Localization of the IgG effector site for monocyte receptors.

Abstract

A peptide consisting of 10 amino acids derived from the CH3 region of human IgG was shown to bind to monocytes and to inhibit rosette formation of antibody-coated erythrocytes with human monocytes. Two myeloma proteins of the IgG1 and IgG3 subclass, both with known deletions in the CH2 region of the gamma chain, showed unimpaired ability to bind to monocytes. These experiments suggest that the isolated peptide represents the primary site of attachment of IgG to monocytes.Antibody which is cytophilic for monocytes and macrophages has been detected in the serum of guinea pigs, rabbits, mice, and man (1-3). Erythrocytes coated with cytophilic antibody readily form rosettes with mononuclear cells at room temperature and thus provide a detection system for cytophilic antibody. In lower species, cytophilic antibody has been found among the 7S IgG2 class (1). Human cytophilic antibody is in the IgG class, among which the IgG1 and IgG3 subclasses contain all the antibodies with cytophilic properties (4, 5). Attempts to localize the effector site for monocyte binding have conclusively demonstrated its presence in the Fc fragment (3). Furthermore, digestion of Fc fragments with pepsin (6) and trypsin (7) has led to the suggestion that the effector site is located in the CH3 region of the gamma chain. The present study was undertaken to attempt further precise localization of the effector site for monocyte binding in the Fc fragment of IgG. MATERIALS AND METHODSFractionation of IgG Peptides. Pooled human IgG was digested with papain (8), and Fab and Fc fragments were separated by electrophoresis on cellulose in Tris-borate buffer at pH 9.2. Fc fragments were allowed to react with 10% cyanogen bromide in 70% formic acid prior to dialysis of the reaction mixture. Peptides in the dialysate were chromatographed into seven fractions on paper with a butanol: pyridine:acetic acid:H20 mixture in a ratio of 15:10:3:12. Peptide fractions, which were functionally active, were labeled with [125I Jiodinated 3-(4-hydroxy)phenylpropionic acid N-hydroxysuccinimide ester (9). Radiolabeled peptides were further fractionated by chromatography on paper and thin-layer silicic acid in the same solvent system. Aminoacid analysis of acid-hydrolyzed peptides was carried out by the method of Moore and Stein (10).Rosette Formation on Mlonocyte Mlonolayers. Human peripheral blood was drawn into heparin (100 units/4 ml), and (v/v). Fourtenths milliliter of EA was added to each monolayer and incubated for 2 hr at room temperature. Unbound EA were removed from the monolayers by aspiration of the supernatant medium, and the monolayers were then assayed for radioactivity after being washed four times with Hanks' solution. Inhibition of erythrocyte rosette formation on monocyte monolayers was effected by prior incubation of monolayers with IgG or peptides derived from IgG for 15 min at 370 prior to addition of EA (3).IgG Myeloma Proteins. Two myeloma proteins with known deletions of amino-acid residues from their H chains were obtained from Dr. Li...