Receptors for C3b on the neutrophil surface associate with the cytoskeleton-containing cell residue subsequent to cell lysis with non-ionic detergent. Cytoskeletal-CR1 interactions.
Radioiodinated Fab' and F(ab')2 fragments were prepared from 57F monoclonal antibody which is specific for the C3b receptor (CR1) on human cells. We find that both Fab' and F(ab')2 fragments bind to CR1 on human neutrophils and then dissociate, at 0 degree C, with half-lives of 172 and 35 min, respectively. In addition to binding to cell surface CR1, both antibody fragments bind specifically to the isolated non-ionic detergent-insoluble cellular residue (NDIR) which remains after cell lysis and solubilization in Triton X-100. We also find that Fab' and F(ab')2 antibody fragments remain associated with the NDIR after the radiolabeled antibody fragments are allowed to bind to cell surface CR1, the unbound fragments removed, and the cells subsequently lysed in non-ionic detergent. Because the cytoskeletal matrix (together with the cell nucleus) makes up a substantial portion of the NDIR, these results suggest that some fraction of cell surface CR1 may be associated in vivo with the cytoskeletal matrix. However, post-lysis association of cell surface-CR1-antibody fragment complexes to the NDIR occurs. Examination of the binding kinetics of this interaction reveals that less than 10% of cell surface CR1 exists bound to the NDIR prior to cell lysis. While the nature of post-cell lysis association of CR1 with the NDIR is unknown, several modulating effects are noted. For example, binding of bivalent cross-linking agents to CR1 on cells followed by a 37 degrees C incubation is known to induce internalization of cell surface CR1. We find that binding of F(ab')2 antibody fragments under these conditions causes a 50% decrease in association of this 57F fragment to the NDIR. Pretreatment of the cells at 37 degrees C with the chemotactic peptide N-formyl-methionyl-leucylphenylalanine similarly caused a 50% decrease of (Fab'-labeled) CR1 association with the NDIR cell fraction. These results support the hypothesis that chemotactic peptide serves to enhance CR1-mediated adherence to C3b-bearing targets by increasing the density of CR1 on the cell surface rather than by inducing cytoskeletal-dependent, detergent-stable, CR1 redistribution on the cell surface.
D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody / Q000187:drug effects; D002458:Cell Fractionation; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D009504:Neutrophils / Q000187:drug effects / Q000648:ultrastructure*; D011485:Protein Binding; D011951:Receptors, Complement / Q000032:analysis / Q000378:metabolism*; D017463:Receptors, Complement 3b; D013501:Surface-Active Agents / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D003902:Detergents; D007140:Immunoglobulin Fab Fragments; D007457:Iodine Radioisotopes; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D013501:Surface-Active Agents