were distinguished in two patients suffering from glycogen storage disease type 1b. In normal neutrophils, a different time course of Ca2+ mobilization of neutrophil subpopulations immediately after stimulation with fMLP was detected. In addition, after stimulation with a low concentration of IL-8/NAP-1 (10(-10) M) two subsets of neutrophils appeared; one of them showed an increase in [Ca2+]i, while the other did not. These results indicate heterogeneity in the neutrophil signal transduction process involved in Ca2+ mobilization. Therefore, flow cytometric analyses can resolve changes in single-cell [Ca2+]i distribution patterns, which is important for the understanding of [Ca2+]i in neutrophil heterogeneous activation processes.

Aniline Compounds↗
PubMed1992

Effects of a 5-lipoxygenase inhibitor, AA861, on lipoxygenase metabolism and superoxide anion generation by human polymorphonuclear leukocytes--potentiation of superoxide anion generation by LTB4.

We studied the influence of a selective 5-lipoxygenase inhibitor, AA861, on the generation of the superoxide anion (O2-) and the lipoxygenase metabolites by human polymorphonuclear leukocytes (PMN). PMN produce O2- in a dose-dependent manner following stimulation with arachidonic acid (AA), leukotriene B4 (LTB4), or C5a. When PMN were stimulated with one of those three agents in the presence of high doses of AA861 (1-10 micrograms/ml), a significant reduction of O2- release was observed. In contrast, the generation of O2- by PMN stimulated by LTB4 was potentiated at lower concentrations of AA861 (0.025-0.25 micrograms/ml). However, stimulation with AA or C5a did not influence O2- generation in the presence of AA861 at the same concentration range. Furthermore, treating the PMN with the cyclooxygenase inhibitor, acetylsalicylic acid, did not potentiate the generation of O2- by stimulation with LTB4 over a wide range of concentrations. Quantification of lipoxygenase metabolites by reverse-phase high-performance liquid chromatography revealed that a high concentration of AA861 (0.5-5 micrograms/ml) completely inhibited the production of LTB4 and its omega-oxidative metabolites by PMN following stimulation with 100 microM AA, but only partially inhibited that of 5-hydroxyeicosatetraenoic acid (5-HETE). AA861 at a concentration of 5 micrograms/ml significantly increased the production of 15-HETE by PMN following the same stimulation. AA861 did not influence catabolism of LTB4 added to the reaction mixture to its omega-oxidative products by PMN over a wide range of concentrations. These findings suggest that the inhibition of 5-lipoxygenase metabolism may stimulate 15-lipoxygenase in human PMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗
PubMed1991

Conversion of leukotriene A4 by neutrophils and platelets from patients with atopic dermatitis.

The generation of arachidonic acid-derived inflammatory mediators from unstimulated and stimulated neutrophils (PMN) and platelets in the presence of exogenous LTA4 has been studied in patients with atopic dermatitis (AD) as well as in healthy volunteers. PMN were stimulated with the interleukins IL-3, IL-8, C5a, and the Ca-ionophore A23187. In addition, NaF and thrombin were used to stimulate platelets. The release of leukotriene (LT)B4, 20-COOH- and 20-OH-LTB4, cysteinyl-leukotrienes and 12-HETE was measured. The proinflammatory mediator release from PMN and platelets of patients with AD was significantly higher as compared to the control group. The spontaneous conversion of LTA4 by PMN and platelets was markedly enhanced in patients with AD. Different results with receptor-specific and non-specific stimuli (Ca-ionophore A23187) in the presence of exogenous LTA4 were obtained. The results indicate a higher state of activation for enzymes involved in leukotriene formation. Furthermore, the production of 12-HETE by platelets from patients with AD was enhanced in unstimulated and stimulated cells. Our data emphasize that neutrophils and platelets may play an important role in the pathogenesis of AD by an increased responsiveness to receptor-specific stimuli and cell-cell interaction via LTA4.

Adolescent↗