Quantitative aspects of signal transduction by the receptor with high affinity for IgE.

Identification of the major components, how these interact with each other, and the modifications that follow in the sequence of events triggered by the receptor with high affinity for IgE, is progressing rapidly. A new challenge is to understand these interactions quantitatively. We present the fundamentals of the mechanistic model we are testing through mathematical modeling. The object is to see if the predictions of the model fit with the experimental results.

The influence of actin microfilaments on signaling by the receptor with high-affinity for IgE.

Aggregation of the receptors with high-affinity for IgE (FcRI) stimulates a variety of cellular responses, but excessive aggregation inhibits such responses. Actin filaments have been implicated in the inhibitory phenomenon because disrupting the filaments enhances the cellular reactions stimulated by the aggregated receptors. To clarify further the molecular mechanism and physiological importance of the actin-mediated inhibition, we assessed the effect of inhibitors of actin polymerization on the initial signaling events of mast cells alternatively stimulated by nitrophenyl ligands that dissociate slowly (high-affinity) or rapidly (low-affinity) from receptor-bound anti-dinitrophenyl IgE. The inhibitors amplified the phosphorylation of FcRI and of Syk induced by addition of either ligand but at physiological temperatures, the augmentation of the response to the low-affinity ligand was especially exaggerated. The effect of actin is on the earliest events, and although the molecular mechanism(s) by which the filaments regulate the intensity of proximal signaling remains unclear, several possibilities have been excluded. That the inhibitors only minimally augment the responses stimulated by preformed dimers of IgE, and in general show smaller effects with more limited aggregation, suggests that the actin-mediated "down-regulation" may be more prominent in laboratory experiments than under physiological circumstances.

The high affinity receptor for IgE, FcepsilonRI.

Progress in our understanding of the structure and function of the receptor with high affinity for IgE (FcepsilonRI) is already being applied in attempts to mitigate allergic reactions, specifically by using specific anti-IgEs to prevent sensitization. Investigations of the complex network of intracellular signals generated by FcepsilonRI and related receptors are proceeding. Although achieving a 'complete' description of the events will be a daunting task, the effort is likely to identify additional targets for therapeutic intervention along the way.

Molecular versatility of antibodies.

As immunology developed into a discrete discipline, the principal experimental efforts were directed towards uncovering the molecular basis of the specificity exhibited by antibodies and the mechanism by which antigens induced their production. Less attention was given to how antibodies carry out some of their effector functions, although this subject presents an interesting protein-chemical and evolutionary problem; that is, how does a family of proteins that can bind a virtually infinite variety of ligands, many of which the species producing that protein has never encountered, reproducibly initiate an appropriate response? The experimental data persuasively suggested that aggregation of the antibody was a necessary and likely sufficient initiating event, but this only begged the question: how does aggregation induce a response? I used the IgE:mast cell system as a paradigm to investigate this subject. Data from our own group and from many others led to a molecular model that appears to explain how a cell 'senses' that antigen has reacted with the IgE. The model is directly applicable to one of the fundamental questions cited above, i.e. the mechanism by which antigens induce the production of antibodies. Although the model is conceptually simple, incorporating the actual molecular events into a quantitatively accurate scheme represents an enormous challenge.

Synthesis and secretion of monocyte chemotactic protein-1 stimulated by the high affinity receptor for IgE.

In prior studies aggregation of the high affinity receptors for IgE, Fc epsilon RI, on a rat mast cell line, RBL-2H3, stimulated transcription of the gene for monocyte chemotactic protein-1 (MCP-1) and secretion of the protein. Unexpectedly, those delayed events appeared much less constrained by kinetic proofreading than had been documented for other receptor-initiated responses. The results of the present experiments are consistent with the proposal that the biosynthesis and secretion of MCP-1 result from a soluble messenger formed in the reaction cascades initiated by the receptor, and that Ca(2+) could serve as that messenger. Interestingly, whereas receptor-mediated signals were required for transcription of the gene for MCP-1 and secretion of the chemokine, such signals were not required for the intervening step of translation of its mRNA.

Investigation of early events in Fc epsilon RI-mediated signaling using a detailed mathematical model.

Aggregation of Fc epsilon RI on mast cells and basophils leads to autophosphorylation and increased activity of the cytosolic protein tyrosine kinase Syk. We investigated the roles of the Src kinase Lyn, the immunoreceptor tyrosine-based activation motifs (ITAMs) on the beta and gamma subunits of Fc epsilon RI, and Syk itself in the activation of Syk. Our approach was to build a detailed mathematical model of reactions involving Fc epsilon RI, Lyn, Syk, and a bivalent ligand that aggregates Fc(epsilon)RI. We applied the model to experiments in which covalently cross-linked IgE dimers stimulate rat basophilic leukemia cells. The model makes it possible to test the consistency of mechanistic assumptions with data that alone provide limited mechanistic insight. For example, the model helps sort out mechanisms that jointly control dephosphorylation of receptor subunits. In addition, interpreted in the context of the model, experimentally observed differences between the beta- and gamma-chains with respect to levels of phosphorylation and rates of dephosphorylation indicate that most cellular Syk, but only a small fraction of Lyn, is available to interact with receptors. We also show that although the beta ITAM acts to amplify signaling in experimental systems where its role has been investigated, there are conditions under which the beta ITAM will act as an inhibitor.