F-met-leu-phe Research Articles

Anti-inflammatory effects of LPS, MDP and FMLP on carrageenan pleurisy in the rat

Bacterial products fmet-leu-phe (FMLP), muramyl dipeptide (MDP) and lipopolysaccharide (LPS) were assayed for their ability to alter the inflammatory response to lambda carrageenan-induced pleurisy in Hooded Surgery rats. Continuously infused FMLP, or one initial i.v. dose of FMLP, MDP or LPS either ablated or partially suppressed the pleurisy. Total circulating leucocytes and neutrophils were suppressed by 55-65% when compared to the normal circulating leucocyte response to carrageenan pleurisy, excepting the protocol incorporating a single i.v. dose of FMLP where suppression was intermediate at 30%. There were also significant changes in the expression of FMLP receptors on circulating neutrophils. MDP and LPS induced a receptor number increase of 2 and 1.7 times initial value respectively, whilst a continuous FMLP infusion caused a receptor decrease to 0.3 times the initial value. The introduction of bacterial products at an alternative site to that of the pleurisy had an anti-inflammatory effect and the pleurisy was reduced.

Effect of fluticasone propionate on neutrophil chemotaxis, superoxide generation, and extracellular proteolytic activity in vitro.

Corticosteroids are widely used in the treatment of many inflammatory conditions but the exact mode of action on neutrophil function is uncertain. Fluticasone propionate is a new topically active synthetic steroid which can be measured in body fluids and which undergoes first pass metabolism. The effects of fluticasone propionate on the function of neutrophils isolated from normal, healthy control subjects and on the chemotactic activity of sputum sol phase were assessed. Preincubation of neutrophils with fluticasone propionate reduced the chemotactic response to 10(-8) mol/l F-Met-Leu-Phe (FMLP) and to a 1:5 dilution of sputum sol phase in a dose dependent manner. Furthermore, when fluticasone propionate was added to sputum from eight patients with stable chronic obstructive bronchitis the chemotactic activity of a 1:5 dilution of the sol phase fell from a mean (SE) value of 22.2 (1.21) cells/field to 19.6 (0.89), 17.1 (0.74), and 11.9 (0.6) cells field at 1 mumol/l, 10 mumol/l, and 100 mumol/l, respectively. In further experiments fluticasone propionate preincubated with neutrophils inhibited fibronectin degradation by resting cells and by cells stimulated by FMLP (15.2% inhibition of resting cells, 5.1% inhibition of stimulated cells with 1 mumol/l fluticasone propionate, 24% and 18.7% inhibition respectively at 100 mumol/l fluticasone propionate. Fluticasone propionate had no effect on generation of superoxide anion by resting or stimulated cells. These results indicate that fluticasone propionate has a direct suppressive effect on several aspects of neutrophil function and may suggest a role for this agent in the modulation of neutrophil mediated damage to connective tissue.

Dextran sulphate inhibits neutrophil emigration and neutrophil-dependent plasma leakage in rabbit skin.

Rolling of neutrophils in postcapillary venules is believed to be an obligatory step in the adhesion of neutrophils to endothelial cells, preceding firm attachment and emigration. This rolling on the endothelial surface can be blocked by sulphated polysaccharides. Therefore, dextran sulphate or dextran T500 was injected intravenously (i.v., 25 mg/kg) and neutrophil infiltration and plasma leakage in skin were measured in response to intradermal (i.d.) injection of inflammatory mediators in the presence of calcitonin gene-related peptide (CGRP). Neutrophil accumulation and oedema formation induced by the neutrophil chemoattractants, f-Met-Leu-Phe (FMLP) or C5adesArg, were completely suppressed in the presence of dextran sulphate. Neutrophil emigration in response to interleukin-1 (IL-1) was not affected by dextran sulphate. These results suggest that neutrophil recruitment induced by FMLP and C5adesArg, but not by IL-1, is mediated via sulphated glycans, perhaps on endothelial cells.

Quinine-Dependent Antibodies to Neutrophils React With a 60-Kd Glycoprotein on Which Neutrophil-Specific Antigen NB1 Is Located and an 85-Kd Glycosyl-Phosphatidylinositol–Linked N-Glycosylated Plasma Membrane Glycoprotein

AbstractWe have previously described a 24-year-old woman with quinine-dependent antibodies that reacted with neutrophils, red blood cells (RBCs), platelets, and T lymphocytes. The drug-dependent neutrophil antibody was found to react with 85- and 60-Kd neutrophil membrane molecules. In these studies, we further characterized these molecules and found that both were glycosyl-phosphatidylinositol (GPI)-linked and contained sialic acid residues and N-linked carbohydrate side chains, but neither contained O-linked carbohydrates. The protein backbone of the 60-Kd molecule was 45 Kd, and the 85 Kd glycoprotein (GP) was made up of 33- and 31-Kd proteins. While some GPI-anchored neutrophil GPs are released by stimulated neutrophils, neither the 85- nor the 60-Kd GP was released by neutrophil stimulated with C5a, f-met-leu-phe (FMLP), or phorbol myristate acetate (PMA). Neutrophil-specific antigen NB1 is located on a 58- to 64-Kd GP. To determine if the quinine-dependent antibody and anti-NB1 recognize the same GP, immunoprecipitation studies were performed with the quinine-dependent antibody using neutrophils with varying NB1 phenotypes. The 60-Kd GP was detected on NB1-positive neutrophils from 11 of 12 donors tested, but not on NB1-negative neutrophils from two donors tested. After solubilized 125l-labeled neutrophils were absorbed with anti-NB1, the quinine-dependent antibody immunoprecipitated the 85-Kd GP, but not the 60-Kd GP. These results indicate that anti-NB1 and the quinine-dependent antibody identified the same GP. The 85-Kd GP was detected on neutrophils from all 14 donors tested. The electrophoretic mobility of the 85-Kd GP was similar to the electrophoretic mobility of the major 125l-labeled neutrophil protein.

Quinine-dependent antibodies to neutrophils react with a 60-Kd glycoprotein on which neutrophil-specific antigen NB1 is located and an 85-Kd glycosyl-phosphatidylinositol-linked N-glycosylated plasma membrane glycoprotein

We have previously described a 24-year-old woman with quinine-dependent antibodies that reacted with neutrophils, red blood cells (RBCs), platelets, and T lymphocytes. The drug-dependent neutrophil antibody was found to react with 85- and 60-Kd neutrophil membrane molecules. In these studies, we further characterized these molecules and found that both were glycosyl-phosphatidylinositol (GPI)-linked and contained sialic acid residues and N-linked carbohydrate side chains, but neither contained O-linked carbohydrates. The protein backbone of the 60-Kd molecule was 45 Kd, and the 85 Kd glycoprotein (GP) was made up of 33- and 31-Kd proteins. While some GPI-anchored neutrophil GPs are released by stimulated neutrophils, neither the 85- nor the 60-Kd GP was released by neutrophil stimulated with C5a, f-met-leu-phe (FMLP), or phorbol myristate acetate (PMA). Neutrophil-specific antigen NB1 is located on a 58- to 64-Kd GP. To determine if the quinine-dependent antibody and anti-NB1 recognize the same GP, immunoprecipitation studies were performed with the quinine-dependent antibody using neutrophils with varying NB1 phenotypes. The 60-Kd GP was detected on NB1-positive neutrophils from 11 of 12 donors tested, but not on NB1- negative neutrophils from two donors tested. After solubilized 125I- labeled neutrophils were absorbed with anti-NB1, the quinine-dependent antibody immunoprecipitated the 85-Kd GP, but not the 60-Kd GP. These results indicate that anti-NB1 and the quinine-dependent antibody identified the same GP. The 85-Kd GP was detected on neutrophils from all 14 donors tested. The electrophoretic mobility of the 85-Kd GP was similar to the electrophoretic mobility of the major 125I-labeled neutrophil protein.

Heat-aggregated IgA prepared from patients with IgA nephropathy increases calcium mobilization and superoxide production of human neutrophils in vitro.

IgA nephropathy, characterized by predominant mesangial IgA deposition, is the commonest glomerulonephritis worldwide. It is envisaged that circulating IgA plays a primary role in the glomerular injury of IgA nephropathy. In this study, we examined the pathophysiologic effect of IgA and IgG isolated from IgA nephritic patients on the signal transduction and oxidative metabolism of human neutrophils. Heat-aggregated forms, monomers, and F(ab')2 fragments of IgA and IgG were prepared from sera of 11 IgA nephritic patients and 11 healthy controls. Signal transduction was studied by measuring calcium mobilization and oxidative metabolism by measuring superoxide production. Different forms of IgA and IgG from patients with IgA nephropathy did not induce a significant increase in calcium mobilization directly. Nonetheless, neutrophils preincubated with heat-aggregated IgA or IgG from IgA nephritic patients demonstrated a significant rise in calcium mobilization upon subsequent stimulation by a chemotactic peptide, FMet-Leu-Phe (FMLP). Heat-aggregated IgA or IgG pretreatment of neutrophils increased FMLP-induced calcium mobilization in a dose-dependent manner. Aggregated IgA or IgG prepared by heat aggregation from IgA nephritic patients induced a significantly greater superoxide production from neutrophils than immunoglobulins from healthy controls. Similarly, heat-aggregated IgA and IgG induced superoxide production in a dose-dependent manner. Our data suggest that heat-aggregated forms of IgA and IgG exert an upregulatory effect on signal transduction and oxidative metabolism in human neutrophils. These findings indirectly support the view that neutrophils could be activated in IgA nephropathy and may potentially be participating in the inflammatory process of glomerular and interstitial injury.

The role of the neutrophil and formed elements of the blood in an in vitro model of reperfusion injury

Using The globally ischaemic isolated guinea-pig heart we conducted studies to assess the role of activated neutrophils (PMNs) and the role of the endothelium in reperfusion injury. Reperfusion injury was induced by a 20 min period of global ischaemia followed by a 30 min reperfusion with Krebs' buffer supplemented with f-Met–Leu–Phe (fMLP) and heparinized blood. Ischaemia alone or blood alone resulted in a complete recovery in contractile function measured by developed pressure, fMLP (500 μM) and blood, administered to normoxic hearts did not affect contractile function. The combination of 100 μM fMLP and blood beginning at reperfusion and continuing for 30 min decreased the recovery in contractile function (max. 33 ± 6% reovery) while buffer and 100 pM fMLP resulted in a complete recovery in function. In hearts infused with buffer and neutropenic blood incubated with 100 μM fMLP a complete recovery in function was observed. Isolated peritoneal neutrophils, 7–70 × 105 PMN/ min, incubated with 100 μM fMLP and Krebs' solution decreased contractile function in a concentration-related manner (max. 44 ± 11% recovery). Platelets, plasma or red blood cells alone incubated with fMLP did not decrease recovery in developed pressure. Platelets and PMN incubated with 100 μM fMLP did not, while red blood cells and PMN did, elicit a reduction in recovery in contractile function (34 ± 4% recovery). A 20 min period of global ischaemia destroys the functional integrity of the endothelium (response to Ach). Pre-treatment of the heart with sufficient H2O2 to functionally damage the endothelium, followed by infusion of Krebs' solution supplemented with blood and 100 μM fMLP also elicited a reduction in recovery of contractile function (42 ± 15% recovery). In summary, partially activated neutrophils play a major role in reperfusion injury and there exists a cooperativity between the RBC and PMN in this model.

Recruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent cell locomotion.

Chemotactic stimulation of neutrophils results in translocation of CD11b/CD18 (Mac-1) from intracellular storage pools to the cell surface. Though results from several laboratories indicate that the newly arrived surface Mac-1 is not involved in the adherence induced by the initial stimulus, the present study addresses the hypothesis that this Mac-1 plays a role in subsequent adherence-dependent functions. The response of human neutrophils to changing concentrations of a chemotactic stimulus was evaluated by determining the amount of newly arrived surface Mac-1, and Mac-1-dependent adhesion and locomotion. Small step-wise increases in the concentration of f-Met-Leu-Phe (FMLP) resulted in proportional stepwise increases in surface Mac-1 that plateaued within 2-4 min. This newly arrived Mac-1 supported adhesion to protein-coated surfaces only when the cells were exposed to an additional increase in the FMLP stimulus level. Adherence-dependent cellular locomotion was evaluated in chambers that allowed rapid changes in the stimulus concentration. Repeated small increments in the stimulus level at 200-s intervals resulted in significantly longer migration paths than a single-step increase in the stimulus. The results support the hypothesis that small increments in the chemotactic stimulus bring Mac-1 to the cell surface, and this newly mobilized Mac-1 is available for adherence-dependent locomotion with subsequent increases in the concentration of the stimulus.

Platelet activating factor amplifies human neutrophil adherence to bovine endothelial cells: evidence for a lipoxygenase dependent mechanism.

Platelet activating factor (PAF) is a potent lipid mediator that induces the release of leukotrienes and prostaglandins from various cells and tissues. We examined the capacity of PAF alone and in combination with soluble stimuli to enhance eicosanoid synthesis and adherence of human neutrophils. Neutrophils were preincubated with PAF and washed before exposure to the soluble stimuli F-Met-Leu-Phe (FMLP), calcium ionophore A23187, and phorbol myristate acetate. Preincubation of neutrophils with 1 microM PAF enhanced the release of both LTB4 and LTC4 in response to each of the three agonists, in contrast with the unprimed neutrophils. Priming was specific for PAF since lyso-PAF was inactive. Priming concentrations of PAF also augmented the adherence of neutrophils to endothelium in the presence of the soluble agonists A23187, phorbol myristate acetate, and FMLP. The priming effect of PAF on eicosanoid release and neutrophil adherence was shown to have similar time- and dose-dependent effects. Further, the priming effects of PAF on adherence could be reversed by preincubation of neutrophils with the lipoxygenase inhibitors nordihydroguiaretic acid and 5,8,11,14-ETYA but not by preincubation with the cyclooxygenase inhibitor indomethacin. These data demonstrate that PAF amplifies neutrophil adherence to endothelium through a lipoxygenase dependent mechanism.

Release of oxygen radicals by articular chondrocytes: A study of luminol-dependent chemiluminescence and hydrogen peroxide secretion

We previously established that normal articular chondrocytes, like macrophages, express class II major histocompatibility antigens, present antigen, and induce mixed and autologous lymphocyte stimulation. In a recent study using the trapped indicator 2',7'-dichlorofluorescein diacetate, we were able to measure levels of intracellular hydrogen peroxide within normal articular chondrocytes (J Immunol 245:690-696, 1990). In the present study, we utilized the technique of chemiluminescence and the biochemical method of quantitating hydrogen peroxide release to measure the production of reactive oxygen intermediates by articular chondrocytes. Chondrocytes, in suspension or adherent to coverslips, showed luminol-dependent chemiluminescence that was dependent on the number and viability of cells. There was a dose-dependent increase in chemiluminescence in response to soluble stimuli, such as phorbol myristate acetate (PMA), concanavalin A (ConA), and f-Met-Leu-Phe (FMLP). Azide inhibited chemiluminescence, suggesting that the light emission in chondrocytes is myeloperoxidase dependent. The antioxidant, catalase, inhibited chemiluminescence but superoxide dismutase had no effect, suggesting that luminol-dependent chemiluminescence in chondrocytes mostly measured hydrogen peroxide. Chemiluminescence was also observed in fragments of live cartilage tissue, indicating that chondrocytes that are cartilage matrix bound can generate the respiratory burst response. Using the scopoletin oxidation assay, we confirmed the release of increasing amounts of hydrogen peroxide by chondrocytes exposed to interleukin-1, rabbit interferon, and tumor necrosis factor alpha. Tumor necrosis factor alpha had both priming and enhancing effects on reactive oxygen intermediate production by chondrocytes. Reactive oxygen intermediates have been shown to play a significant role in matrix degradation. We suggest that reactive oxygen intermediates produced by chondrocytes play an important role in the degradation of matrix in arthritis.

Signal transduction in neutrophil activation Phosphatidylinositol 3-kinase is stimulated without tyrosine phosphorylation

Treatment of human neutrophils with the peptide f-Met-Leu-Phe (FMLP) results in neutrophil activation concomitant with stimulation of phosphatidylinositol (PtdIns) 3-kinase activity as measured by production of PtdIns-3,4,5-P 3 in [ 32P]orthophosphate labeled cells. Antiphosphotyrosine immunoprecipitates were assayed for PtdIns 3-kinase activity; essentially no activity was present in lysates from either stimulated or unstimulated cells. The 85 kDa regulatory subunit of PtdIns 3-kinase, which normally serves as a substrate for tyrosine kinases, was not detected by SDS-PAGE or Western blot analysis in antiphosphotyrosine immunoprecipitates. In addition, no radioactive band corresponding to PtdIns 3-kinase was observed by SDS-PAGE following antiPtdIns 3-kinase immunoprecipitations. However, immunoprecipitates using polyclonal antibodies against PtdIns 3-kinase showed high PtdIns 3-kinase activity in neutrophil lysates and the 85kDa subunit of PtdIns 3-kinase was detected in Western blots; no differences in activity were observed in FMLP-stimulated and unstimulated cells. These results suggest that, in contrast to polypeptide growth factor signal transduction systems, the activation of PtdIns 3-kinase by FMLP does not require tyrosine phosphorylation.

Superoxide anion production and intracellular free calcium levels in resting and stimulated polymorphonuclear leukocytes obtained from healthy and arteriosclerotic subjects of various ages

It has been established that phagocytic cells are integral components of advanced arteriosclerotic plaques but their role in plaque formation remains unclear. Therefore, toxic agents, such as superoxide anion produced by polymorphonuclear leukocytes (PMNLs) were studied in a clinically defined group of arteriosclerotic patients suffering from obliterative arteriosclerosis of the lower legs. Owing to a close correlation between O2- generation and calcium, the intracellular free calcium concentrations of PMNLs were measured in a resting state and after stimulation with various agents, for example, opsonized zymosan (OZ), the chemotactic peptide f-met-leu-phe (FMLP), and the calcium ionophore A23187. Healthy aged-matched controls were employed. The patients were divided into two age groups: 30-59 years and 60-80 years. We found that in the younger group of arteriosclerotic patients, superoxide anion production and intracellular free calcium concentrations were increased even in the resting state, and only a slight increase was observed after stimulation compared with healthy controls. Granulocyte responses seemed to be similar, independent of the patient's age, to those found in healthy elderly subjects. Arteriosclerosis appears to be associated with an early aging process expressing marked alterations that are greater than those associated with normal aging.

Priming of human neutrophil functions by tumor necrosis factor: enhancement of superoxide anion generation, degranulation, and chemotaxis to chemoattractants C5a and F-Met-Leu-Phe.

Recombinant human tumor necrosis factor-alpha (rTNF) stimulated increased generation of superoxide anion (O2-) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2-2200 units/ml) for 10 min enhanced the subsequent generation of O2- in response to C5a and f-Met-Leu-Phe (FMLP). Recombinant TNF did not enhance O2- generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001-50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001-0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100-10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.

Regulation of phosphatidic acid phosphohydrolase activity during stimulation of human polymorphonuclear leukocytes.

Phosphatidic acid phosphohydrolase (PPH) activity was determined in human polymorphonuclear leukocytes (PMNs) by measuring the hydrolysis of [32P]phosphatidic acid (PA) added to cell sonicates. Enzyme activity was localized primarily to a soluble fraction. Soluble and particulate activities required magnesium and were inhibited by calcium, N-ethylmaleimide, sphingosine, and propranolol. The activity in unstimulated PMNs was 0.64 +/- 0.11 nmol of PA hydrolyzed.mg protein-1.min-1 in particulate and 4.20 +/- 0.42 in soluble fractions. Stimulation of PMNs with 1 microM f-Met-Leu-Phe (FMLP) for 10 min caused a slight decrease in soluble activity and a small increase in the activity of particulate fractions. Preincubation with 10 microM cytochalasin B for 5 min before FMLP stimulation markedly enhanced both of these changes. The effect of FMLP plus cytochalasin B was rapid (less than 10 s), whereas the calcium ionophore A23187 (1 microM) and phorbol myristate acetate (100 ng/ml) caused slower and smaller changes in enzyme activity. These results indicate that after chemoattractant stimulation; PPH activity decreases in the soluble fraction and increases in the particulate fraction suggesting that PPH may participate in signal transduction in the PMN.

Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-triphosphate, diacylglycerol and intracellular calcium

Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-triphosphate (IP 3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 μM concentration. The IP 3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP 3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3–1.0 μM, did not affect the initial mobilization of FMLP-induced intracellular Ca 2+ (Ca 2+ i), although a sustained elevation of cytosolic Ca 2+ level was observed within 5 min. This effect could not be suppressed, even by 1 μM phorbolmyristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (≤ 100 nM) were unable to affect FMLP-induced IP 3 production, DG accumulation and Ca 2+ i, the PMA-inhibited initial Ca 2+ i signal and IP 3 formation triggered by FMLP were almost completely restored. At higher concentrations (≥ 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-biphospinate (PIP i) breakdown, thus causing accumulation of IP 3 and DG and an elevation of Ca 2+ i level. Whereas IP 3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.

Inhibition of histamine release from human FcεRI+ cells by nimesulide

We have previously demonstrated that pharmacological concentrations of non-steroidal anti-inflammatory drugs (NSAID) such as indomethacin, acetylsalicylic acid, and meclofenamic acid enhance IgE-mediated histamine release (HR) from human basophils. We have now examined the effects of nimesulide (NIM), a new NSAID, on mediator release from human basophils. Preincubation (10 min at 37°C) of basophils with pharmacological concentrations (10−6−10−3M) of NIM resulted in a concentration-dependent decrease of HR induced by anti-IgE, the Ca2+-ionophore A23187 and the formylated tripeptide f-Met-Leu-Phe (FMLP). Maximal inhibition of anti-IgE-induced HR was achieved after preincubation with 10−4M NIM and ranged between 14.5% and 44.5% (mean±SEM, 29.7±4.5%). The drug had a marked inhibitory effect on HR from basophils induced by A23187 (80.6±5.5%), FMLP (63.5±10.3%), 12-O-tetradecanoyl-phorbol-13-acetate (57.0±8.7%) and bryostatin 1 (65.7±7.6%). NIM also inhibited the IgE-mediated synthesis of peptide leukotriene C4 from basophils.

Interactions of Granulocyte-Macrophage Colony-Stimulating Factor (CSF), Granulocyte CSF, and Tumor Necrosis Factor a in the Priming of the Neutrophil Respiratory Burst

AbstractExposure of neutrophils to a range of cytokines augments their response to subsequent agonist-induced activation of the respiratory burst. We have examined the effects of several of these factors, both singly and in combination, on the priming of f-met-leu-phe (FMLP) and complement C5a-stimulated neutrophil H2O2 production, using a whole blood flow cytometric assay designed to minimize artefactual activation. Both granulocyte-macrophage colony-stimulat-ing factor (GM-CSF) and tumor necrosis factor a (TNFα) produced a similar degree of priming of the FMLP-stimulated burst in vitro (558% ± 86%, n = 41, and 581% ± 95%, n = 21, of the response seen with FMLP alone, respectively), but with markedly different kinetics (half-maximal response 20 minutes and 7 minutes, respectively). Preincubation with granulocyte colony-stimulating factor (G-CSF) alone caused only modest priming (202% ± 39%, n = 14). Priming with cytokine combinations of the FMLP-stimulated burst showed that the combinations of G-CSF and TNFα and GM-CSF and TNFα are highly synergistic, with recruitment of neutrophils unresponsive to priming by single agents. Priming with the combination of GM-CSF and G-CSF was not significantly different to priming with GM-CSF alone. Similar results were obtained using C5a as the respiratory burst stimulus. Significant priming of the FMLP-stimulated respiratory burst was seen in vivo in patients receiving an infusion of GM-CSF (332% ± 50% of preinfusion response to FMLP, P < .005, n = 8). Priming was also seen in patients receiving G-CSF (152% ± 58%, n = 5), although this did not reach conventional significance levels (.05 < P < .1). Although GM-CSF infusion caused priming in vivo, this was 48% less than predicted by preinfusion in vitro responses. This result was not due to inadequate GM-CSF levels as addition of further GM-CSF ex vivo did not correct the response. However, these neutrophils were still able to respond appropriately to ex vivo priming with TNFα, with a doubling in H202 production.© 1992 by The American Society of Hematology. 0006-497119217903-0031$3.0010

Interactions of granulocyte-macrophage colony-stimulating factor (CSF), granulocyte CSF, and tumor necrosis factor alpha in the priming of the neutrophil respiratory burst

Exposure of neutrophils to a range of cytokines augments their response to subsequent agonist-induced activation of the respiratory burst. We have examined the effects of several of these factors, both singly and in combination, on the priming of f-met-leu-phe (FMLP) and complement C5a-stimulated neutrophil H2O2 production, using a whole blood flow cytometric assay designed to minimize artefactual activation. Both granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF alpha) produced a similar degree of priming of the FMLP-stimulated burst in vitro (558% +/- 86%, n = 41, and 581% +/- 95%, n = 21, of the response seen with FMLP alone, respectively), but with markedly different kinetics (half-maximal response 20 minutes and 7 minutes, respectively). Preincubation with granulocyte colony-stimulating factor (G-CSF) alone caused only modest priming (202% +/- 39%, n = 14). Priming with cytokine combinations of the FMLP-stimulated burst showed that the combinations of G-CSF and TNF alpha and GM-CSF and TNF alpha are highly synergistic, with recruitment of neutrophils unresponsive to priming by single agents. Priming with the combination of GM-CSF and G-CSF was not significantly different to priming with GM-CSF alone. Similar results were obtained using C5a as the respiratory burst stimulus. Significant priming of the FMLP-stimulated respiratory burst was seen in vivo in patients receiving an infusion of GM-CSF (332% +/- 50% of preinfusion response to FMLP, P less than .005, n = 8). Priming was also seen in patients receiving G-CSF (152% +/- 58%, n = 5), although this did not reach conventional significance levels (.05 less than P less than .1). Although GM-CSF infusion caused priming in vivo, this was 48% less than predicted by preinfusion in vitro responses. This result was not due to inadequate GM-CSF levels as addition of further GM-CSF ex vivo did not correct the response. However, these neutrophils were still able to respond appropriately to ex vivo priming with TNF alpha, with a doubling in H2O2 production.

Dynamic changes in neutrophil cytoskeleton during priming and subsequent surface stimulated functions.

The naturally occurring inositol polyphosphate, inositol hexakisphosphate (InsP6, phytic acid) primes human neutrophils for enhanced responses to stimulatory agonists. Unlike other primers InsP6 has no effect at high doses (up to 500 microM) on the neutrophil's basal rate of superoxide production (O2-). Pretreatment of the cells for 2 min with InsP6 (100 microM) substantially enhances fMet-Leu-Phe (FMLP) (10(-7) M)-induced respiratory burst. In investigations of the dynamic aspect of neutrophil actin during priming and stimulation with FMLP the content of F-actin has been measured with and NBD (nitrobenz-2-oxa-1,3-diazole)-phallacidin assay. The induced assembly of F-actin is rapid (peaks at approximately 45 s) followed by depolymerization. In contrast, priming with InsP6, with or without FMLP stimulation, results in a sustained assembly of F-actin as visualized with a rhodamine-phalloidin staining technique. These changes in macromolecular status and distribution of F-actin during InsP6 priming occur in the absence of any other demonstrable functional responses and thus InsP6 may be a useful tool to follow early events in neutrophil functions or to monitor the presence of unprimed and preprimed subpopulations in the circulation. Since InsP6 is present in high concentrations in neutrophils (up to 100 microM), its release from damaged or effete cells may have a modulatory role on neutrophil functions.

Oxidative metabolism of circulating granulocytes in adult respiratory distress syndrome

Among the different mechanisms involved, polymorphonuclear leukocytes (PMNs) may play a central role in the pathogenesis of adult respiratory distress syndrome (ARDS). PMNs were evaluated in 15 patients with ARDS, in 21 at risk of developing ARDS (AR), and in 36 controls (C). Spontaneous and opsonized zymosan (OZ), phorbol myristate acetate (PMA), and F-Met-Leu-Phe (F-M-L-P)-stimulated oxygen radical production was measured by luminol- and lucigenin-enhanced chemiluminescence (CL). Spontaneous CL activity of PMNs from ARDS patients was significantly greater than that from the PMN control (luminol CL, 2.8 ± 0.6 vs. 0.8 ± 0.1 mV, p <0.001; lucigenin CL, 2.0 ± 0.6 vs. 0.30 ± 0.04 mV, p <0.001), and the CL value from AR patients (luminol CL, 1.3 ± 0.2 mV, p <0.001 vs. C; Lucigenin CL, 0.8 ± 0.1 mV, p <0.001 vs. C) was found to be between the ARDS and C patients. The peak of PMA-stimulated CL occurred earlier and it was significantly higher in ARDS patients than in AR patients (p <0.05) and controls (p <0.001). When the CL response was elicited with F-M-L-P, no difference among the three groups was found. When stimulated with OZ, the peak CL generated by PMNs from ARDS patients was significantly depressed compared with controls (luminol CL, 26.7 ± 1.8 vs. 40.9 ± 2.3 mV, p <0.01; lucigenin CL, 5.0 ± 0.4 vs. 7.4 ± 0.5 mV, p <0.005) with a similar result being obtained from AR patients (luminol CL, 32.1 ± 2.5 mV, p <0.01 vs. C). Plasma from ARDS and AR patients showed a defective opsonizing capacity, suggesting in vivo complement consumption in both patient groups. No correlation between the severity of hypoxemia, the cause of ARDS, the outcome, and the different PMN functions could be established. Our results are in agreement with a determinant role of PMNs in the development of ARDS. The opposite metabolic responses may explain both the pulmonary injury and the increased susceptibility to infections observed in patients at risk of or with ARDS.