Mobile Haptens in Lipid Bilayers Cause Large-Scale Clustering of IgE Receptors

Abstract

Rat basophilic leukemia (RBL) mast cells express IgE receptors on the membrane that, when aggregated, initiate biochemical events that lead to the exocytosis of inflammatory mediators (degranulation). Low concentrations of monovalent haptens in fluid supported lipid bilayers have been found to cause degranulation, but microscopically visible aggregation has not been previously observed. To investigate possible receptor aggregation at higher hapten concentration, RBL cells were loaded with a fluorescent anti-DNP IgE and then deposited onto fluid supported lipid bilayers containing DNP-lipids at up to 50 mole %. Total internal reflection fluorescence microscopy was used to image the cells; large clusters were observed at DNP concentrations ≥ 10 mol %. The characteristic cluster sizes and densities were analyzed with image correlation spectroscopy (Petersen et al., BJ 65:1135. 1993), with the improvement that the autocorrelation value g(0,0) was explicitly corrected for shot noise and CCD camera read noise.Monovalent ligands in fluid membranes cause receptor clustering in T-cells and B-cells, in cytoskeleton-dependent processes. The high DNP concentration required for large-scale aggregation in RBLs suggests that degranulation signaling and large-scale aggregation may be two distinct cellular responses that depend differently on hapten concentration and presentation.This research was supported by Sandia National Laboratories. Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-ACO4-94AL85000. KS is supported by ARO grant (W911NF-05-1-0464).