Signal-specific activation and regulation of human neutrophil Fc gamma receptors

FcgammaRs with the ITIM domain have been shown to regulate the inflammatory signal delivered by the ITAM-containing FcgammaRs. In this study, we demonstrate that the function of human neutrophil FcgammaR type IIA (CD32A) is regulated in a distinct manner by different cell activation signals at the ligand-binding stage. Activation of neutrophils with fMLP up-regulated the ligand-binding function of CD32A, whereas PMA-mediated activation completely abolished ligand binding without altering CD32A expression. Furthermore, PMA treatment also abolished CD16B-dependent ligand binding irrespective of the level of expression. The effect of PMA was cell type specific, because the ligand-binding function of CD32A expressed on cultured cells such as K562 and CHO-CD32A transfectants was not affected by PMA. Interestingly, phorbol 12,13-dibutyrate, another phorbol ester, and IL-8 up-regulated CD32A-dependent ligand-binding function. These results demonstrate that regulation of CD32A-dependent ligand binding in human neutrophils is not only cell type specific but also activation signal specific. Moreover, these results suggest the possibility that signals delivered to neutrophils by various inflammatory stimuli can exert opposing effects on the function of human FcgammaRs, representing a novel inside-out regulatory mechanism of FcgammaR ligand binding.     

Isolation and partial characterization of membrane protein constituents of human neutrophil receptors for chemotactic formylmethionyl peptides

Plasma membranes of human neutrophils were solubilized in buffer containing a nonionic detergent and applied to a formylmethionylleucylphenylalanine (fMet-Leu-Phe)-Sepharose column that was washed and eluted with the chemotactic peptide fMet-Leu-Phe. Analysis of the eluate by filtration on Bio-Gel P150 in sodium dodecyl sulfate (NaDodSO4) buffer and by NaDodSO4-polyacrylamide gel electrophoresis revealed three predominant membrane proteins of approximate molecular weight 94 000 (MP-1), 68 000 (MP-2), and 40 000 (MP-3), of which MP-2 accounted for 74--93% of the total protein. Purified MP-1 and MP-2 contained an above average content of hydrophobic amino acids, while MP-2 and MP-3 had an above average content of acid and/or amide amino acids and a below average content of basic amino acids. MP-2 and MP-3, but not MP-1, bound [3H]fMet-Leu-Phe in equilibrium dialysis chambers. Both MP-2 and MP-3 exhibited high-affinity sites with a valence of 0.2--0.3 and mean KA values of 9 x 10(8) and 2 x 10(7) M-1, respectively, and low-affinity sites with a valence of 0.3--0.5 and mean KA values of 3 x 10(7) and 2 x 10(6) M-1 (n = 3). The specificity of the binding of fMet-Leu-Phe was suggested by the failure of MP-2 and MP-3 to bind lipid chemotactic factors and to adhere to a Sepaharose column to which had been coupled chemotactic fragments of the fifth component of complement. A series of synthetic formylmethionyl peptides exhibited the same rank order of potency as inhibitors of the binding of [3H]fMet-Leu-Phe by MP-2 and as stimuli of neutrophil chemotaxis. Membrane proteins isolated by fMet-Leu-Phe-Sepharose affinity chromatography may represent constituents of specific human neutrophil receptors for chemotactic peptides.     

Deactivation of human neutrophil chemotaxis by chemoattractants: effect on receptors for the chemotactic factor f-Met-Leu-Phe

Normal human peripheral blood PMN were exposed to varying concentrations of partially purified chemotactic complement fragments (C5fr) and a chemotactic peptide N-formyl methionylleucylphenylalanine (f-Met-Leu-Phe). This exposure resulted in a decreased chemotactic response termed deactivation of chemotaxis. Deactivation was found to be nonpreferential for the deactivating stimulus when high concentrations of either f-Met-Leu-Phe (10(-6) M) or C5fr (20 micrograms/ml) were used. When PMN were incubated with lower concentrations of C5fr (10 micrograms/ml), there was preferential deactivation towards C5fr. Similarly, preferential deactivation of chemotaxis was observed when PMN were incubated with 10(-6) M f-Met-Leu-Phe, but this was transient and cells were nonpreferentially deactivated 60 min after the initial exposure to f-Met-Leu-Phe. The availability of receptors for tritiated f-Met-Leu-Phe was examined by Scatchard analyses and measurement of reversible f-Met-Leu-[3H]Phe binding to C5fr and f-Met-Leu-Phe-deactivated PMN. When PMN f-Met-Leu-Phe receptors were studied immediately after exposure to concentrations of C5fr causing either preferential or nonpreferential deactivation, there was increased receptor availability compared with control PMN. In contrast, PMN deactivated with high concentrations of f-Met-Leu-Phe 10(-6) M) had a transient decrease in the number of receptors followed 1 hr later by an increase in the number of receptors. This was similar to the functional correlate of preferential deactivation of chemotaxis immediately after incubation with f-Met-Leu-Phe followed by nonpreferential deactivation in these same PMN. The data indicate that preferential deactivation of chemotaxis may be associated with a preferential decrease (down-regulation) of chemoattractant receptors and that nonpreferential deactivation is associated with an increase in chemoattractant receptors.     

Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.     

Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts

Although fibroblasts are important in providing a structural framework for most tissues, they also appear to be active participants in the inflammatory process via the production of specific mediators. The production of inflammatory mediators by fibroblasts is especially important in relation to their strategic location within connective tissue as they may act as a cellular communication bridge between the interstitium and vasculature. In this paper, we demonstrate that fibroblasts may participate in these inflammatory reactions by the production of a neutrophil chemotactic factor (NCF) with characteristics similar to a recently isolated and cloned monocyte-derived NCF. Either tumor necrosis factor-alpha-, interleukin-1 alpha-, or interleukin-1 beta-stimulated fibroblasts showed both a time- and dose-dependent increase in steady-state levels of NCF mRNA and secretion of chemotactic activity. In contrast, lipopolysaccharide and interleukin-6 failed to induce fibroblast-derived NCF. The expression of fibroblast-derived NCF mRNA was first detectable by 30 min poststimulation, whereas chemotactic activity was significantly observed 3-4 h postchallenge. Heat-inactivated monokine (100 degrees C) failed to induce NCF mRNA expression, suggesting that only the active proteins are capable of inducing NCF. Gel filtration analysis using high pressure liquid chromatography indicated peak chemotactic activity with an approximate molecular mass of 8000 daltons. This peak of NCF activity was found to be relatively stable to both heat and trypsin inactivation. Specificity of the fibroblast-derived neutrophil chemotactic activity was demonstrated with inhibition of chemotaxis by the addition of neutralizing antibody directed against recombinant human neutrophil chemotactic factor. These data provide evidence that monokine-treated fibroblasts can synthesize a potent chemotactic agent with molecular and physicochemical characteristics similar to monocyte-derived NCF and that this factor may contribute to neutrophil-mediated disease processes.     

Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide

The activity of glycogen phosphorylase, an enzyme that is activated by both cAMP and calcium, was used as an indicator of the state of the cytoplasm after chemotactic stimulation of polymorphonuclear leukocytes (neutrophils). The activity of the enzyme showed a clear dependence on cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a 4-5-fold increase in activity of phosphorylase a. In the absence of external Ca2+, A23187 caused only brief transient activation of phosphorylase; probably reflecting release of sequestered intracellular Ca2+. Addition of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold activation of the enzyme. The dose-dependence of activation by FNLLP showed a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The phosphorylase activity peaks by 90 s and then declines, returning to basal levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with 10(-7) M peptide. This finding suggests that the cells do not need to maintain elevated cytoplasmic calcium levels to exhibit stimulated locomotion. Thus, if calcium continues to modulate the motility, there either must be highly localized changes that are not detected in measures of the total cytoplasm, or the sensitivity to calcium must be variable such that basal levels are sufficient to maintain locomotion. Cells loaded with the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of external Ca2+ had elevated phosphorylase levels before addition of FNLLP. Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it clearly alters some aspects of the neutrophil physiology. Phosphorylase a appears to be a sensitive, nonperturbing indicator of the cytoplasmic calcium levels.     

The activation of human neutrophil gelatinase

To study the mechanisms of activation of human neutrophil gelatinase, the enzyme has been purified using a combination of chromatography on a DEAE-Sephacel and a gelatin-peptide-Sepharose column. On reducing SDS-polyacrylamide-gel electrophoresis the purified gelatinase ran as a single band of about 94,000 Da, and had a specific activity of 5624.4 units/mg of enzyme protein. When latent gelatinase was treated with trypsin, cathepsin G, neutrophil elastase, HgCl2 or urea, its activity was enhanced and the enzyme was processed and converted into species of the lower molecular mass. Upon activation, the protein band of 94,000 Da of reduced latent gelatinase underwent a decrease of about 6,000-12,000 Da. Formation of the species of lower molecular mass during urea activation could be blocked by the addition of EDTA.     

Substance P primes human neutrophil activation: a mechanism for neurological regulation of inflammation

The neuropeptide substance P (SP), a member of the tachykinin family, has stimulatory effects on various cell types at nanomolar concentrations. SP has also direct effects on polymorphonuclear leukocytes (PMNs). However, unlike other cells, stimulation of PMNs requires extremely high concentrations of the peptide (greater than 10 microM), suggesting that direct PMN activation by SP is not physiologically relevant. By measuring primed stimulation of PMNs, we now demonstrate potent synergistic effects of nanomolar doses of SP on the migratory and cytotoxic functions of human PMNs stimulated by fMLP and C5a. This synergism between SP and chemotactic peptides reveals a new regulatory activity of SP and suggests that neurogenic stimuli may prepare neutrophils for an exaggerated inflammatory response to other phlogistic mediators.     

Monokine-induced gene expression of a human endothelial cell-derived neutrophil chemotactic factor

Monokines have been increasingly recognized as communication signals that interact with both immune and non-immune cells during inflammation. Specifically, interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) possess potent effector activities on various cell types. We present novel data demonstrating that human endothelial cells are a major source of a neutrophil chemotactic factor (NCF) synthesized upon stimulation with either IL-1 alpha, IL-1 beta, or TNF-alpha; but not with interleukin-6 (IL-6). Northern blot analysis demonstrated that 20 ng/ml of either IL-1 or TNF-alpha could induce endothelial cells to express significant levels of NCF mRNA, while IL-6 was not active in this system. These data demonstrate that monokines play an important role in mediating acute inflammation via induction of an endothelial cell-derived NCF.     

Extracellular acidification induces human neutrophil activation

In the current work, we evaluated the effect of extracellular acidification on neutrophil physiology. Neutrophils suspended in bicarbonate-buffered RPMI 1640 medium adjusted to acidic pH values (pH 6.5-7.0) underwent: 1) a rapid transient increase in intracellular free calcium concentration levels; 2) an increase in the forward light scattering properties; and 3) the up-regulation of surface expression of CD18. By contrast, extracellular acidosis was unable to induce neither the production of H2O2 nor the release of myeloperoxidase. Acidic extracellular pH also modulated the functional profile of neutrophils in response to conventional agonists such as FMLP, precipiting immune complexes, and opsonized zymosan. It was found that not only calcium mobilization, shape change response, and up-regulation of CD18 expression but also production of H2O2 and release of myeloperoxidase were markedly enhanced in neutrophils stimulated in acidic pH medium. Moreover, extracellular acidosis significantly delayed neutrophil apoptosis and concomitantly extended neutrophil functional lifespan. Extracellular acidification induced an immediate and abrupt fall in the intracellular pH, which persisted over the 240-s analyzed. A similar abrupt drop in the intracellular pH was detected in cells suspended in bicarbonate-supplemented PBS but not in those suspended in bicarbonate-free PBS. A role for intracellular acidification in neutrophil activation is suggested by the fact that only neutrophils suspended in bicarbonate-buffered media (i.e., RPMI 1640 and bicarbonate-supplemented PBS) underwent significant shape changes in response to extracellular acidification. Together, our results support the notion that extracellular acidosis may intensify acute inflammatory responses by inducing neutrophil activation as well as by delaying spontaneous apoptosis and extending neutrophil functional lifespan.     

Identification of phosphoproteins associated with human neutrophil granules following chemotactic peptide stimulation

Regulated exocytosis of neutrophil intracellular storage granules is necessary for neutrophil participation in the inflammatory response. The signal transduction pathways that participate in neutrophil exocytosis are complex and poorly defined. Several protein kinases, including p38 MAPK and the nonreceptor tyrosine kinases, Hck and Fgr, participate in this response. However, the downstream targets of these kinases that regulate exocytosis are unknown. The present study combined a novel inhibitor of neutrophil exocytosis with proteomic techniques to identify phosphopeptides and phosphoproteins from a population of gelatinase and specific granules isolated from unstimulated and fMLF-stimulated neutrophils. To prevent loss of granule-associated phosphoproteins upon exocytosis, neutrophils were pretreated with a TAT-fusion protein containing a SNARE domain from SNAP-23 (TAT-SNAP-23), which inhibited fMLF-stimulated CD66b-containing granule exocytosis by 100±10%. Following TAT-SNAP-23 pretreatment, neutrophils were stimulated with the chemotactic peptide fMLF for 0 min, 1 min, and 2 min. Granules were isolated by gradient centrifugation and subjected to proteolytic digestion with trypsin or chymotrypsin to obtain peptides from the outer surface of the granule. Phosphopeptides were enriched by gallium or TiO2 affinity chromatography, and phosphopeptides and phosphorylation sites were identified by reversed phase high performance liquid chromatography-electrospray ionization-tandem MS. This resulted in the identification of 243 unique phosphopeptides corresponding to 235 proteins, including known regulators of vesicle trafficking. The analysis identified 79 phosphoproteins from resting neutrophils, 81 following 1 min of fMLF stimulation, and 118 following 2 min of stimulation. Bioinformatic analysis identified a potential Src tyrosine kinase motif from a phosphopeptide corresponding to G protein coupled receptor kinase 5 (GRK5). Phosphorylation of GRK5 by Src was confirmed by an in vitro kinase reaction and by precursor ion scanning for phospho-tyrosine specific immonium ions containing Tyr251 and Tyr253. Immunoprecipitation of phosphorylated GRK5 from intact cells was reduced by a Src inhibitor. In conclusion, targets of signal transduction pathways were identified that are candidates to regulate neutrophil granule exocytosis.     

Selective neutrophil desensitization to chemotactic factors

In the presence of extracellular calcium and magnesium, a series of chemotactic oligopeptides and C5a caused aggregation of human polymorphonuclear neutrophils (PMNs). This cellular response developed rapidly and began to reverse 2 min after exposure to the chemotactin. In the absence of the bivalent cations, none of the chemotactins stimulated the aggregation response. If cells were first exposed to a chemotactin and then treated with calcium and magnesium, aggregation was detected only after addition of the cations, and the magnitude of the response fell sharply as the interval between the addition of chemotactin and addition of cations was lengthened: when this interval exceeded 2 min, aggregation was barely detectable. This loss of reactivity persisted even when cells were re-exposed to fresh chemotactic factor and washed between the first and second exposures. In all instances, however, loss of cellular reactivity was highly selective: cells preincubated with any chemotactic oligopeptide were hyporesponsive to subsequent stimulation with an oligopeptide but remained fully responsive to C5a; cells preincubated with C5A were hyporesponsive to C5a but retained their responsitivity to the oligopeptides. Because this selectivity parallels the known specificities of these chemotactic factors for their receptors in or on the neutrophil, desensitization may reflect functional loss of receptors after stimulation. Alternatively, this selectivity may indicate that morphologically identical neutrophils contain subpopulations of cells with varying reactivities to receptor-bound chemotactic factors. In either event, desensitization may be useful in functionally defining chemotactic factors and their respective receptors. The rapidity of development of desensitization suggests that it may operate to limit or moderate various in vitro and in vivo neutrophil responses to chemotactic factors.     

Co-expression of chemotactic ligand receptors on human peripheral blood monocytes

Directed migration of monocytes is dependent upon interaction of cell surface receptors and specific chemotactic ligands. To determine whether circulating human monocytes express multiple chemotactic ligand receptors or whether subpopulations of monocytes exist with a single receptor specificity, nonoverlapping fluorescent probes for two chemotactic ligands, N-formyl methionyl leucyl phenylalanine (FMLP) and C5a, were developed to simultaneously evaluate the expression of receptors for these ligands on individual monocytes. The subsequent incubation with different fluorochrome labeled C5a and FMLP probes and monoclonal antibodies specific for antigenic determinants on distinct subsets of mononuclear cells followed by analysis with dual parameter flow microfluorometry indicated that cells that express C5a and FMLP receptors are the OKM1, Mac-1, and Fc gamma receptor positive population. Furthermore, it was demonstrated that approximately 90% of peripheral blood monocytes expressed FMLP receptors, and the majority of FMLP+ cells were also C5a receptor positive. In addition, a parallel spectrum of chemotactic ligand receptor density from low to high levels was demonstrated for both C5a and FMLP. Additional analysis revealed that the density of chemotactic ligand receptors on resting peripheral blood monocytes did not correlate with monocyte maturation levels measured by HLA-DR expression. Elucidation of the monocyte chemotactic receptor-ligand interactions that lead to migration and/or activation may provide insight into the regulation of monocyte function in inflammation.     

Development of a new method of analysing chemotactic deactivation of human neutrophil granulocytes

Measurement of chemotactic migration of human neutrophil granulocytes (PMN) induced by chemotaxins serves as a simple and reliable method for assessing the expression of chemotaxin receptors. Incubation of PMN with a certain chemotaxin leads to a diminished chemotactic migration towards this chemotaxin. This is called chemotactic deactivation. We developed a new deactivation chamber to determine chemotaxis and chemotactic deactivation of human PMN. This novel chamber is a modification of the commercially available acrylic 48-well microchemotaxis chamber consisting of an upper block with wells drilled all the way through the block and a blind-well lower block. Both blocks are separated by a polycarbonate membrane. PMN from the wells in the upper block migrate through the pores of the membrane into the wells of the lower block containing the chemoattractants. Migrated PMN on the lower side of the PC membrane were quantified after staining by measuring specific light absorbance. The chemotactic activity is quantified as a ratio of stimulated migration and random migration (chemotactic index=CI). For our novel chamber, only the upper blocks of this commercial chamber were connected like a sandwich, including a polyvinylpyrrolidone-free polycarbonate membrane with a pore size of 3 microm. The wells in the upper compartment were filled with 5 x 10(4) PMN and deactivating chemotaxin. The lower block was then filled with the chemotactic stimulus and the chamber was then incubated in humidified air with 5% CO2 atmosphere at 37 degrees C. The influence of cell concentration, incubation time, chemotactic factor concentration, pore size and alkaline treatment of polycarbonate membranes on migrational activity of PMN have been investigated. The technique was rigorously standardized in order to optimize the assay conditions. The method is relatively simple, sensitive and fast. The determination of chemotaxis and deactivation are performed in the same chamber, thus avoiding cell loss due to nonspecific adherence in other incubation tubes. The chamber can be used to characterize the chemotactic activity of chemoattractants of unknown structure via known and unknown receptors. This new chamber can be very helpful in detecting unknown chemotactic stimuli, which are not detectable by, for example, antibodies.     

Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons.     

Heme induces neutrophil migration and reactive oxygen species generation through signaling pathways characteristic of chemotactic receptors

Hemolysis or extensive cell damage can lead to high concentrations of free heme, causing oxidative stress and inflammation. Considering that heme induces neutrophil chemotaxis, we hypothesize that heme activates a G protein-coupled receptor. Here we show that similar to heme, several heme analogs were able to induce neutrophil migration in vitro and in vivo. Mesoporphyrins, molecules lacking the vinyl groups in their rings, were not chemotactic for neutrophils and selectively inhibited heme-induced migration. Moreover, migration of neutrophils induced by heme was abolished by pretreatment with pertussis toxin, an inhibitor of Galpha inhibitory protein, and with inhibitors of phosphoinositide 3-kinase, phospholipase Cbeta, mitogen-activated protein kinases, or Rho kinase. The induction of reactive oxygen species by heme was dependent of Galpha inhibitory protein and phosphoinositide 3-kinase and partially dependent of phospholipase Cbeta, protein kinase C, mitogen-activated protein kinases, and Rho kinase. Together, our results indicate that heme activates neutrophils through signaling pathways that are characteristic of chemoattractant molecules and suggest that mesoporphyrins might prove valuable in the treatment of the inflammatory consequences of hemorrhagic and hemolytic disorders.     

Linear gramicidin activates neutrophil functions and the activation is blocked by chemotactic peptide receptor antagonist

The activation of functional responses in rabbit peritoneal neutrophils by gramicidin and the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine methyl ester, was studied. Gramicidin activated superoxide generation, lysosomal enzyme release and a decrease in fluorescence of chlortetracycline-loaded cells, as for the chemotactic peptide. The maximum intensities of the responses by gramicidin were lower than that by chemotactic peptide. Responses by both these peptides could be inhibited by t-butyloxycarbonyl-methionyl-leucyl-phenylalanine, a chemotactic peptide receptor antagonist. Gramicidin gave responses at low doses comparable to that of the chemotactic peptide.     

Detergent solubilization of human neutrophil leukotriene B4 receptors

Specific leukotriene B4 (LTB4) receptors in human neutrophils were solubilized by treatment of "receptor fraction" membranes with the zwitterionic detergent (3-[(3-cholamidopropyl)-dimethylammonio]1-propane sulfonate (CHAPS). The soluble receptors were assayed by polyethylene glycol (PEG) precipitation coupled with Millipore filtration. The solubilized receptors retained all of the characteristics of the receptor sites in intact neutrophils. The binding of LTB4 was rapid, reversible and stereospecific. Mathematical modeling analysis revealed biphasic binding of [3H] LTB4 indicating two classes of binding sites. The high affinity binding site had a dissociation constant of 1.93 nM and Bmax of 281 fmoles/mg protein; the low affinity binding site had a dissociation constant of 78.92 nM and Bmax of 2522 fmoles/mg protein. Competitive binding experiments with structural analogs of LTB4 demonstrate that the interaction between LTB4 and its binding site is stereospecific and correlates with the relative biological activity of the analogs. These data suggest that it may be possible to purify the LTB4 receptor from human neutrophil membranes.