Fc Receptors on Human Lymphocytes Detected with Double-Coating Indirect Rosette Formation

Abstract

The Fc receptor (FcR) on human lymphocytes is studied by using soluble immune complexes in antigen excess, prepared between ferritin and rabbit anti-ferritin, and a modification of Coombs’ antiglobulin reaction (double-coating indirect rosette formation). 12% of human blood lymphocytes are shown to have Fc receptors. The FcR on lymphocytes is sensitive to pronase treatment, partially sensitive to trypsin digestion, and is inactivated after short glutaraldehyde fixation of lymphocytes. By cell fractionation experiments, FcR-positive lymphocytes were enriched in cell populations enriched with B cells.

Are Fcγ Receptors Naturally Clustered on Normal Human B Lymphocytes?

“…Anti-ferritin (anti-Fer) and anti-sheep red blood cells were prepared in alalb4b4 rabbits as described before [4], F(ab'); fragments and 7S fractions of antisera were prepared as de scribed [7]. Apoferritin was prepared from crystallized Fer (Sigma, St. Louis, Mo.)…”

“…The ligands were monovalent in terms of numbers of Fc pieces available per immune-complexed molecule. In contrast, in our earlier studies of FcR on lymphocytes, soluble immune com plexes were prepared in the same excess of the antigen, but were ob tained after chromatography on Sephadex G-200 [3,7]. Cytochala sin B (CB; Sigma) and colchicine (COL; Sigma) were prepared as described [6].…”

“…Lymphocytes were pre pared from healthy human peripheral blood by using carbonyl iron and Ficoll-Hypaque gradients [7,11]. For electron-microscopic studies, lymphocytes were similarly prepared without incubating defibrinated blood with carbonyl iron [3], To study the effects of cytoskeletal reagents on receptor distribution, 0.1 ml of lymphocytes and 0.1 or 0.2 ml of the cytoskeletal reagents were added into a test tube holding 0.8 or 0.7 ml of RPMI 1634 containing 2% fetal calf serum (FCS; North American Biological, Miami, Fla.) [6].…”

“…Binding of the ligand for FcR on human lymphocytes was detected by using a modified Coombs mixed antiglobulin reaction which we called DIRF [4,7,12]. In brief, human lymphocytes were coated at 0°C (ice bath) with the ligand for FcR; the coated lymphocytes formed rosettes (indirect rosettes) with sheep erythrocytes sensitized with a subhemagglutinating dose of F(ab')2 fragments of b4 rabbit anti-SRBC with interposing bridges of F(ab')2 anti-b4 at 4°C in FCS-PBS or FCS-PBS-R. Lymphocytes with four or more adhering indi cator cells were expressed as a percentage of total counted lympho cytes.…”

“…In the current studies, lymphocyte donors were selected from the group whom we studied previously [7]. These donors showed similar percentagesof FcR-positive lymphocytes and B cells.…”