Antibody cross-linking of human CD9 and the high-affinity immunoglobulin E receptor stimulates secretion from transfected rat basophilic leukaemia cells

Abstract

Previous studies have shown that antibody cross-linking of the tetraspanin protein CD9 stimulates the degranulation of platelets and eosinophils, although the mechanism of activation is unclear. In this work we transfected human CD9 into the rat basophilic leukaemia (RBL-2H3) cell line and studied the stimulation of secretion from these cells in response to a panel of anti-CD9 antibodies. Intact immunoglobulin G1 (IgG1) antibodies activated transfected cells whereas F(ab')2 fragments of antibody and an intact IgG2a did not. Stimulation of secretion was inhibited by co-incubation with monomer murine immunoglobulin E (IgE) but not with an IgG1 isotype control, indicating that the response involves the endogenous high-affinity IgE receptor (FcepsilonRI). The anti-CD9 antibody activation curve was biphasic, and supraoptimal antibody concentrations stimulated little or no degranulation, indicating that multivalent binding of human CD9 molecules is necessary for the formation of an active complex with rat FcepsilonRI. Immunoprecipitation of FcepsilonRI under mild detergent conditions co-precipitated CD9, suggesting the presence of pre-existing complexes of CD9 and FcepsilonRI that could be activated by antibody cross-linking. These data are further evidence that tetraspanins are involved in FcepsilonRI signalling and may reflect the participation of tetraspanins in the formation of complexes with other membrane proteins that use components of Fc receptors for signal transduction.

Figure 1

Antibody binding and activation of rat basophilic leukaemia (RBL-2H3) cells transfected with human CD9. RBL-2H3 cells transfected with human CD9 were incubated with anti-CD9 antibodies at 4° for measurement of binding by indirect immunofluorescence (squares, left axes). RBL-2H3 cells labelled with [³H]5-hydroxytryptamine (5-HT) were incubated with antibodies at 37° for the measurement of cell activation (triangles, right axes), as described in the Materials and methods. The binding data is the mean of one experiment performed in duplicate, typical of two similar experiments. Cell activation data is expressed as the mean ± SEM of three experiments performed in duplicate. The open symbols on (a) show the binding and cell activation measured using F(ab′)₂ fragments of ALMA 1.

Figure 2

Cell activation by anti-CD9 antibodies is inhibited by murine immunoglobulin E (IgE) but not by murine immunoglobulin G1 (IgG1). Rat basophilic leukaemia (RBL-2H3) cells labelled with [³H]5-hydroxytryptamine (5-HT) were co-incubated with different concentrations of either murine IgE or IgG1 and 67 nm anti-CD9 antibody (ALMA 1), and cell activation was measured as described in the Materials and methods. The results show 5-HT release relative to the effect of ALMA 1 alone (= 100) and are expressed as the mean ± SEM of three experiments performed in duplicate.

Figure 3

Co-precipitation of CD9 and FcεRI from solubilized cell membranes. Immunoglobulin E (IgE)/FcεRI complexes were immunoprecipitated by anti-κ light-chain antibodies (Ab; 0–100 µg) immobilized on protein A–agarose beads from either resting rat basophilic leukaemia (RBL) cells (a) or from cells activated with polyvalent antigen (b) prior to solublization in 1% CHAPS. Western blots were developed using ALMA 1 anti-CD9 monoclonal antibodies (mAb) and visualized by enhanced chemiluminescence. The results shown are representative of four similar experiments.

Figure 4

Models of anti-CD9-mediated Fc receptor activation. Supraoptimal concentrations of anti-CD9 immunoglobulin G1 (IgG1) inhibit deganulation of transfected rat basophilic leukaemia (RBL) cells. In (a), increasing competition for limiting CD9 epitopes decreases the proportion of complexes between CD9 and FcεRI that are capable of stimulating degranulation. In (b), the active complex is a large network of Fc receptors linked by CD9 dimers. The size of these networks is diminished at high antibody concentrations, leading to inhibition of the antibody response.