Desensitization uncouples the formyl peptide receptor-guanine nucleotide-binding protein interaction in HL60 cells.

Formyl peptide receptors on differentiated HL-60 cells were desensitized to formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated superoxide production in a concentration-dependent manner, similar to that previously described for neutrophils. Membranes isolated from desensitized (DM) and normal (NM) HL-60 cells were used to compare receptor numbers and affinities between NM and DM and compare the ability of receptors on DM and NM to interact normally with their guanine nucleotide regulatory proteins (G proteins). Exposure of differentiated HL-60 cells to 10(-7) M FMLP for 10 min before membranes were isolated resulted in a 75% reduction in receptor number, without alteration of dissociation constants. The remaining receptors on DM did not interact normally with their G proteins, as demonstrated by 1) the failure of guanine nucleotides to alter FMLP binding, 2) the inability of FMLP to stimulate guanosine-5'-O-(3-thiotriphosphate) binding, and 3) the attenuation of FMLP stimulation of GTPase activity. These results were not due to a reduction in G protein content of DM, as determined by Western blot analysis with an antibody that recognized alpha 40 and by pertussis toxin-catalyzed [32P]ADP-ribosylation of membrane G proteins in NM and DM. The failure of FMLP receptors on DM to interact with their G proteins was not due to differences in receptor number between NM and DM. Increasing the Mg2+ concentration partially restored the FMLP receptor-G protein interaction in DM. We conclude that desensitization of the formyl peptide receptor is associated with both loss of membrane receptors and a functional alteration in the receptor-G protein interaction, which can be partially reversed by increased concentrations of Mg2+.

Synergism between chemotactic peptide and platelet-activating factor in stimulating thromboxane B2 and leukotriene B4 biosynthesis in human neutrophils.

Formyl-Met-Leu-Phe (FMLP) and platelet-activating factor (PAF) were capable of stimulating thromboxane B2 (TXB2) and leukotriene B4 (LTB4) syntheses in human neutrophils, albeit in a relatively poor degree. A combination of FMLP and PAF, however, was synergistic in stimulating TXB2 and LTB4 syntheses. Phorbol myristate acetate (PMA) appeared to attenuate PAF- but not FMLP-induced arachidonate metabolism. These results suggest that cooperative action of FMLP and PAF on arachidonate release and metabolism does exist and that PMA-mediated protein kinase C activation may regulate FMLP and PAF actions in a different manner.

Human neutrophil chemiluminescence and f-Meth-Leu-Phe receptor exposure in bacterial infections.

Polymorphonuclear leukocytes were isolated from 12 patients with acute bacterial infections and the ability of the chemoattractant formylmethionyl-leucyl-phenylalanine (FMLP) to bind and induce a metabolic response in these cells was investigated. Cells isolated from the patients showed a significantly increased metabolic response in a luminol enhanced chemiluminescence assay compared to cells, isolated and analyzed in parallel, from healthy controls i.e. the patient cells were primed. The primed state was, as calculated by Scatchard analysis, accompanied by a significantly increased number of FMLP receptors exposed on the cell surface while the receptor binding affinity remained unchanged. There was, however, no correlation between the degree of priming and the degree of receptor upregulation. Furthermore, it was found that stimulation also with phorbol myristate acetate (PMA), a substance lacking specific cell surface receptors on the PMNL, gave rise to an increased metabolic response in the primed cells. These results indicate that the priming activity induced by a bacterial infection can only partly be explained by receptor modulation and that other mechanisms must also be considered. This study was approved by the Ethics Committee of the Medical Faculty, University of Linköping.

Reconstitution and characterization of the human neutrophil N-formyl peptide receptor and GTP binding proteins in phospholipid vesicles.

We have developed a unilamellar phospholipid vesicle system which contains the N-formyl peptide receptor and GTP binding proteins. Several detergents were investigated but only two, octyl glucoside (35 mM) and deoxycholate (7.5 mM), were capable of extracting N-formyl peptide receptor from neutrophil membranes in a form which remained functionally active upon reconstitution into phospholipid vesicles. Extracted proteins were reconstituted into phosphatidylcholine vesicles by passage over a Sephadex G-50-80 column. The reconstituted formylpeptide receptor could bind [3H]FMLP (3H-labeled fMet-Leu-Phe) and [125I]FMLPL-SASD (125I-labeled N-formylmethionylleucylphenylalanyl-N epsilon-(2-(p-azidosalicylamido)ethyl- 1,3'-dithiopropionyl)lysine) while the endogenous G protein could bind [35S]GTP gamma S. Furthermore, the functional interaction of the two proteins was preserved. Addition of the nonhydrolyzable guanine nucleotide, GTP gamma S, shifted the N-formyl peptide receptor from a high- to a low-affinity binding state for ligand. The development of this in vitro reconstitution system should provide a basis to study the mechanism of interaction of the N-formyl peptide receptor and the G protein.

Activation of the neutrophil respiratory burst by chemoattractants: regulation of the N-formyl peptide receptor in the plasma membrane.

The N-formyl peptide receptor mediates a number of host defensive responses of human neutrophils that result in chemotaxis, secretion of hydrolytic enzymes, and superoxide generation. Inappropriate activation or defective regulation of these responses can result in pathogenic states responsible for inflammatory disease. The receptor is a 50 to 70-kD, integral plasma membrane glycoprotein with intracellular and surface localization. Its abundance in the membrane is regulated by membrane flow and recycling processes. Cytoskeletal interactions are believed to control its organization in the plane of the membrane and interaction with other proteins. The receptor's most important interaction is with guanyl nucleotide binding proteins that serve as signal transduction partners ultimately leading to activation of effector responses. Because the interaction of the receptor with G proteins is necessary for transduction, control of this interaction may be at the root of understanding the molecular control of responses in these cells. This review briefly summarizes some of the molecular properties, dynamics, and interactions of this receptor system in human neutrophils and discusses how these characteristics may pertain to the activation and control of superoxide generation.

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on intracellular pH in mature granulocytes.

We studied the effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on the internal pH of granulocytes using the fluorescent probe BCECF. GM-CSFrh did not directly alter the resting pH of granulocytes isolated from the peripheral blood; however, when the cells were preincubated for 90 minutes with the growth factor and then activated with the chemotactic peptide N-formyl met leu phe (fMLP), they exhibited both an acceleration in the initial rate of acidification and a marked delay in realkalinization. The kinetic changes both in initial acidification and in subsequent realkalinization induced by GM-CSFrh priming were not prevented by protein synthesis inhibitors and were observed in granulocytes harvested from patients with both sex-linked and autosomal recessive chronic granulomatous disease (CGD). By directly quantitating H+ ion secretion, by monitoring the effects of sodium repletion on intracellular pH, and through use of the sodium channel inhibitors amiloride and dimethyl amiloride and the Na+/K+-ATPase inhibitor ouabain, we showed that the altered kinetics of intracellular acidification and alkalinization following fMLP stimulation of GM-CSFrh-primed granulocytes could not be accounted for by changes in transmembrane proton exportation regulated by the Na+/H+ antiport channel. Although the initial acidification following fMLP was abrogated by 2-deoxy-D-glucose in both GM-CSFrh-pretreated and GM-CSFrh-untreated granulocytes, retardation of the subsequent phase of alkalinization was observed in GM-CSFrh-primed cells even after inhibition of both glycolytic and mitochondrial metabolism. Our data indicate that the increased cytosolic acidification following fMLP stimulation in granulocytes "primed" with GM-CSFrh does not result from disordered proton excretion but instead from increased release of intracellular free acid which is only partially coupled to glucose catabolism or to the generation of superoxide anion (O2-).

Regulation of the affinity state of the N-formylated peptide receptor of neutrophils: role of guanine nucleotide-binding proteins and the cytoskeleton.

Previous studies have indicated that the receptor for N-formylated peptides present on human neutrophils can exist in several ligand-dissociation states at least one of which is sensitive to guanine nucleotides. Human neutrophil membranes rich in cell surface enzyme markers have been isolated from cells pretreated at 37 degrees C with 5 nM fluoresceinated chemotactic peptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein; Fl-peptide) or a buffer control and analyzed for receptor-ligand dissociation states using a previously published fluorescence assay for estimating ligand binding and dissociation rates (Sklar, L. A., et al. 1984. J. Biol. Chem. 259:5661-5669). Fractionation of crude microsomes derived from homogenates of unstimulated cells by ultracentrifugation on linear D2O gradients yielded two plasma membrane-rich fractions termed fast and slow microsomes. Analysis of Fl-peptide dissociation rates from receptor present in fast membrane fractions of unstimulated cells yielded data that could be best fit by assuming that the receptor exists in three distinct ligand-dissociation states. The intermediate ligand-dissociation state (state B) accounted for 47% of the total and was converted to the fastest ligand-dissociation state (state A) by incubation of membranes with GTP or GTP-gamma-S. The remainder of the receptor (17%) present in unstimulated membranes was in a state from which ligand was virtually nondissociable (state C). This form of the receptor was insensitive to GTP-gamma-S. When cells were stimulated with Fl-peptide, most of the receptor present in slow and fast membranes was of the state C type. In contrast to unstimulated cells, slow membranes derived from cells exposed to Fl-peptide contained the majority of the recoverable receptor indicating that receptor was transferred to a physically isolatable membrane domain after ligand binding to the intact cell. The ligand-induced formation of state C in both fast and slow microsome fractions was inhibited by treatment of cells with dihydrocytochalasin B. However, the drug had no effect on translocation of the receptor to slow membranes. Pertussis toxin treatment of intact cells had no effect on ligand-induced formation of state C in either fraction even though other cellular responses were inhibited. Both slow and fast membranes contained a 41-kD G protein as assayed by immunoblot analysis. The data suggest that ligand induces a segregation of receptor-ligand complexes into a membrane domain in which the receptor is functionally uncoupled from the 41-kD neutrophil G protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Removal of human polymorphonuclear leukocyte surface sialic acid inhibits reexpression (or recycling) of formyl peptide receptors. A possible explanation for its effect on formyl peptide-induced polymorphonuclear leukocyte chemotaxis.

Removal of surface sialic acid specifically inhibits human polymorphonuclear leukocyte (PMN) chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP). Neuraminidase-treated (NT)-PMN bound and internalized [3H]FMLP (used as receptor marker) as well as normal PMN. NT-PMN, however, retained more [3H]FMLP-associated radioactivity than normal PMN. Subcellular fractionation studies demonstrated that NT-PMN retained more sedimentable (100,000 X G for 180 min) [3H]FMLP-associated radioactivity within light Golgi-containing fractions than normal PMN. Furthermore, NT-PMN exhibited a defect in their ability to reexpress (or recycle) a population of FMLP receptors. Abnormal receptor recycling was associated with inhibition of FMLP-induced PMN chemotaxis. Thus, it appears that recycling of formyl peptide receptors may be necessary for optimal PMN chemotactic responses to FMLP. We postulate that removal of PMN surface sialic acid inhibits FMLP-induced PMN chemotaxis by blocking the reexpression (or recycling) of a population of formyl peptide receptors, perhaps by preventing trafficking of desialated receptors through a light Golgi pathway.

Localization of chlorotetracycline fluorescence in human polymorphonuclear neutrophils.

Chlorotetracycline (CTC) has been used in many cells as a probe for membranous calcium. In polymorphonuclear neutrophils (PMN), the changes in CTC fluorescence upon stimulation are considered to monitor an early event in the activation process. Using quantitative video-enhanced microscopy, we report that in resting cells about 80% of the CTC signal emanates from the perinuclear region of the cell, indicating that internal structures are labeled with CTC. Approximately 20% of the total CTC fluorescence is taken up in a compartment sensitive to mitochondrial inhibitors, which is not present in neutrophils depleted of nucleus and granules or cytoplasts. Upon stimulation PMN loaded with CTC exhibit a rapid, biphasic decrease in fluorescence that is dose dependent. The second phase of the response is not seen in neutrophil cytoplasts. These results suggest that internal stores of CTC are responsive upon stimulation and could account for the later decrease in CTC fluorescence, whereas the early phase of CTC changes represents the plasma membrane response.

In vitro studies of human monocyte migration across endothelium in response to leukotriene B4 and f-Met-Leu-Phe.

Relatively little is known about monocyte emigration from the vasculature or about the factors that regulate this process. In this study, a human in vitro model of a blood vessel wall was used for examination of monocyte transendothelial migration. Umbilical vein endothelial cells were grown to confluency on amnion connective tissue, and human monocytes were stimulated to cross the monolayer in response to the chemoattractants leukotriene B4 or f-Met-Leu-Phe. The pattern and time course of monocyte migration were similar for the two chemotactic factors. In both cases, approximately 40-50% of the adherent monocytes extended single or multiple pseudopods into the apical endothelial surface. This indenting behavior was also observed in the absence of chemotactic factors. It was not affected by the medium (M199 or Gey's) or method of monocyte isolation. Neutrophils also displayed this behavior, but only about half as many neutrophils as monocytes indented the endothelial surface. The integrity of the endothelium remained intact as the monocytes traversed the monolayer. When the monocytes reached the basal surface of the endothelium, they frequently wedged themselves between the basal surface of the endothelium and its basal lamina. The monocytes then invaded the basal lamina and accumulated in the connective tissue. In response to both f-Met-Leu-Phe and leukotriene B4, monocyte migration across the endothelium began as early as 10 minutes. The average rate of accumulation in the connective tissue peaked at 30 minutes; and by 60 minutes, 25-35% of the monocytes had traversed the monolayer. Approximately two to three times as many monocytes traversed the endothelium under conditions of chemotaxis as under conditions of chemokinesis or random migration. These studies provide the basis for understanding the process of monocyte migration out of the bloodstream and lay the foundation for the study of their differentiation into macrophages in the connective tissue.

Tumor necrosis factor is chemotactic for monocytes and polymorphonuclear leukocytes.

Human recombinant tumor necrosis factor (TNF) induced migration across polycarbonate and nitrocellulose filters of human peripheral blood monocytes and polymorphonuclear leukocytes, TNF was active in inducing migration at concentrations less than 1 U/ml, and maximal responses (observed at greater than 100 U/ml) were comparable to those elicited by standard reference chemoattractants (FMLP, 10 nM; activated human serum, 5%). Checkerboard analysis performed by seeding different concentrations of TNF above and below the filter revealed that maximal induction of migration required a positive concentration gradient between the lower and upper compartments and that TNF elicited an actual chemotactic response in phagocytes. An anti-TNF rabbit antiserum and anti-TNF mouse monoclonal antibody abolished the chemotactic activity of TNF. Recombinant lymphotoxin was also chemotactic for phagocytes, and its activity was blocked by an anti-lymphotoxin antiserum. Human umbilical vein endothelial cells and blood large granular lymphocytes did not respond chemotactically to TNF under conditions in which appropriate reference chemoattractants were active. The chemotactic activity of TNF may serve to recruit phagocytic cells from the blood compartment to amplify resistance against noxious agents.

In vivo chemotaxis of rat leucocytes to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP).

FMLP was potently chemotactic in vivo to rat leucocytes. Doses of 1 microgram attracted net leucocytes into experimental cotton pellets in 6 h. Maximal chemotactic activity occurred at 0.01 mg. Doses greater or less than 0.01 mg attracted less net leucocytes into experimental pellets. The effect of time on chemotaxis of 0.05 mg FMLP showed that between 3 and 36 h, chemotactic index remained above unity and rose maximally to over 1.5. Maximal chemotactic index occurred at 6 h followed by maximal leucocytic infiltration. FMLP showed early chemoattraction in vivo to rat leucocytes. Leucocytic migration into control pellets rose from 3 h, reached maximum levels at 24 h and remained almost at this level at 36 h. L-methionyl-L-leucyl-L-phenylalanine (MLP), studied as a negative control, showed a chemotactic index and chemotactic differential that rose and fell together between 3 and 36 h. The kinetics of migrating leucocyte populations in response to FMLP showed absolute polymorphonuclear leucocytes at 3 h and over 90% mononuclear leucocytes at 36 h.

Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-Met-Leu-Phe receptors.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates the function of mature neutrophils by priming for enhanced chemotaxis and oxidative metabolism in response to N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). Our studies establish a relationship between f-Met-Leu-Phe receptor number and affinity and neutrophil chemotaxis and oxidative metabolism. A brief (5- to 15-min) exposure to physiologic concentrations of GM-CSF (10 pM to 100 pM) enhances f-Met-Leu-Phe-induced neutrophil chemotaxis by 85%, correlating with a rapid threefold increase (46,000/cell to 150,000/cell) in high-affinity neutrophil f-Met-Leu-Phe receptors. More prolonged incubation (1 to 2 hr) of neutrophils with GM-CSF is accompanied by a change to low-affinity f-Met-Leu-Phe receptors (Kd = 29 nM to Kd = 99 nM) concomitant with priming for enhanced neutrophil oxidative metabolism. Moreover, enhanced chemotactic responses to f-Met-Leu-Phe are no longer evident after more prolonged incubation of neutrophils with GM-CSF. These results show that a single lymphokine (GM-CSF) induces sequential changes in neutrophil f-Met-Leu-Phe receptor number and affinity that may enhance different physiologic responses.

Transient catecholamine modulation of neutrophil activation: kinetic and intracellular aspects of isoproterenol action.

Modulation of neutrophil activation by catecholamines may reflect regulatory mechanisms that couple beta-adrenergic and N-formyl peptide receptors to antagonistic biochemical pathways. We examined kinetic and mechanistic aspects of the inhibition by catecholamines of neutrophil activation by formyl peptides. Inhibition of oxidant production by isoproterenol (ISO) was detected as low as 3 nM, had an ID50 of 10(-7) M, and could be blocked and reversed by propranolol. Recovery of cell function occurred over a period of minutes when the concentration of ISO was less than 10(-6) M. These observations are discussed in terms of the interaction of ISO with the adrenergic receptors. The site of catecholamine action is addressed. ISO neither influences formyl peptide-receptor interaction nor does it inhibit oxidant production by phorbol ester. These results suggest an impairment of intracellular signalling processes that couple the formyl peptide-receptor binding to cell activation. We observed inhibition of intracellular Ca++ elevation by ISO only at low formyl peptide concentrations. This inhibition is consistent with a partial inhibition of phosphoinositide metabolism, which was observed. Several other cell responses, including actin polymerization and right angle light scatter, are minimally inhibited by 10(-6) M ISO indicating that the cell activation process is not entirely obliterated. The presence of catecholamine and formyl peptide results in a synergistic elevation of cAMP. The intracellular targets of ISO action may be regulated by cAMP dependent kinases and could follow a branchpoint in the activation sequence that leads distinctly to oxidase activation and cytoskeletal activation.

Coexistence of a chemotactic factor and a retroviral P15E-related chemotaxis inhibitor in human tumor cell culture supernatants.

Two sets of seemingly contradictory evidence have been reported concerning the effects of tumor cell products on the regulation of monocyte migration in vitro and presumably the extravasation of macrophages into tumors in vivo. The present study was designed to explore the relationship between chemotactic and anti-chemotactic products related to tumor cells: a tumor-derived chemotactic factor (TDCF) and retroviral P15E-related inhibitor(s) of chemotaxis. Culture supernatants of the human 8387 sarcoma and SW626 ovarian carcinoma were depleted of P15E-related antigens with immobilized anti-P15E monoclonal antibodies. This treatment produced a significant and consistent increase of the polarizing and chemotactic activity in the tumor cell supernatants. The material eluted from Sepharose-bound anti-P15E antibodies was devoid of chemotactic and polarizing activity and suppressed the polarization and migration of monocytes in response to chemoattractants. These results demonstrate the coexistence in culture supernatants of two human tumor cell lines of factors with opposite influences on monocyte chemotaxis. The data suggest that the entry of monocytes into neoplastic tissue may be regulated by the interplay of chemotactic and anti-chemotactic principals produced by tumor cells.

PMN heterogeneity: long-term stability of fluorescent membrane potential responses to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine in healthy adults and correlation with respiratory burst activity.

Polymorphonuclear neutrophils (PMNs) were isolated from 24 healthy adults 20 to 61 years of age and the proportion of cells that demonstrated depolarization responses to the synthetic chemotaxin N-formyl-methionyl-leucyl-phenylalanine (FMLP) were enumerated using the lipophilic fluorescent cyanine dye 3,3'-di-pentyl-oxacarbocyanine [di-O-C(5)(3)] and flow cytometry. The membrane potential responses were correlated to the cells' respiratory burst capabilities as measured by nitroblue tetrazolium (NBT) reduction and/or superoxide production in response to FMLP; 40.2% +/- 15.1% (mean +/- SD) of cells depolarized to FMLP. The mean SE for duplicate determinations 1 hour apart was 3.8% (range 0.4% to 13.6%, n = 15). There was no correlation between the percentage of depolarizing PMNs and the yield of cells, the percentage of immature cells, or the circulating WBC count. There was no difference in the average age of men (34.9 +/- 9.9 years, n = 11) v women (33.8 +/- 8.5, n = 13) (mean +/- SD) studied, or in the percentage of depolarizing PMNs when men and women were compared (42.2 +/- 10.6% v 43.1 +/- 13.3%, respectively). However, there was a significant increase in the percentage of depolarizing PMNs with increasing age of women (r = .61, P less than .025) but not men (r = .03, P greater than .05). Analysis of variance revealed significantly greater person-to-person variability in the membrane potential response than in day-to-day variability in the same person (P less than .0005). The percentage of depolarizing PMNs in response to FMLP was significantly correlated with the percentage of NBT-positive cells from both purified PMNs and from whole blood (r = .849, P less than .0005, r = .857, P less than .05, respectively), and with the amount of superoxide produced, expressed as a percentage of that amount produced by cytochalasin B (cyto-B)-pretreated cells (r = .565, P less than .01). The data indicate that PMNs from healthy adults demonstrate a heterogeneous membrane potential response to the chemotaxin FMLP that correlates with the cells' oxidative responsiveness and that intersubject differences can be detected. In addition, the proportion of responsive PMNs increases with increasing age in women.

Characterization of the plasma membrane bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein coupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors.

We have characterized the GTPase activity of the Ni-like guanine-nucleotide-binding regulatory protein in rabbit neutrophil plasma membranes. The low Km (3.64 +/- 0.87 X 10(-7) M) GTPase copurified with the formyl peptide receptor in the plasma membrane fraction obtained by discontinuous sucrose density gradient centrifugation. The Vmax (23.9 +/- 2.91 pmol/mg/min) and Km of the unstimulated enzyme were similar to those reported for Ni in other cell types. The activity of the unstimulated enzyme was both magnesium and sodium dependent and linear over the first 4 min of the assay. The chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and leukotriene B4 (LTB4) stimulated the GTPase in purified neutrophil plasma membrane preparations, whereas other secretagogues, such as A23187 and PMA, were without effect. Lineweaver-Burk analysis showed an fMLP-induced increase in Vmax (31.94 +/- 4.80 pmol/mg/min) (33.1 +/- 9.5%) but not in Km. The dose-response curve for fMLP stimulation showed an ED50 of 4.1 +/- 1.0 X 10(-8) M and an overall 22.2 +/- 3.1% maximal stimulation. C5a (30 micrograms/ml) increased the activity of the GTPase 21.3 +/- 5.7% and 10(-7) M LTB4 produced a 32.2 +/- 5.4% increase. Activated pertussis toxin treatment of neutrophil plasma membranes inhibited by 72.5 +/- 14.3% the stimulation of GTPase activity induced by fMLP; however, activated cholera toxin had no effect on the inhibition of fMLP stimulation, suggesting a direct role for an Ni-like protein in the coupling process. In contrast to the lack of inhibition of fMLP stimulation by activated cholera toxin treatment of plasma membranes, both pertussis toxin and to a lesser extent cholera toxin treatment reduced fMLP, C5a, and LTB4 stimulation of the GTPase in sonicates prepared from pretreated whole cells. Pertussis toxin inhibited fMLP stimulation of the GTPase by 75 +/- 7%, C5a stimulation was inhibited by 83 +/- 13%, and LTB4 stimulation was inhibited completely. Sonicates prepared from neutrophils treated similarly with cholera toxin showed a smaller inhibition of GTPase activity (50 +/- 4% and 14 +/- 9% for fMLP and LTB4, respectively) with the exception of C5a, where CT inhibition (81 +/- 32%) equaled pertussis toxin inhibition. Similarly, pertussis toxin completely inhibited the release of the granule enzyme N-acetyl-glucosaminidase by all three chemoattractants, whereas cholera toxin, except with C5a stimulation, had little or no effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Monocyte dysfunction in severe trauma: evidence for the role of C5a in deactivation.

Consecutive severely traumatized patients (n = 16) requiring intensive care underwent serial monitoring of complement activation and monocyte migratory function with the chemoattractant activated serum (C5a) and formyl-methionyl-leucyl-phenylalanine (FMLP). Complement was found to be activated, and chemotaxis to C5a was correspondingly depressed maximally at a mean 5 to 7 days after injury (p = less than 0.01). The migratory response to FMLP was within the normal range throughout. Conversely, in a consecutive series of patients undergoing aortoiliac bypass grafting (n = 11), there was no evidence of complement activation, and monocyte migratory function remained normal for both C5a and FMLP. These data suggest that in patients with severe trauma, the activation of complement, particularly the fifth component (C5a), reduces the migratory responsiveness of circulating monocytes to C5a. This reduction in a host-response mechanism may explain the propensity to infection and poor wound healing seen in patients with severe trauma and also indicates that C5a, thought to be the major in vivo chemoattractant for leukocytes, has profound systemic actions.