Biomedical subjects

C A Parkos

Publications and source records attributed to C A Parkos.

At least 19 recordsLinked to original sources

PubMed2006

Raf 1 represses expression of the tight junction protein occludin via activation of the zinc-finger transcription factor slug.

Although dysregulation of tight junction (TJ) proteins is observed in epithelial malignancy, their participation in epithelial transformation is poorly understood. Recently we demonstrated that expression of oncogenic Raf 1 in Pa4 epithelial cells disrupts TJs and induces an oncogenic phenotype by downregulating expression of the TJ protein, occludin. Here we report the mechanism by which Raf 1 regulates occludin expression. Raf 1 inhibited occludin transcription by repressing a minimal segment of the occludin promoter in concert with upregulation of the transcriptional repressor, Slug without influencing the well-documented transcriptional repressor, Snail. Overexpression of Slug in Pa4 cells recapitulated the effect of Raf 1 on occludin expression, and depletion of Slug by small interfering RNA abrogated the effect of Raf 1 on occludin. Finally, chromatin immunoprecipitation assays and site-directed mutagenesis demonstrated a direct interaction between Slug and an E-box within the minimal Raf 1-responsive segment of the occludin promoter. These findings support a role of Slug in mediating Raf 1-induced transcriptional repression of occludin and subsequent epithelial to mesenchymal transition.

Animals↗

PubMed2001

The role of CD47 in neutrophil transmigration. Increased rate of migration correlates with increased cell surface expression of CD47.

CD47, a cell surface glycoprotein, plays an important role in modulating neutrophil (PMN) migration across endothelial and epithelial monolayers. Here we show that anti-CD47 monoclonal antibodies (mAbs) delay PMN migration across collagen-coated filters or T84 epithelial monolayers toward the chemoattractant formylmethionylleucylphenylalanine (fMLP). Despite delayed transmigration by anti-CD47 mAbs, the numbers of PMN migrating across in either condition were the same as in the presence of control non-inhibitory mAbs. Cell surface labeling and immunoprecipitation demonstrated upregulation of CD47 to the PMN cell surface with kinetics similar to those of the transmigration response. Subcellular fractionation studies revealed redistribution of CD47 from intracellular compartments that co-sediment with secondary granules to plasma membrane-containing fractions after fMLP stimulation. Experiments performed to investigate potential signaling pathways revealed that inhibition of tyrosine phosphorylation with genistein reversed the anti-CD47-mediated PMN migration delay, whereas inhibition of phosphatidylinositol 3-kinase only partially reversed anti-CD47 effects that correlated with a rapid increase in PMN cell surface CD47. Analysis of the contribution of epithelial-expressed CD47 to PMN transmigration revealed that PMN migration across CD47-deficient epithelial monolayers (CaCO2) was significantly increased after stable transfection with CD47. These results suggest that cell surface CD47 and downstream tyrosine phosphorylation signaling events regulate, in part, the rate of PMN migration during the inflammatory response.

Antibodies, Monoclonal↗

PubMed2001

Novel G protein-coupled responses in leukocytes elicited by a chemotactic bacteriophage displaying a cell type-selective binding peptide.

Recently, we identified a neutrophil-binding phage displaying a novel peptide motif, GPNLTGRW. It was determined that this peptide, when displayed on bacteriophage (FGP phage), elicits a transient increase in cytosolic calcium. Here, we show that FGP phage stimulate neutrophil chemotaxis and induce a pertussis toxin-sensitive rise in cytosolic calcium in monocytes as well as in neutrophils. In contrast to the calcium response elicited by classical chemoattractants fMLP and IL-8, the FGP phage-elicited response in neutrophils is dependent on extracellular calcium and is mediated by receptor-activated, divalent cation channels. Consistent with G protein-coupled receptor signaling, FGP phage effect homologous and reciprocal heterologous desensitization with fMLP- and IL-8-stimulated calcium responses. Like non-G protein-coupled responses, the FGP-elicited calcium transient is abolished with phosphoinositide-3-kinase inactivation. Nonetheless, specific binding of GTP to neutrophil membranes follows stimulation with FGP phage, further supporting involvement of G proteins. However, FGP phage neither bind to nor elicit a calcium response from transfectant cells harboring known candidate G protein-coupled receptors. These data together suggest that the elicited responses are mediated by a novel G protein-coupled receptor or represent novel responses of a known receptor.

Animals↗

PubMed2001

Junction adhesion molecule is a receptor for reovirus.

Virus attachment to cells plays an essential role in viral tropism and disease. Reovirus serotypes 1 and 3 differ in the capacity to target distinct cell types in the murine nervous system and in the efficiency to induce apoptosis. The binding of viral attachment protein sigma1 to unidentified receptors controls these phenotypes. We used expression cloning to identify junction adhesion molecule (JAM), an integral tight junction protein, as a reovirus receptor. JAM binds directly to sigma1 and permits reovirus infection of nonpermissive cells. Ligation of JAM is required for reovirus-induced activation of NF-kappaB and apoptosis. Thus, reovirus interaction with cell-surface receptors is a critical determinant of both cell-type specific tropism and virus-induced intracellular signaling events that culminate in cell death.

Animals↗

PubMed2001

Neutrophil transmigration in inflammatory bowel disease is associated with differential expression of epithelial intercellular junction proteins.

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.

Adherens Junctions↗

PubMed2001

Reduced expression of junctional adhesion molecule and platelet/endothelial cell adhesion molecule-1 (CD31) at human vascular endothelial junctions by cytokines tumor necrosis factor-alpha plus interferon-gamma Does not reduce leukocyte transmigration under flow.

The combination of tumor necrosis factor (TNF)-alpha plus interferon (IFN)-gamma has been shown previously to promote redistribution of platelet/endothelial cell adhesion molecule-1 (PECAM-1) (CD31), junctional adhesion molecule (JAM), and VE-cadherin away from lateral junctions of human umbilical vein endothelial cell monolayers. In parallel, neutrophil transmigration was significantly reduced. Because PECAM-1 and JAM have been implicated in leukocyte transmigration, the observed redistribution by cytokine activation was presumed to represent the mechanism causing decreased transmigration under static conditions. The current results confirm that culture of human umbilical vein endothelial cells with TNF-alpha plus IFN-gamma caused a decrease in surface-expressed and junctional-localized JAM and PECAM-1, but did not cause decreased leukocyte transmigration in an in vitro flow assay. Furthermore, blocking monoclonal antibody to PECAM-1 still significantly reduced monocyte transmigration, demonstrating that it retains a functional role even though its levels were reduced and redistributed away from junctions, whereas a panel of monoclonal antibodies to JAM failed to reduce leukocyte transmigration. Given the alterations in junction protein location, permeability function was assessed. IFN-gamma alone or TNF-alpha plus IFN-gamma significantly increased permeability, but TNF-alpha alone did not, suggesting lack of correlation between transmigration and loss of permeability. In conclusion, cytokine activation induced loss and redistribution of PECAM-1 and JAM away from lateral junctions, but per se does not negatively regulate either neutrophil or monocyte transmigration under flow.

Cell Adhesion↗

PubMed2001

A carbohydrate neoepitope that is up-regulated on human mononuclear leucocytes by neuraminidase treatment or by cellular activation.

The expression of cell-surface antigens can delineate specific leucocyte developmental or functional stages. For example, certain membrane glycoproteins are expressed selectively on leucocyte subsets only after activation. Leucocyte activation can also induce changes in carbohydrate epitopes expressed on surface antigens. In the present studies, we report on a novel monoclonal immunoglobulin M antibody (mAb 13.22) that recognizes a unique carbohydrate epitope expressed on human leucocyte membrane proteins. Characterization of mAb 13.22 specificity by immunoblotting showed that it recognized proteins of MW approximately 95 000 and 150 000, including both CD18 and CD11b. The mAb 13.22 epitope was removed by N-glycosidase F but not by endoglycosidase H or fucosidase, demonstrating that it is an N-linked carbohydrate antigen. Interestingly, immunoblot staining was enhanced after neuraminidase treatment, suggesting that the antibody epitope might also be partially masked by sialic acid. In resting leucocytes, the mAb 13.22 antigen was expressed strongly on neutrophils, while dull staining was present on monocytes, and no lymphocyte staining was observed. In marked contrast, treatment of leucocytes with neuraminidase resulted in exposure of a mAb 13.22 neoepitope on a subset of lymphocytes (primarily T lymphocytes and natural killer cells) as well as up-regulated staining more than 18-fold on monocytes. Activation of lymphocytes in culture with phytohaemagglutinin or concanavalin A also unmasked the mAb 13.22 neoepitope on approximately 37% of the CD45RO+ lymphocytes. Furthermore, analysis of leucocytes collected from the synovial fluid of patients with rheumatoid arthritis showed that approximately 18% of the lymphocytes present expressed the mAb 13.22 neoepitope. Taken together, our results suggest that the mAb 13.22 carbohydrate neoepitope could represent a physiologically relevant marker that is up-regulated on leucocyte subsets during the inflammatory response.

Antibodies, Monoclonal↗

PubMed2001

Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia.

BACKGROUND & AIMS: Tight junctions are crucial determinants of barrier function in polarized intestinal epithelia and are regulated by Rho guanosine triphosphatase. Rho kinase (ROCK) is a downstream effector of Rho. METHODS: A specific inhibitor of ROCK, Y-27632, was used to examine the role of ROCK in the regulation of tight junctions in model intestinal (T84) cells by electrophysiologic, biochemical, morphologic, and molecular biologic approaches. RESULTS: ROCK inhibition induced reorganization of apical F-actin structures and enhanced paracellular permeability but did not alter the distribution or detergent solubility of tight junction proteins. Confocal microscopy showed colocalization of a subpool of ROCK with the tight junction protein zonula occludens 1. Inhibition of ROCK function by a dominant negative mutant of ROCK also produced reorganization of apical F-actin structures without disruption of tight junctions. ROCK inhibition in calcium switch assays showed that ROCK is necessary for the assembly of tight and adherens junctions. Upon calcium repletion, occludin, zonula occludens 1, and E-cadherin failed to redistribute to the intercellular junctions; assembly of the apical F-actin cytoskeleton was prevented; and barrier function failed to recover. CONCLUSIONS: We suggest that ROCK regulates intact tight junctions via its effects on the F-actin cytoskeleton. ROCK is also critical for assembly of the apical junctional proteins and the F-actin cytoskeleton organization during junctional formation.

Actins↗

PubMed2001

Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins.

The anaerobic bacterium Clostridium difficile is the etiologic agent of pseudomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021-16026, 1998). We utilized purified reference toxins of C. difficile, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting "raft-like" membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis.

Actins↗

PubMed2001

Neutrophil transepithelial migration: regulation at the apical epithelial surface by Fc-mediated events.

Neutrophil (PMN) transepithelial migration is a major effector of epithelial defense in inflammatory diseases involving mucosal surfaces. However, major receptor-ligand interactions between epithelial cells and PMN remain incompletely characterized. To better define the molecular events involved in PMN interactions with epithelial cells, we produced a monoclonal antibody called g82 that inhibited PMN transepithelial migration in the physiological basolateral-to-apical direction. The g82 antigen localized to the apical surface of human colonic epithelium and was significantly upregulated under inflammatory conditions. Immunoprecipitation revealed two polypeptides of M(r) 207 and 32 kDa. F(ab')(2) fragments from g82 IgG had no effect on transmigration, suggesting Fc dependence. Further experiments confirmed dependence on the PMN Fc receptor CD32A and that the observed effects were secondary to a failure of PMN to detach from the apical epithelial surface. These Fc-mediated events were epitope specific since binding, isotype-matched antibodies did not affect detachment. These results identify a new mechanism for retention of PMN at the apical epithelial surface following transepithelial migration. This pathway may be important in pathogen clearance and mucosal pathophysiology associated with autoimmunity.

Animals↗

PubMed2000

Modulation of epithelial and endothelial paracellular permeability by leukocytes.

The tight junction acts as a regulated barrier for diffusion of ions, larger solutes, and migration of leukocytes through the paracellular space. Barrier function varies with the tissue type, and positively correlates with the number and complexity of tight junction strand formation. Polarized epithelia and brain capillary endothelium display a high degree of barrier function and tight junction formation. Conversely, vascular endothelium in other tissues has fewer, loosely organized tight junctions strands and greater permeability. Through cytoskeletal association, the tight junction and the adherens junction form a functional unit termed the apical junction complex. The development and stabilization of the tight junction is dependent upon the adherens junction. It has become apparent that paracellular permeability is altered during the migration of leukocytes across the apical junction complex and that the apical junction complex plays an important role in the regulation of leukocyte transmigration (or extravasation) through the endothelium and epithelium. Evidence suggests that important cell-cell adhesive events between transmigrating leukocytes and the apical junction complex and subsequent signaling events result in the facilitation of the passage of cells through the paracellular space. Possible mechanisms for the regulation of barrier function pertaining to leukocyte transmigration are discussed.

Animals↗

PubMed2000

CD11b/CD18-coated microspheres attach to E-selectin under flow.

Neutrophils can attach to E-selectin under flow. Proposed ligands for E-selectin carry SLe(x)-type glycans. The leukocyte beta2 integrins are glycosylated with SLe(x). Thus, we speculated that beta2 integrins could support attachment to E-selectin. To test this hypothesis, we coated 10-microm-diameter microspheres with purified CD11b/CD18 (alphaMbeta2) and investigated the adhesion of the resulting alphaMbeta2 microspheres to E-selectin. Under in vitro flow conditions, the alphaMbeta2 microspheres attached to Chinese hamster ovary cells expressing E-selectin (CHO-E) and 4-h interleukin-1beta-activated human umbilical vein endothelial cells (HUVEC). At a shear stress of 1.8 dynes/cm2, the attachment events were eliminated by pretreatment of the cellular monolayers with a mAb to E-selectin. alphaMbeta2 microspheres did not attach to untransfected CHO cells or unactivated HUVEC at 1.8 dynes/cm2. Taken together, the results strongly suggest that the CD11b/CD18-E-selectin bond has sufficient biophysical properties to mediate attachment of neutrophil-sized particles to E-selectin under flow.

Animals↗

PubMed2000

Neutrophil migration across intestinal epithelium.

Transmigration of neutrophils across epithelial surfaces is the hallmark of inflammatory mucosal diseases of diverse organs. In disorders such as Crohn's disease, ulcerative colitis, pyelonephritis, and bronchitis, for example, neutrophil transmigration correlates with clinical disease activity, is associated morphologically with injury to the epithelium, and is central to disease pathophysiology. The mechanisms by which neutrophils transmigrate across epithelia are, therefore, of considerable significance for numerous pathologic states. In this paper, we discuss current evidence that defines these mechanisms in intestinal epithelium, emphasizing the structural constituents determining adhesive interactions and a subset of the complex regulatory signals between neutrophils and epithelium.

Animals↗

PubMed2000

Tight junctions are membrane microdomains.

Tight junctions (TJ) of polarized epithelial cells regulate barrier function at mucosal surfaces. Structural proteins of TJs include hyperphosphorylated occludin (HO) and the peripheral membrane protein, ZO-1. Since TJs are dynamically regulated, and lipid-modified signal transduction proteins localize to TJs, we considered the possibility that the TJ itself is composed of microdomains with unique structure. Differential detergent extraction and isopycnic sucrose density gradients were utilized to isolate TJ-enriched membranes from a polarized intestinal epithelial cell line, T84. Here we report that major pools of hyperphosphorylated occludin (HO) and ZO-1 are found in raft-like membrane microdomains with characteristics of the previously described detergent-insoluble glycolipid rafts (DIGs). Properties of such gradient fractions included Triton X-100 (TX-100) insolubility, light scattering at 600 nm, buoyant density of approximately 1.08 g/cm(3) and increased cholesterol content compared to high density fractions. Similar results were obtained using natural epithelium. Unlike the TJ proteins HO and ZO-1, other basolateral transmembrane proteins including E-cadherin, c-met and &bgr; 1 integrin were not increased in DIG-like fractions. Immunoprecipitation studies revealed coprecipitation of a pool of occludin with caveolin-1, a scaffolding protein abundant in DIGs. Coprecipitation results were supported by immunofluorescence and immunogold labeling studies demonstrating caveolin-1 localization in the apical membrane and focal colocalization with occludin in TJs. TJ disassembly by calcium chelation resulted in displacement of TJ proteins from the 'raft-like' compartment. Our findings suggest that raft-like compartments play an important role in the spatial organization of TJs and probably in regulation of paracellular permeability in epithelial cells.

Caveolin 1↗

PubMed2000

Human junction adhesion molecule regulates tight junction resealing in epithelia.

Epithelial cells form a highly selective barrier and line many organs. The epithelial barrier is maintained by closely apposed cell-cell contacts containing tight junctions, the regulation of which is incompletely understood. Here we report the cloning, tissue localization and evidence for a role in epithelial barrier regulation of an immunoglobulin superfamily member that likely represents the human homolog of murine junction adhesion molecule (JAM). Analysis of the primary structure of human JAM, cloned from T84 epithelial cells, predicts a transmembrane protein with an extracellular domain that contains two IgV loops. Monoclonal antibodies generated against the putative extracellular domain were reactive with a 35-39 kDa protein from both T84 epithelial cells and human neutrophils. By immunofluorescence, JAM mAbs labeled epithelial cells from intestine, lung, and kidney, prominently in the region of tight junctions (co-localization with occludin) and also along lateral cell membranes below the tight junctions. Flow cytometric studies confirmed predominant JAM expression in epithelial cells but also revealed expression on endothelial and hematopoietic cells of all lineages. Functional studies demonstrated that JAM specific mAbs markedly inhibited transepithelial resistance recovery of T84 monolayers after disruption of intercellular junctions (including tight junctions) by transient calcium depletion. Morphologic analysis revealed that, after disassembly of cell-cell junctions, anti-JAM inhibition of barrier function recovery correlated with a loss of both occludin and JAM, but not ZO-1, in reassembling tight junction structure. Reassembly of the major adherens junction component E-cadherin was not affected by JAM specific mAbs. Our findings suggest that JAM plays an important role in the regulation of tight junction assembly in epithelia. Furthermore, these JAM-mediated effects may occur by either direct, or indirect interactions with occludin.

Amino Acid Sequence↗

PubMed1999

Cell-specific peptide binding by human neutrophils.

Analysis of peptide binding to human neutrophils (PMN) using phage display techniques has revealed cell-specific motifs reactive with the PMN surface. Phage libraries displaying either linear 9-mer or cyclic 10-mer and 6-mer peptides were incubated with normal human neutrophils followed by elution of bound phage with low pH (pH 2.2) and non-ionic detergent. Three rounds of selection generated several related peptide sequences that bound with high avidity to PMN. Using the linear 9-mer library, PMN-binding phage expressed peptides with the motif (G/A)PNLTGRW. The binding of phage bearing this motif was highly specific since no binding was observed on lymphocytes, fibroblasts, epithelial, or endothelial cells. Functional assays revealed that phage bearing the sequence FGPNLTGRW induced a pertussis toxin-sensitive increase in PMN cytosolic calcium analogous to that observed with Galphai coupled receptors. Other prominent motifs identified included phage bearing the consensus DLXTSK(M/L)X(V/I/L), where X represents a non-conserved position. Phage with this motif bound exclusively to a sub population of human PMN that comprised approximately 50% of the total and did not elicit a calcium response. The binding of such phage to PMN was prevented by co-incubation with competing peptides displaying identical or similar sequences (IC50 range from 0.6 micromol/L to 50 micromol/L for DLXTSK and GPNLTG, respectively). We speculate that these techniques will be useful in identifying functional cell-specific binding motifs and contribute to the development of new therapeutic and diagnostic strategies in human disease.

Amino Acid Sequence↗

PubMed1999

Non-serum-dependent chemotactic factors produced by Candida albicans stimulate chemotaxis by binding to the formyl peptide receptor on neutrophils and to an unknown receptor on macrophages.

Serum-free culture filtrates of six Candida species and Saccharomyces cerevisiae were found to contain chemoattractants for human polymorphonuclear leukocytes (PMNs) and a mouse macrophage-like cell line, J774. The chemotactic factors differed for the PMN and J774 cells, however, in terms of heat stability, kinetics of liberation by the yeast cells, and divalent cation requirements for production. The chemoattractant in Candida albicans culture filtrates appeared to act through the formyl peptide receptor (FPR) of PMNs, since it was found to induce chemotaxis of Chinese hamster ovary (CHO) cells that were expressing the human FPR but did not induce chemotaxis of wild-type CHO cells. The C. albicans culture filtrates also induced migration of PMNs across confluent monolayers of a human gastrointestinal epithelial cell line, T84; migration occurred in the basolateral-to-apical direction but not the reverse direction, unless the epithelial tight junctions were disrupted. J774 cells did not migrate toward the formylated peptide (fMet-Leu-Phe; fMLF), and chemotaxis toward the C. albicans culture filtrate was not inhibited by an FPR antagonist (t-butoxycarbonyl-Met-Leu-Phe), suggesting that a different receptor mediated J774 cell chemotaxis. In conclusion, we have identified a receptor by which a non-serum-dependent chemotactic factor (NSCF) produced by C. albicans induced chemotaxis of PMNs. Additionally, we have shown that NSCF was active across epithelial monolayers. These findings suggest that NSCFs produced by C. albicans and other yeast species may influence host-pathogen interactions at the gastrointestinal tract mucosal surface by inducing phagocytic-cell infiltration.

Animals↗

PubMed1998

Organization and mobility of CD11b/CD18 and targeting of superoxide on the surface of degranulated human neutrophils.

A monoclonal IgM, specifically recognizing both CD11b and CD18 of human neutrophils, was used to examine the organization and mobility of CD11b/CD18 in the plasma membrane of human neutrophils degranulated by dihydrocytochalasin B (dhCB) treatment and fMet-Leu-Phe (fMLF) stimulation. Subcellular fractionation analysis of untreated or dhCB-treated control neutrophils indicated that 20% of CD11b/CD18 cosedimented with plasma membrane and the remainder with specific granules. In contrast, fMLF stimulation of dhCB-treated cells caused a major reorganization of CD11b/CD18, in which 60-70% of CD11b/CD18 sedimented in dense plasma membrane fractions that were also enriched in superoxide-generating NADPH oxidase activity. Similarly pretreated neutrophils were fixed, immunogold labeled, and examined by scanning electron microscopy. Immunogold particles were distributed uniformly over the symmetrically ruffled surface of unstimulated neutrophils. On dhCB-treated cells, immunogold was mostly uniformly distributed on a smooth membrane with a small percentage of particles lining up into linear arrays. After fMLF + dhCB stimulation, CD11b/CD18 gold label was more abundant on the cell surface and formed large aggregates on polarized membrane protrusions. However, when cells were adhered to an albumin-coated quartz surface and stimulated with fMLF in the presence of dhCB, immunogold was excluded on the articulated and rounded cell body but concentrated on the periphery of adherent lamellae. Fluorescence photobleaching recovery indicated that in unstimulated cells 38 +/- 3% of CD11b/CD18 was mobile (R) with a diffusion constant D of 3.1 +/- 0.3 x 10(-10) cm2/s. Treatment with dhCB raised R and D 24 and 74%, respectively. Stimulation using 1 microM fMLF with dhCB lowered D and R to near control levels. Since NADPH oxidase and CD11b/CD18 cosediment in high-density plasma membrane domains after fMLF + dhCB stimulation, we speculate that a stimulus-induced reorganization of CD11b/CD18 and NADPH oxidase to common membrane domains may occur in fMLF + dhCB-degranulated neutrophils.

CD18 Antigens↗