Internalization pathway of C3b receptors in human neutrophils and its transmodulation by chemoattractant receptors stimulation.

Abstract

On the surface of phagocytes, C3b receptors (CR1) bind C3b-coated particles and promote their ingestion after activation by appropriate stimuli such as lymphokines or the chemoattractant formyl methionyl leucyl phenylalanine (fMLP) and fibronectin. The aims of the present study were 1) to define at the electron microscopic level the nature of the process responsible for CR1 internalization and 2) to dissect the mechanism by which a physiological activator (fMLP) stimulates this process. CR1 was visualized either by the immunogold technique or by quantitative electron microscopic autoradiography using a monoclonal anti-CR1 antibody. Both techniques revealed that after anti-CR1 binding, CR1 cluster on the neutrophil surface in a time-, temperature-, and antibody-dependent fashion, but do not concentrate in coated pits. CR1 internalization requires receptor cross-linking (does not occur in the presence of Fab fragments of anti-CR1) and intact microfilaments. It results in the association of the internalized material with large flattened vacuoles, organized in stacks. Together with the surface localization of CR1 close to cytoplasmic projections (ruffles), these observations suggest that uptake of CR1 occurs through a macropinocytotic process. Eventually, CR1 concentrate in lysosomal structures. fMLP markedly stimulates this pattern of CR1 internalization without affecting their clustering or their lack of association with coated pits. Stimulation by fMLP is inhibited by pertussis toxin, unaffected by preventing receptor-triggered cytosolic free calcium [Ca2+]i elevations, and mimicked by phorbol myristate acetate. Taken together our data demonstrate 1) that, in neutrophils, CR1 is internalized via a coated pit independent macropinocytotic process, dependent on intact microfilaments and receptor cross-linking; 2) that, in the same cells, fMLP is internalized via the classical coated pits pathway; and 3) that fMLP amplifies CR1 uptake possibly via protein kinase C stimulation.

CD14-dependent Endotoxin Internalization via a Macropinocytic Pathway

“…After two washes in 4°C phosphate-buffered saline, cells were warmed and kept 5 or 20 min in a 37°C water bath in the presence or in the absence of 2 g/ml Escherichia coli K12 LCD25 LPS (List Biological Laboratories Inc., Campbell, CA)/10% fetal bovine serum. Cells were then fixed for 30 min at 20°C with 2.5% glutaraldehyde diluted in pH 7.4 phosphate buffer, dehydrated, and processed for electron microscopy as described elsewhere (30). Thin sections were examined in a Philips electron microscope 301, and gold particles were quantitatively analyzed.…”

Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis.

“…Unbound secondary antibody was removed by washing twice with ice-cold PBS, and cells were warmed for 5 min at 37°C to allow endocytosis. Cells were finally fixed 30 min at room temperature with 2.5% glutaraldehyde in phosphate buffer, dehydrated, and processed for electron microscopy as described previously (Carpentier et al, 1991). Thin sections were examined in a Philips EM 300 (Philips), and gold particles were quantitatively analyzed on cells considered well preserved.…”

“…Heterologous regulation (i.e., receptor cross-talk) occurs when one receptor generates a second messenger that regulates the internalization of another receptor. Examples for the latter include the increased internalization of type 1 complement receptor after stimulation of the Nformylmethionylleucylphenylalamine (f-MLP) receptor (presumably via protein kinase C; Carpentier et al, 1991) and cyclic AMP (cAMP) enhanced internalization of adherence-receptors in neurons (through an increase in the number of clathrin-coated pits; Hu et al, 1993). cAMP has also been implicated in the regulation of CD4 capping and endocytosis after receptor multimerization in human T lymphocytes (Kammer et al, 1988).…”

Neutrophilic Inflammatory Response and Oxidative Stress in Premenopausal Women Chronically Exposed to Indoor Air Pollution from Biomass Burning

“…Neutrophils in peripheral blood may be activated or primed with regard to superoxide production, chemotaxis, and increased expression of surface molecules such as CD35 which mediates the binding and phagocytosis of C3b-coated particles and immune complexes [28]. The study of Smith et al [29] suggested that upregulation of CD11b expression may be associated with neutrophils degranulation.…”