No 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

Background In human deciduous teeth, odontoclastic resorption takes place at the pulpal surface of the coronal dentine prior to shedding, and this resorption shows clear time‐related histological changes (Sahara et al., 1992). Methods Using this phenomenon as an observation system, we examined the cytodifferentiation of human odontoclasts by light and electron microscopy. For a histochemical marker of odontoclast differentiation and function, tartrate‐resistant acid phosphatase (TRAP) activity was determined by light and electron microscopic enzyme histochemistry. Results As root resorption neared completion, TRAP‐positive mononuclear cells were initially detected in the pulp chamber. They had abundant mitochondria, small lysosomes, and moderately developed rough endoplasmic reticulum throughout their cytoplasm. In these mononuclear cells, TRAP activity was localized in compartments of the biosynthetic pathway, i.e., in cisternae of the endoplasmic reticulum and Golgi lamellae, as well as small lysosomes. The TRAP‐positive mononuclear cells first made contact with the predentine surface by their elongated cellular processes. After attachment, they spread out along the predentine surface and developed specialized membrane structures, clear zones, and ruffled borders. Next, they fused with each other on the predentine surface and formed typical multinucleate odontoclasts. After termination of their resorption function, the odontoclasts lost their ruffled borders and became detached from the resorbed surface. Most of the detached odontoclasts had numerous large pale vacuoles and secondary lysosomes and appeared to be in the process of degeneration. Conclusions The present study demonstrates that: (1) odontoclasts differentiated from TRAP‐positive mononuclear cells, which presumably originate from circulating progenitor cells, (2) membrane specialization of odontoclasts, i.e., development of a clear zone and ruffled border, is induced following their contact with the resorption surface, (3) multinucleation of odontoclasts takes place only after their attachment to the resorption surface, (4) mature multinucleate odontoclasts can resorb predentine as well as dentine in the same way as osteoclasts resorb bone, and (5) at the end of the resorption, odontoclasts gradually lose their ruffled borders and become detached from the resorbed surface. © 1996 Wiley‐Liss, Inc.

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological changes after IgE-mediated secretion. The present study was undertaken to examine if the morphological changes were dependent on activation of the Fc receptor. Therefore, the cells were stimulated to release histamine by two different mechanisms: activation of the Fc receptor by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE-or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement ofthe cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed on the surface of the cells undergoing IgEmediated release. The surface changes were not as pronounced with the ionophore. The distribution of the cytoskeletal elements was examined by immunofluorescence using FI'It-phalloidin and antibodies against vimentin and tubulin. In unstimulated cells stain was localized at the cell