Publisher Summary

This chapter discusses the molecular mechanisms of antibody action and describes the use of cross-linking reagents. Antibodies have no ability to alter the antigen with which they combine directly; they change the ultimate fate of the antigen only by interacting with effector system. When the high molecular weight product of cross-linking with the cleavable reagent is rerun under conditions where the crosslinks are broken, a new component is observed that has a molecular weight of about 30,000. The cross-linked product can be observed after reacting either intact cells or cells solubilized with nonionic detergents. Some of the serotonin that can be released by the higher oligomers is completely resistant to release by dimers. Dimers by themselves are adequate to stimulate secretion of at least a portion of the releasable serotonin. Trimers are more active than dimers, while the higher oligomers are even more effective.

I INTRODUCTION

Immunologists have traditionally delighted in clumping things together. This is reflected in the by-now classical methods of precipitin analysis and agglutination as well as in the new clumping methods which are constantly being developed. Lately, ‘rosette’ formation has become a popular technique among immunologists, and most recently, the use of ‘patching’ and ‘capping’ and of cell fusion to make ‘hybridomas’ demonstrates that the tradition lives on.

It is interesting to speculate that the immunologist’s early use of aggregation as a favored analytical tool stemmed from a subconscious prescience that aggregation was of fundamental importance to what the immunologist was interested in studying; namely, the mechanisms of immune responsiveness. Whether one accepts this psycho-immunologic thesis or not, the significance of aggregation in immunologic mechanisms cannot be disputed. At the cellular level, direct cell to cell interactions have been implicated in a variety of phenomena; e.g., T cell interaction with macrophages and B cells, and T cell killing. Similarly, the significance of aggregation in antibody-mediated mechanisms is well recognized. After some brief comments on the latter subject, we shall describe how still another technique of clumping – the use of chemical cross-linking reagents – has permitted us to explore some basic questions in the system with which we are working.

Antibodies have no ability to alter the antigen with which they combine directly; they change the ultimate fate of the antigen only by interacting with effector systems. In some instances, this leads to a direct and prompt change in the antigen’s prospects, as in phagocytosis. Alternatively, a much more indirect pathway is chosen, such as stimulation of antibody-forming precursor cells. The mechanisms by which antibodies mediate these effects have received considerable attention, especially in recent years, as our detailed knowledge of antibody structure has increased. Several plausible models have been proposed and discussed in reviews [1–3]. A recent workshop, the first to discuss this topic comprehensively [4], did not come up with a consensus report. Nevertheless, with only one significant exception – the observations of Koshland and her colleagues [5,6] – all of the data on activation of effector systems by antibodies point to the critical requirement for aggregation of antibody; specifically, of the Fc regions of the antibodies. Whether the findings of Koshland’s group (that apparently univalent antigens induce complement fixation by IgM) are a valid exception is uncertain. Univalent ligands assuredly don’t cause intermolecular aggregation, but it is less clear whether the special antigens required to mediate the effects studied by these investigators cause intramolecular aggregation of the Fc pentamers in IgM.

In no system is the need for aggregation of antibody more persuasively supported than in the IgE-mast cell system [7–9]. In this instance, antigen reacts with cell-bound IgE (alternatively, preformed IgE-antigen complexes become cell-bound), resulting in the triggering of noncytotoxic degranulation. Any procedure which serves to aggregate the surface IgE will cause stimulation, although hyper-aggregation is inhibitory. The reason why aggregation of the IgE is critical has recently been elucidated by the use of antibodies directed to the cellular membrane component (receptor for IgE) to which the IgE is bound [10,11]. These studies showed that what is important is that the receptor becomes aggregated. Even cells grown in vitro in the total absence of IgE can be stimulated to secrete with bivalent antireceptor antibodies [11].

II NEW STUDIES

We shall now describe two studies recently completed in our laboratory which have used chemical cross-linking procedures as a probe for studying how receptor aggregation may mediate mast cell (or basophil) secretion [12,13]. The reagents used in these studies (Fig. 1) are somewhat more useful than the one traditionally used by immunologists – bisdiazobenzidine. The latter compound is sufficiently carcinogenic to make its use banned in the United States; in addition, the newer reagents are more stable, specific, and versatile [14]. The versatility arises because one can vary the length between the reactive groups as well as prepare analogs which can be cleaved under mild conditions. The latter property has been particularly useful for some of our work.