Lipid monolayer-coated solid surfaces do not perturb the lateral motion and distribution of C3b receptors on neutrophils.

Abstract

We have used epifluorescence and photobleaching techniques to study the lateral distribution and motion of fluorescein-conjugated Fab fragments of anti-C3b receptor antibody bound to human neutrophils when the cells rest on various solid supports (microscope slides or cover slips). Supports composed of quartz, glass, or alkylated glass induce cellular adhesion, spreading, and an extensive lateral redistribution of C3b receptors (but not HLA antigens). The neutrophil C3b receptors become patchy, and the patches apparently undergo nonrandom translational motion. Many patches are found on the upper surfaces of the cells removed from the region of cell membrane-glass contact. In contrast, neutrophils supported by lipid monolayer-coated glass do not adhere or spread, and the C3b receptor remains uniform and diffuses freely (D = 2 x 10 -1° cm2/s).Previous studies of the lateral motion and distribution of the complement (C3b) receptor on peripheral blood leukocytes using fluorescent antireceptor antibody and antireceptor antibody fragments have indicated that these receptors are highly clustered (3, 9). Indirect measurements have also suggested that the C3b receptor is immobile on normal resting macrophages (4, 7, 8). Because cell surface receptors usually have a diffuse distribution in the absence of receptor-ligand interactions, we suspected that the clustered distribution of the C3b receptors might be a consequence of cell triggering. Concurrent studies in this laboratory have recently shown that macrophages (5), basophils (13), and neutrophils (D. G. Hafeman, M. Seul, and H. M. McConnell, unpublished observation) do not bind to lipid monolayer--coated alkylated glass surfaces in the absence of specific antibody-mediated monolayer--cell membrane interaction. We therefore undertook a study to determine whether interaction of cells with solid supports could influence the distribution and motion of the C3b receptor on human neutrophils. We find that the distribution and motion of the C3b receptor is strongly affected by the nature of the substrate on which the cells are examined. In contrast, HLA antigens are relatively unaffected by substrate contact. MATERIALS AND METHODS Preparation of CellsWhole blood was drawn from healthy human donors. Fibrin and platelets were removed by gentle swirling of the blood with 3-ram-diameter solid glass beads (one bead/ml) for 10 min. Blood was diluted with 1 vol of 0.85% NaCI. An aliquot of 6% dextran in 0.85% NaC1 (T-500; Pharmacia Fine Chemicals, Piscataway, btJ) was added to give a final dextran concentration of 0.6%. The erythrocytes were allowed to sediment at 1 g for 40 rain at 24°C. The supernatant was layered onto a cushion of Ficoll-Hypaque (Pharmacia) and granulocytes and mononuclear leukocytes were separated by centrifugation at 400 g for 40 min. Each cell fraction was washed twice in Ca +*-and Mg++-free Earle's balanced salt solution with 0.2% gelatin and buffered with 25 mM HEPES (pH 7.4). All ceils were resuspended at 5 x 10e/ml in cell buffer (CB; 2.0 mM CaCI2...

Inflammatory roles of P-selectin.

“…PMNs allowed to settle onto planar phospholipid membranes that contained no incorporated protein remained rounded, as predicted from an earlier study showing that surface receptors are not redistributed (50). PMNs on membranes containing glycophorin or W6/32, the control proteins, also were round.…”

Neutrophil C3bi receptors: Formation of membrane clusters during cell triggering requires intracellular granules

“…External but not internal fluorescence is quenched by this procedure Wed, 1977). A second method of quenching employing 10 mM cupric sulfate at pH 5 has also been employed (Hafeman et al, 1982). Similar results were obtained by both procedures.…”

“…The clinical importance of CR3 is indicated by recent studies showing that granulocytes of certain patients prone to bacterial infections were deficient in this receptor (Anderson et al, 1984;Curnutte and Boxer, 1985;Arnaout et al, 1982). Although the cell surface properties of CR1 have been carefully studied (Fearon, 1984;Hafeman et al, 1982;Jack and Fearon, 1984;Petty et al, 1980), little is known about the cell surface properties of CR3 and the mechanisms that regulate its topographic distribution on the plasma membrane. have stated that CR3 is uniformly distributed on the plasma membrane of untreated monocytes or cells treated with 4-phorbol 12-myristate 13-acetate (PMA) or supernates from cell suspensions (2 x 10 cells/ml) fibronectin.…”

Impaired T cell capping and receptor regeneration in active systemic lupus erythematosus. Evidence for a disorder intrinsic to the T lymphocyte.

“…To verify that caps were localized to the external surface of the membrane and that fluorescent vesicles were intracellular, 10 mM isotonic cupric sulfate replaced the medium bathing the cells. Fluorescence emitted by a source on the external surface of the cell is quenched by the cupric ion, whereas that from an intracellular source is not quenched (Dr. Dean Hafeman, personal communication; reference 22). For purposes of enumeration, capped cells included those exhibiting either capping or endocytosis.…”