Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation.

Abstract

. The mAb AA4 binds to novel derivatives of the ganglioside GD1b on rat basophilic leukemia (RBL-2H3) cells . Some of the gangliosides are located close to the high affinity IgE receptor (FCcRI), and binding of mAb AA4 inhibits FCERI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes . They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles . Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC . However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the FcERI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4°C or in the absence of extracellular calcium . Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events .phate by mobilizing intracellular calcium pools . Receptor activation also results in calcium influx, activation of phospholipase A2, the phosphorylation of a number of intracellular proteins (7), and the rearrangement of the cytoskeleton before mediator release (35,37,48) .The rat basophilic leukemia cell line, RBL-2113, has been widely used as a model to study 48,49) . A number ofmAbs have been raised against surface molecules on these cells (3,5,29,50) . These mAb were selected for their ability to either stimulate or inhibit histamine release in the RBL-2113 cells. The majority of the antibodies were directed against the Fcc-RI ; however, two antibodies, mAb ADl and AA4, bind other cell surface components . The mAb ADl binds to a novel 50-60-kD protein on the surface of the RBL-2113 cells and modulates histamine release (31). The other antibody, mAb AA4, binds to unique a-galactosyl derivatives of the ganglioside GD, b (18). These gangliosides are closely related to the FceRl, both spatially and functionally (5) . Binding ofboth the intact mAb AA4 and its Fab fragments to cells inhibits the binding

Mast Cell-specific Gangliosides and Fc∊RI Follow the Same Endocytic Pathway From Lipid Rafts in RBL-2H3 Cells

“…In unstimulated cells, although MAb AA4 binding partially activates RBL-2H3 cells (Oliver et al 1992), little internalization of the gangliosides occurs.…”

“…When the gangliosides are crosslinked by MAb AA4, the mast cells are partially activated but do not degranulate (Guo et al 1989;Oliver et al 1992;Swaim et al 1994). The gangliosides recognized by MAb AA4 coimmunoprecipitate with the receptor (Stephan et al 1991).…”

Alterations in Granule Matrix and Cell Surface of Focal Adhesion Kinase-Deficient Mast Cells

“…Mouse IL-3 and stem cell factor (SCF) were from Biosource International (Camarillo, CA). mAbs AA4 and the anti-Fc⑀RI␤ Abs have been described previously (24,25). Chondroitin 4-sulfate and dermatan sulfate were purchased from Miles Laboratories (Elkhart, IN).…”

“…By 4 wk, 98 -99% of cells in all the cultures had the distinctive surface markers for mast cells: by immunolabeling, the cells all expressed the ␤ subunit of Fc⑀RI (99.6 Ϯ 0.6%), and the rodent mast cell-specific gangliosides (24) recognized by mAb AA4 (98.7 Ϯ 0.5%) and bound IgE (99.3 Ϯ 0.7%). By FACS analysis, the binding of IgE was similar on cells from each of the three genotypes.…”

Differential expression of integrin subunits on adherent and nonadherent mast cells

“…However, studies of mast cell adhesion in situ have been limited by lack of mast cellspecific markers, since mast cells in early stages of maturation cannot be differentiated from other cell types on the basis of their morphology alone. Using the monoclonal antibody (mAb) AA4 (18) that recognizes two derivatives of the ganglioside G D1b (19), which are unique to the surface of rodent mast cells (20), pure populations of mast cells can be isolated (21).…”