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

Abstract

Video-intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescein- or rhodamine-labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double-labeling studies were conducted by first labeling with anti-CR3 followed by attachment to LPS/serum-treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum-treated slides. To determine the contribution of granule-associated CR3, we have studied neutrophils defective in receptor up-regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3-modified surfaces. The patient's neutrophils were defective in CR3 up-regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum-treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3-bearing granules at local sites of adhesion.

Cell surface distribution of Fc receptors II and III on living human neutrophils before and during antibody dependent cellular cytotoxicity

“…Similarly, other workers have inferred that FcRs are uniformly distributed on unstimulated or fixed cells (Sajnani et al, 1976;Romans et al, 1976;Walter et al, 1980). Additional cell surface molecules such as HLA antigens, lipid probes, and CR3 are uniformly distributed during resting conditions (Petty et al, 1980a(Petty et al, , 1987. However, other neutrophil receptors (e.g., histamine and CR1) appear clustered during…”

Histamine inhibits cell spreading and C3bi receptor clustering and diminishes hydrogen peroxide production by adherent human neutrophils

“…Furthermore, it has been shown that this clustered arrangement of CR3 may result from up-regulation of CR3 from an intracellular pool(s) (Fig. 4E,F; Petty et al, 1987). Neutrophils pretreated with M histamine failed to display fluorescent CR3 clusters but instead showed diffuse CR3-labeling on over 95% of the adherent cells (Fig.…”

Deficiency of the specific granule proteins, R-binder/transcobalamin I and lactoferrin, in plasma and saliva: A new disorder

“…The prime example is the congenital neutrophil disorder known as speci®c granule de®ciency (SGD) [Gallin, 1985;Malech and Nauseef, 1997]. Its multiple neutrophil abnormalities include low levels of lactoferrin, R binder, and the other speci®c granule proteins, as well as proteins from other granules, such as defensin [Ganz et al, 1988] and gelatinase [Petty et al, 1987]. However, the defect in SGD is limited to the neutrophil; R binder and lactoferrin levels are normal in secretions [Ambruso et al, 1984;Lomax et al, 1989].…”