Molecules, Cells, and Parasites in Immunology

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MOLECULAR MECHANISMS OF ANTIBODY ACTION: USE OF CROSS-LINKING REAGENTS*

HENRY METZGER, HELEN HARTMANN, DAVID HOLOWKA and CLARE FEWTRELL,     Section on Chemical Immunology, Arthritis and Rheumatism Branch, National Institute of Arthritis Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

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.

FIG. 1 Chemical cross-linking reagents used in the experiments discussed in this paper. The chemistry of these reagents is reviewed in Ref. 14.

A Effect of IgE Oligomer Size in the Triggering of Mast Cells

The first study made use of cross-linking reagents to analyze the extent of receptor aggregation that is required for optimal stimulation of mast cell degranulation. Our original work in this area involved the use of dimethylsuberimidate (DMS) to prepare well-defined stable oligomers of rat IgE [15]. These were then used to elicit passive cutaneous anaphylactic reactions in the rat. These experiments led us to conclude that dimers of IgE could generate so-called ‘unit signals’. It might take many such signals to trigger a cell completely, but the individual signal could be generated by as small a unit as a receptor dimer.

What was not entirely clear from these studies was whether higher oligomers might yield stronger signals. While the in vivo data suggested that they did not, the in vitro data were less definitive. Furthermore, a recent investigation using oligomers of human IgE on human peripheral blood basophils studied in vitro has suggested that all oligomers may not be equal [A. Sobotka and L. Lichtenstein, submitted for publication].

In an attempt to resolve this question in our system, rat IgE was reacted with DMS and separated into monomers, dimers, trimers, and higher oligomers by gel filtration (Fig. 2A). We principally studied rat basophilic leukemia (RBL) cells [16], which can be grown as solid tumors in animals or in cell culture and are a unique resource for detailed structural analyses such as those described below. The cells were allowed to incorporate radioactive serotonin and the stimulated release of the latter by the oligomers was then assayed (Fig. 2B). Our experiments will be published in complete form elsewhere [12], and only the most significant findings will be summarized here:

FIG. 2 Separation of IgE oligomers and assay of their relative activities. A A trace amount of iodinated IgE was added to nonradioactive IgE (21 mg, 60 mg/ml) and the solution reacted with a 16-fold molar excess of DMS for 1 h at 30°C in 0.2 M Tris buffer, pH 8.6. The preparation was applied to sequential Sephadex G-200 and Ultrogel Ac A 22 columns, and the radioactivity in the effluent was measured. The peaks are labeled to indicate the relative effluent volumes for monomer (M), dimer (D), trimer (T), and higher oligomers (H). B Analysis of column fractions (numbers on the right) for their capacity to stimulate the release of incorporated tritiated serotonin (³H.5HT) from RBL cells. The test material was incubated with 2 × 10⁶ cells/ml for 1 h at 37°C. The medium contained 1.8 mM Ca++ but not D2O.

(1) Dimers by themselves were adequate to stimulate secretion of at least a portion of the releasable serotonin, although this was often barely observable in the absence of D2O (Fig. 2B).

(2) Trimers were more active than dimers, while the higher oligomers were even more effective (Fig. 2B, Table I).

TABLE I

Serotonin Release by Oligomers of IgEa

aNet secretion equalled 38 % of releasable mediator. This table is adapted from Ref. 12. The 2H3 secreting subline of RBL cells was incubated with tritiated serotonin and after washing was reacted with varying concentrations of monomeric or oligomeric IgE in the presence of 30 0/0 D2O in a Ca++-containing salt solution. The percent of cell-bound labeled serotonin released in 1 h was measured and the net release calculated by subtraction of the percent release obtained with unstimulated cells. The amount of each bound oligomer was calculated by using data from binding studies (to determine the relative amounts of each oligomer in each preparation) and the previously measured kinetic constants for the system. These calculations and the assumptions on which they are based are described fully in Ref. 12.

bEn, m is an oligomer which contains n molecules of IgE per molecule of oligomer. Because of inactivation due to the chemical cross-linking, only some of the individual IgE molecules are capable of binding to the receptor. The active IgE molecules are denoted by the subscript m, and these values are based upon binding studies performed with each preparation.

(3) Relatively few higher oligomers were required to stimulate the secretion of a substantial fraction of the releasable mediator.

(4) The shapes of the dose-response curves to the various oligomers were very different. In addition, it seemed as though some of the serotonin which could be released by the higher oligomers was completely resistant to release by dimers. In other words, the higher oligomers appear to give not simply more signals than dimers, but different