Trivalent antigens for degranulation of mast cells.

Degranulation of basophils and mast cells plays a central role in allergic reactions. Degranulation is a response to cell surface receptor aggregation caused by association of receptors with antibodies bound to multivalent antigens. Tools used in studying this process have included small-molecule divalent antigens, but they suffer from weak signaling apparently due to small aggregate size. We have prepared trivalent antigens that allow formation of larger aggregates and potent responses from mast cells.

Selective inhibition of the Fc epsilon RI-induced de novo synthesis of mediators by an inhibitory receptor.

Aggregation of the type 1 Fc-epsilon receptors (Fc-epsilon-RI) on mast cells initiates a network of biochemical processes culminating in secretion of both granule-stored and de novo-synthesized inflammatory mediators. A strict control of this response is obviously a necessity; nevertheless, this regulation is hardly characterized. Here we report that a prototype inhibitory receptor, the mast cell function-associated antigen (MAFA), selectively regulates the Fc-epsilon-RI stimulus-response coupling network and the subsequent de novo production and secretion of inflammatory mediators. Specifically, MAFA suppresses the PLC-gamma2-[Ca2+]i, Raf-1-Erk1/2, and PKC-p38 coupling pathways, while the Fyn-Gab2-mediated activation of PKB and Jnk is essentially unaffected. Hence, the activities of several transcription/nuclear factors for inflammatory mediators (NF-kappaB, NFAT) are markedly reduced, while those of others (Jun, Fos, Fra, p90rsk) are unaltered. This results in a selective inhibition of gene transcription of cytokines including IL-1beta, IL-4, IL-8, and IL-10, while that of TNF-alpha, MCP-1, IL-3, IL-5, or IL-13 remains unaffected. Taken together, these results illustrate the capacity of an immunoreceptor tyrosine-based inhibitory motif-containing receptor to cause tight and specific control of the production and secretion of inflammatory mediators by mast cells.

Co-clustering activating and inhibitory receptors: impact at varying expression levels of the latter.

Clustering the mast cell function-associated antigen (MAFA) has earlier been shown to inhibit mast cells' secretory response to the type 1 Fcepsilon receptor (FcepsilonRI) stimulus. MAFA is a type II membrane glycoprotein first identified on rat mast cells and contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytosolic domain. This inhibition is induced already upon clustering MAFA alone. Still, the inhibitory capacity of MAFA-FcepsilonRI co-clustering has recently been characterized and co-clustered MAFA molecules were found to exhibit a markedly higher inhibition capacity than MAFA-clusters alone. We have now compared the inhibitory capacity of FcepsilonRI co-clustered MAFA on the secretory response of rat mucosal-type mast cells (RBL-2H3 line) expressing different levels of this inhibitory protein. Reacting these cells carrying an IgE class, 2,4 dinitrophenyl (DNP)-specific monoclonal antibody with DNP-conjugated F(ab')2 fragments of non-specific polyclonal mouse IgG causes clustering of the FcepsilonRI-IgE. Reaction of these cells with DNP-conjugated F(ab')2 fragments of the MAFA-specific, monoclonal antibody G63 co-aggregates MAFA together with the FcepsilonRI-IgE thereby producing FcepsilonRI-IgE-MAFA co-clusters. Results of measurements of the secretory responses of RBL-2H3 cells expressing higher or lower MAFA levels than those of unmodified cells provided further support to the notion that co-clustered MAFA molecules exhibit a markedly higher inhibition capacity than MAFA-clusters alone. The molecular basis for this enhanced inhibition is most probably the increased concentration of the inhibitory cell components in the immediate proximity of the co-clustered FcepsilonRI-MAFA.

Regulation of mast cells' secretory response by co-clustering the Type 1 Fcepsilon receptor with the mast cell function-associated antigen.

The mast cell function-associated antigen (MAFA) is a type II membrane glycoprotein first identified on rat mast cells and basophils. Clustering MAFA inhibits these cells' secretory response to the type 1 Fcepsilon receptor (FcepsilonRI) stimulus. To quantitatively characterize this inhibition and its dependence on MAFA-FcepsilonRI co-clustering, we investigated the secretory response of rat mucosal-type mast cells of the RBL 2H3 line carrying an IgE class, 2,4 dinitrophenyl (DNP) specific monoclonal antibody to DNP-conjugated Fab and F(ab')(2) fragments of (1) mouse IgG, and (2) of the MAFA-specific, monoclonal antibody G63. The first reagent clusters FcepsilonRI-IgE complexes into oligomers by reacting with the DNP residues. The DNP conjugated G63 Fab and F(ab')(2) fragments, additionally aggregate MAFA and form FcepsilonRI-IgE-MAFA co-clusters. All experiments using these ligands were performed in the absence or presence of an excess of intact mAb G63, which clusters MAFA molecules. Empirical Hill functions were used to relate the secretory response of mast cells to the equilibrium concentrations of FcepsilonRI-IgE or MAFA clusters and co-clusters calculated as function of the employed ligands concentrations. This analysis of the experimental results indicates that co-clustered MAFA molecules have a markedly higher inhibitory capacity than MAFA-clusters alone. The molecular basis of the enhanced inhibition observed upon co-clustering MAFA with the FcepsilonRI is most probably the increased concentration of the inhibitory cell components in the immediate proximity of the activation coupling elements.

Interaction of a monoclonal IgE-specific antibody with cell-surface IgE-Fc epsilon RI: characterization of equilibrium binding and secretory response.

Aggregation of FcepsilonRI, the high-affinity cell-surface receptor for IgE antibody, is required for degranulation of basophils and mast cells, but not all receptor aggregates elicit this cellular response. The stereochemical constraints on clusters of FcepsilonRI that are able to signal cellular responses, such as degranulation, have yet to be fully defined. To improve our understanding of the properties of FcepsilonRI aggregates that influence receptor signaling, we have studied the interaction of 23G3, a rat IgG(1)(kappa) IgE-specific monoclonal antibody, with IgE-FcepsilonRI complexes on rat mucosal-type mast cells (RBL-2H3 line). We find that 23G3 is a potent secretagogue. This property and the structural features of 23G3 (two symmetrically arrayed IgE-specific binding sites) make 23G3 a potentially valuable reagent for investigating the relationship between FcepsilonRI clustering and FcepsilonRI-mediated signaling events. To develop a mathematical model of 23G3-induced aggregation of FcepsilonRI, we used fluorimetry and flow cytometry to quantitatively monitor equilibrium binding of FITC-labeled 23G3 intact Ab and its Fab' fragment to cell-surface IgE. The results indicate that IgE bound to FcepsilonRI expresses two epitopes for 23G3 binding; that 23G3 binds IgE resident on the cell surface with negative cooperativity; and that 23G3 appears to induce mostly but not exclusively noncyclic dimeric aggregates of FcepsilonRI. There is no simple relationship between receptor aggregation at equilibrium and the degranulation response. Further studies are needed to establish how 23G3-induced aggregation of IgE-FcepsilonRI correlates with cellular responses.