Neutrophil-activating peptide 1/interleukin 8 mRNA expression and protein secretion by human monocytes: effect of cyclosporin A

Neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8) is a recently described cytokine with potent chemotactic activity for human neutrophil granulocytes (PMN) and T cells. In psoriasis, a chronic hyperproliferative and inflammatory skin disorder, PMN and T cells are found as prominent cells in the inflammatory infiltrate of the lesions; however, monocytes were shown to be the first cells invading a newly formed plaque. NAP-1/IL-8 was found to be present in high amounts in the skin and in scale material of psoriatic patients. Psoriasis responds well to systemic treatment with cyclosporin A (CsA), an immunosuppressive peptide. Therefore, we addressed the question of whether the clinical improvement of psoriatic patients during CsA therapy may be due to an inhibition of NAP-1/IL-8 production and secretion from monocytes. Purified human monocytes were stimulated by lipopolysaccharide in the presence or absence of various concentrations of CsA. Production of NAP-1/IL-8 was determined as expression of specific mRNA by fluorescent in situ hybridization. Secreted peptide was measured by bioassay (PMN chemotaxis) and enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies. The results show that CsA neither inhibited mRNA expression for NAP-1/IL-8 nor secretion of the peptide. These findings support the hypothesis that the pharmacological effect of CsA may be restricted to the inhibition of T-cell activation and proliferation.     

Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity

A novel monocyte-derived neutrophil-activating peptide (MONAP) produced by lipopolysaccharide- and phorbol myristate acetate-stimulated human peripheral blood monocytes was purified by sequential ion exchange-high performance liquid chromatography (HPLC), size exclusion HPLC, and reversed phase HPLC. Biologic activities of the purified cytokine were monitored by either an enzyme release assay or a chemotaxis assay, using peripheral human neutrophils. Purified MONAP was found to be homogeneous, giving a single peak on size-exclusion HPLC, reversed-phase HPLC, as well as a single 10-kDa band on silver-stained polyacrylamide gels. Purified MONAP stimulate human neutrophil chemotaxis at an estimated molarity of 5 x 10(-11) M. Half-maximal enzyme release of cytochalasin B pretreated neutrophils occurred at 2 to 3 x 10(-10) M, whereas superoxide anion production elicited by various concentrations of MONAP was found to be low. Isolated human peripheral monocytes, as well as human eosinophils, showed no chemotactic response to MONAP, indicating neutrophil specificity. MONAP activity was separated from thymocyte-stimulating activity by reversed-phase HPLC, indicating nonidentity with interleukin (IL)-1. This was further supported by heat resistance of MONAP, which is in contrast to the heat sensitivity of IL-1. In addition, IL-1 obtained as a by-product during isolation of MONAP did not stimulate human neutrophil chemotaxis.     

Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis.

Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN.     

Regulation by interleukin 2 of interleukin 2 receptors and gamma-interferon synthesis by human thymocytes: augmentation of interleukin 2 receptors by interleukin 2

The role of interleukin 2 (IL 2) on the expression of IL 2 receptors and on the synthesis of gamma-interferon (gamma-IFN) by human thymocytes was investigated. Human thymocytes isolated from specimens obtained during cardiac surgery of infants and children were induced with one or all of the following agents: IL 2, concanavalin A (Con A), and 12-O-tetradecanoylphorbol 13-acetate (TPA). The expression of IL 2 receptors and gamma-IFN titers were determined. The results indicate that thymocytes cultured in complete medium do not express receptors for IL 2, nor did IL 2 by itself induce the expression of IL 2 receptors. Con A induced the expression of IL 2 receptors by a moderate number of the thymocyte population and induced the synthesis of low amounts of gamma-IFN. Preincubation of thymocytes with TPA increased the response to Con A; both the number of thymocytes expressing receptors and the synthesis of gamma-IFN were increased. Addition of IL 2 to these cultures further augmented the expression of IL 2 receptors and gamma-IFN synthesis and resulted in the optimal expression of IL 2 receptors and maximal gamma-IFN synthesis. The expression of IL 2 receptors could be detected within 24 hr and preceded the induction of proliferation; it was therefore probably not due to the clonal expansion of a population of receptor-bearing thymocytes. Conversely, inhibition of IL 2 synthesis with dexamethasone (Dex) by thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes and of gamma-IFN synthesis. Thymocytes activated with TPA and Con A were more resistant to the inhibitory effects of Dex on the expression of IL 2 receptors than thymocytes activated with Con A alone. Maximal inhibition of the expression of IL 2 receptors and of gamma-IFN synthesis was achieved as a result of the synergistic effect of anti-Tac with Dex. Therefore, when IL 2 was prevented from binding to the receptors, and IL 2 synthesis was inhibited, the number of thymocytes expressing IL 2 receptors was sharply reduced and gamma-IFN synthesis was markedly inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)     

A short synthetic peptide fragment of human interleukin 1 with immunostimulatory but not inflammatory activity

Short peptide fragments of human and murine interleukin 1 (IL 1) were synthesized on the basis of their predicted exposure on the surface of the molecule in an attempt to identify the minimal structure responsible for the immunostimulatory activity of IL 1. One of these peptides, a fragment of nine residues of human IL 1 beta (VQGEESNDK, fragment 163-171), showed high T cell activation capacity, as judged by its ability to stimulate murine thymocyte proliferation and to potently induce interleukin 2 production in spleen cells. On the other hand, the 163-171 peptide was devoid of prostaglandin-inducing capacity in vitro and pyrogenic activity in vivo, two inflammatory features peculiar to the entire hu IL 1 beta molecule. Thus we propose that this peptide may represent one of the portions of hu IL 1 beta responsible for its immunostimulatory capacity.     

Granulocyte/macrophage colony-stimulating factor induces interleukin 1 production by human polymorphonuclear neutrophils

The purpose of this study was to determine whether human polymorphonuclear neutrophils (PMN), which share a common cell lineage with macrophages, could produce factors such as IL-1. We show by Northern blot analysis and bioassays that PMN can be induced to accumulate mRNA specific for IL-1 alpha and IL-1 beta indistinguishable in size from IL-1 mRNA synthesized by activated human macrophages and consequently to release IL-1-like activity in their culture supernatants, that could be neutralized by a mAb to IL-1. The granulocyte-macrophage colony-stimulating factor was identified as a major physiologic inducer for PMN-IL-1.     

The biological activities of gro beta and IL-8 on human neutrophils are overlapping but not identical.

Gro beta and IL-8 are two members of the small induced secreted (SIS) cytokine family (C-X-C subgroup) with proinflammatory activities on neutrophils. In order to assess whether or not the interaction with their receptors results in similar biological actions, we compared the two cytokines in five different bioassays. Gro beta showed similar biological activities as IL-8 in tests of chemotaxis, induction of the respiratory burst, and induction of interleukin 6 (IL-6) production. However, for two other biological activities: augmentation of the expression of CD11b on the cell surface and rapid elevation of the intracellular calcium concentration, maximal effects required 100 times more gro beta than IL-8. Taken together, these results suggest that the stimulation of the IL-8 or gro beta receptor evokes three similar responses, but that only the activation of the IL-8 receptor and not that of gro beta results in elevated CD11b expression and calcium mobilization in human neutrophils.     

Expression of interleukin 1 receptors on human peripheral T cells

The expression of interleukin 1 receptors (IL 1R) on human peripheral T cells was studied by the binding assay with 125I-labeled recombinant human interleukin 1 (IL 1) alpha and IL 1 beta and by the flow cytofluorometry with the fluorescein isothiocyanate (FITC)-conjugated IL 1 alpha. Peripheral blood lymphocytes expressed only few IL 1R without any stimulations. When they were stimulated with concanavalin A (Con A), IL 1R-positive cells began to increase by 4 hr, reached the maximum level at 48 hr, and then gradually decreased. The kinetics of the expression of IL 1 alpha R and IL 1 beta R showed the same pattern. Furthermore the binding of 125I-labeled IL 1 alpha to IL 1R on T cells was inhibited by the addition of either cold IL 1 alpha or IL beta, but not by interleukin 2 or interferons. The similar results were observed in the binding of 125I-labeled IL 1 beta. These results suggest that IL 1R on human peripheral T cells reactive for IL 1 alpha and IL 1 beta were identical. By Scatchard plot analysis, the numbers of IL 1R were estimated as 40 and 350 molecules per cell before and after Con A stimulation, respectively, and their Kd values were 3.1 X 10(-10) M and 2.8 X 10(-10) M. When purified T cells alone were stimulated with Con A, IL 1R were only marginally expressed. However, by the addition of monocytes, IL 1R were expressed on T cells in a dose-dependent manner. The maximum response was induced in the presence of 10% monocytes. The maximum IL 1R-positive T cells were approximately 30% by the detection of the flow cytofluorometry with FITC-conjugated IL 1 alpha. This enhancing activity of IL 1R expression on T cells by monocytes was inhibited by the addition of an anti-HLA-DR antibody or by the treatment of monocytes with the anti-HLA-DR antibody and complement. Furthermore T cell proliferative responses induced with IL 1 and Con A were also enhanced by the addition of HLA-DR-positive monocytes. These results suggest that IL 1R are expressed as the result of monocyte-T cell interaction in the early stage of T cell activation, and the expression of IL 1R on T cells and the responsiveness of T cells for IL 1 require the accessory function of HLA-DR-positive monocytes.     

Proliferation of phenotypically immature human thymocytes with and without interleukin 2 receptors

Previous studies have indicated that the human thymus is composed of several discrete compartments. Cortical thymocytes are reactive with the monoclonal antibody anti-T6, whereas most medullary cells, unreactive with anti-T6, stain brightly with anti-T3, which defines mature T cell populations. Only a minor thymocyte population lacks both T3 and T6 but expresses T11 antigens. Within the thymus, several proliferating lymphoblasts are present. In addition a distinct subset shows the capacity to proliferate in response to mitogens. By continuous Percoll density gradient centrifugation, we have obtained a cell fraction comprising the vast majority of cells able to proliferate spontaneously or after PHA stimulation. By a panning procedure performed with anti-T3 and anti-T6 antibodies, three phenotypically distinct thymocyte subsets were separated from this fraction, and their functional capabilities were tested. The spontaneous proliferating activity was found to be mainly attributable to thymocytes unable to respond to mitogen, expressing the cortical T6 marker and lacking receptors for IL 2. T3-positive cells are able to respond to mitogen. However, these thymocytes are incapable of producing the adequate amount of IL 2 required to fully saturate their intrinsic proliferative capability. Surprisingly, the phenotypically least mature intrathymic T lymphocytes (T3 and T6 negative) respond to phytomitogen, at least in part, in an interleukin-dependent manner. It is noteworthy that a large proportion of these T3- and T6-negative thymocytes express IL 2 receptors and class II MHC antigens without in vitro activation. These novel findings have potential implications in the context of current models of differentiation pathways within the human thymus.     

Effect of phorbol myristate acetate on processing of formyl peptide receptors by human neutrophils

We examined the effect of phorbol myristate acetate on the ability of human neutrophils to process formyl peptide receptors. The receptor was affinity-labeled and its extracellular localization assessed over time, by cleavage of extracellular labeled receptor with papain. Neutrophils were capable of internalizing (and/or recycling) affinity labeled formyl peptide receptor in the absence of extracellular calcium. This phenomenon was dependent upon stimulation with phorbol myristate acetate, suggesting a role for protein kinase C in this process.     

Natural and recombinant human interleukin 1-beta is cytotoxic for human melanoma cells

Human peripheral blood monocytes stimulated with LPS were found to release an activity that was cytotoxic for the A375 melanoma. Biochemical and immunological characterization of the activity indicated that IL 1-beta was the cytotoxic agent. Human recombinant IL 1-beta, purified to homogeneity, was directly cytotoxic for A375. Tumor necrosis factor, also released by activated monocytes, was not cytotoxic for the A375 melanoma.     

NMDA receptors interact with flotillin-1 and -2, lipid raft-associated proteins

N-methyl-d-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the brain. Here we demonstrate interactions between the NR2A and NR2B subunits of NMDARs with flotillin-1 (flot-1), a lipid raft-associated protein. When mapped, analogous regions in the far distal C-termini of NR2A and NR2B mediate binding to flot-1, and the prohibitin homology domain of flot-1 contains binding sites for NR2A and NR2B. Although NR2B can also directly bind to flot-2 via a separate region of its distal C-terminus, NMDARs were significantly more colocalized with flot-1 than flot-2 in cultured hippocampal neurons. Overall, this study defines a novel interaction between NMDARs and flotillins.

Structured summary

MINT-7013094: NR2A (uniprotkb:Q00959), NR2B (uniprotkb:Q00960) and Flot2 (uniprotkb:Q9Z2S9) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7013147: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with NR2A (uniprotkb:Q00959) by anti bait coimmunoprecipitation (MI:0006)MINT-7013189: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013033: NR2A (uniprotkb:Q00959) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by two hybrid (MI:0018)MINT-7013178: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013197, MINT-7013210: NR2B (uniprotkb:Q00960) and NR2A (uniprotkb:Q00959) physically interact (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013002: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot1 (uniprotkb:O08917) by two hybrid (MI:0018)MINT-7013117: Flot1 (uniprotkb:Q9Z1E1), NR2B (uniprotkb:Q00960) and NR2A (uniprotkb:Q00959) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7013171: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by anti bait coimmunoprecipitation (MI:0006)MINT-7013017: NR2A (uniprotkb:Q00959) physically interacts (MI:0218) with Flot1 (uniprotkb:O08917) by two hybrid (MI:0018)MINT-7013054: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by two hybrid (MI:0018)MINT-7013129: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with NR2B (uniprotkb:Q00960) by anti bait coimmunoprecipitation (MI:0006)MINT-7013155: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with NR2B (uniprotkb:Q00960) by anti bait coimmunoprecipitation (MI:0006)MINT-7013074: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by two hybrid (MI:0018)MINT-7013162: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with NR2A (uniprotkb:Q00959) by anti bait coimmunoprecipitation (MI:0006)     

Recombinant interferon-gamma induces interleukin 2 receptors on human peripheral blood monocytes

The present report describes the inducibility of IL 2 receptors on human peripheral blood monocytes. Although freshly isolated monocytes are IL 2 receptor negative, approximately one-third of the cells react with the anti-Tac antibody within 18 hr of culture. IFN-gamma is found to double both the number of positive cells and the number of binding sites, whereas IL 2 has no influence on the IL 2 receptor expression on monocytes. Anti-Tac precipitates from monocyte lysates several protein bands of similar m.w. to those previously found with activated T and B cells. Finally, IFN-gamma-induced, but not resting, monocytes are found to bind recombinant IL 2. We conclude that IFN-gamma induces peripheral blood monocytes to express IL 2 receptors similar in structure to those found on activated T and B lymphocytes.     

Demonstration of histamine H2 receptors on human melanoma cells

Histamine induced a concentration-dependent increase in intracellular cyclic-AMP of the two human melanoma cell lines SK23 and DX3.LT5.1; maximal stimulation was obtained with 17.8 microM histamine which consistently produced greater than 50-fold increases in the cyclic AMP content of both cell lines. The dose-response curve for histamine in each culture was progressively displaced to the right with increasing concentrations of the histamine H2 receptor antagonist cimetidine. Ranitidine, another H2 receptor antagonist also prevented the histamine-induced cyclic AMP elevation, but the H1 receptor antagonists mepyramine and tripelennamine had no significant effect. These findings indicate that human melanoma cells express histamine H2 receptors, stimulation of which activates adenylate cyclase with a subsequent rise in intracellular cyclic AMP. Mast cell:melanoma interactions mediated by histamine in vivo might therefore be expected to modify some aspects of melanoma cell behaviour.     

Constitutive ERK activity induces downregulation of tristetraprolin, a major protein controlling interleukin8/CXCL8 mRNA stability in melanoma cells

Most melanoma cells are characterized by the V600E mutation in B-Raf kinase. This mutation leads to increased expression of interleukin (CXCL8), which plays a key role in cell growth and angiogenesis. Thus CXCL8 appears to be an interesting therapeutic target. Hence, we performed vaccination of mice with GST-CXCL8, which results in a reduced incidence of syngenic B16 melanoma cell xenograft tumors. We next addressed the molecular mechanisms responsible for aberrant CXCL8 expression in melanoma. The CXCL8 mRNA contains multiples AU-rich sequences (AREs) that modulate mRNA stability through the binding of tristetraprolin (TTP). Melanoma cell lines express very low TTP levels. We therefore hypothesized that the very low endogenous levels of TTP present in different melanoma cell lines might be responsible for the relative stability of CXCL8 mRNAs. We show that TTP is actively degraded by the proteasome and that extracellular-regulated kinase inhibition results in TTP accumulation. Conditional expression of TTP in A375 melanoma cells leads to CXCL8 mRNA destabilization via its 3' untranslated regions (3'-UTR), and TTP overexpression reduces its production. In contrast, downregulation of TTP by short hairpin RNA results in upregulation of CXCL8 mRNA. Maintaining high TTP levels in melanoma cells decreases cell proliferation and autophagy and induces apoptosis. Sorafenib, a therapeutic agent targeting Raf kinases, decreases CXCL8 expression in melanoma cells through reexpression of TTP. We conclude that loss of TTP represents a key event in the establishment of melanomas through constitutive expression of CXCL8, which constitutes a potent therapeutic target.     

Nuclear factor-kappa B activation by the CXC chemokine melanoma growth-stimulatory activity/growth-regulated protein involves the MEKK1/p38 mitogen-activated protein kinase pathway

Melanoma growth stimulatory activity/growth-regulated protein (MGSA/GRO), a CXC chemokine, plays an important role in inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. Constitutive expression of MGSA/GROalpha in melanoma tumors is associated with constitutive nuclear factor (NF)-kappaB activity. We show here that either exogenous addition or continuous expression of MGSA/GROalpha in immortalized melanocytes enhances NF-kappaB activation, as well as mitogen-activated protein (MAP) kinase kinase kinase (MEKK) 1, MAP kinase kinase (MEK) 3/6, and p38 MAP kinase activation. Expression of dominant negative M-Ras (S27N), dominant negative MEKK1 (K432M), or specific chemical inhibitors for p38 MAP kinase (SB202190 and SB203580) block MGSA/GROalpha-induced NF-kappaB transactivation, demonstrating that Ras, MEKK1, and p38 are involved in the signal pathways of MGSA/GROalpha activation of NF-kappaB. Expression of dominant active Ras or dominant active MEKK1 alone can also stimulate NF-kappaB activation. The expression of dominant negative MEKK1 inhibits the Ras-induced NF-kappaB activation, suggesting that MEKK1 is a downstream target of Ras. Moreover, MGSA/GROalpha induction of NF-kappaB is independent of the MEK1/ERK cascade, because MGSA/GROalpha failed to increase ERK and ELK activation, and specific chemical inhibitors for MEK1 (PD98059) had no effect on MGSA/GROalpha-enhanced NF-kappaB activation. These data demonstrate that NF-kappaB activation is required for MGSA/GROalpha-induced melanocyte transformation through a Ras/MEKK1/p38 cascade in melanocytes.     

N-formyl peptide receptors in human neutrophils display distinct membrane distribution and lateral mobility when labeled with agonist and antagonist

Receptors for bacterial N-formyl peptides are instrumental for neutrophil chemotactic locomotion and activation at sites of infection. As regulatory mechanisms for signal transduction, both rapid coupling of the occupied receptor to cytoskeletal components, and receptor lateral redistribution, have been suggested (Jesaitis et al., 1986, 1989). To compare the distribution and lateral diffusion of the nonactivated and activated neutrophil N-formyl-peptide receptor, before internalization, we used a new fluorescent N-formyl-peptide receptor antagonist, tertbutyloxycarbonyl-Phe(D)-Leu-Phe(D)-Leu-Phe-OH (Boc- FLFLF, 0.1-1 microM), and the fluorescent receptor agonist formyl-Nle- Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK, 0.1-1 microM). Fluorescent Boc-FLFLF did not elicit an oxidative burst in the neutrophil at 37 degrees C, as assessed by chemiluminescence and reduction of p-nitroblue tetrazolium chloride, but competed efficiently both with formyl-methionyl-leucyl- phenylalanine (fMLF) and fnLLFnLYK. It was not internalized, as evidenced by confocal microscopy and acid elution of surface bound ligand. The lateral mobility characteristics of the neutrophil fMLF receptor were investigated with the technique of FRAP. The diffusion coefficient (D) was similar for antagonist- and agonist-labeled receptors (D approximately 5 x 10(-10) cm2/s), but the fraction of mobile receptors was significantly lower in agonist- compared to antagonist-labeled cells, approximately 40% in contrast to approximately 60%. This reduction in receptor mobile fraction was slightly counteracted, albeit not significantly, by dihydrocytochalasin B (dhcB, 5 microM). To block internalization of agonist-labeled receptors, receptor mobility measurements were done at 14 degrees C. At this temperature, confocal microscopy revealed clustering of receptors in response to agonist binding, compared to a more uniform receptor distribution in antagonist-labeled cells. The pattern of agonist- induced receptor clustering was less apparent after dhcB treatment. To summarize, this work shows that activated N-formyl peptide receptors aggregate and immobilize in the plane of the neutrophil plasma membrane before internalization, a process that is affected, but not significantly reversed, by cytochalasin. The results are consistent with a model where arrested receptors are associated mainly with a cytochalasin-insensitive pool of cytoskeletal elements.