The formation of specific, heterophilic conjugates between cells from the P388D1 mouse macrophage line and antibody-coated mouse spleen cells was followed in cell suspensions at 4 degrees C by dual parameter flow cytometry. Intercellular aggregation in this system is mediated by the binding of the Fc portions of IgG antibodies on the spleen cells with Fc receptors (Fc gamma R) on P388D1. We show that the rate of aggregation reaches a plateau with increasing cell concentrations, suggesting that the initial collision between cells is not the rate limiting step of conjugate formation. The rates of aggregation are strongly dependent upon the cell surface densities of both Fc gamma R and antibody. In conjugates, however, only small fractions of available receptors or antibodies are utilized in bond formation. The rate-limiting step of aggregation, therefore, involves the formation of ligand-receptor bonds, and may be the diffusion of antibodies and receptors toward one another in small areas of intercellular contact. Inhibitor studies implicate microfilaments, but not microtubules, divalent cations, or energy-dependent processes as being important in aggregation. Finally, conjugates are stable when diluted into medium alone, but dissociate in media containing protein A, soluble immune complexes, or anti-Fc gamma R antibodies. This suggests that conjugates are stabilized by multiple intercellular ligand-receptor bonds, which constantly break and reform at the cell:cell interface, and that protein A, immune complexes, and anti-Fc gamma R disaggregate the conjugates by preventing the reformation of broken bonds.
- Copyright © 1984 by American Association of Immunologists