Early activation and cell cycle entry of resting B cells after Fab-anti-Ig treatment: role of receptor crosslinking.
Comparison of the effect of goat anti-rabbit Ig (GARIg) and its monovalent fragment (Fab-GARIg) demonstrates that surface Ig (sIg) crosslinking is not necessary to effect G0 to G1 transition in rabbit peripheral blood B cells but is required for induction of DNA synthesis. Five micrograms per milliliter or more of GARIg is sufficient to induce DNA synthesis but up to 50 micrograms/ml of Fab-GARIg is not. However, the monovalent reagent induces microscopically observable cytoplasmic and nuclear changes (blast transformation) in a dose-dependent manner. These differ qualitatively and quantitatively from the morphological changes seen with comparable doses of GARIg; Fab anti-Ig produces "small blasts" whereas complete GARIg induces large blasts. The monovalent reagent, in a wide range of concentrations, is as effective as the complete antibody in modulating sIg from rabbit B cells. Fab-GARIg treatment modulates sIg in a biphasic manner. It clears the high-density sIg within 5 min, whereas the remaining low-density receptors disappear after 4 hr. Cytosolic protein kinase C levels decline equally after treatment with either Fab-GARIg or whole anti-Ig. RNA synthesis, as measured by [3H]uridine incorporation, increases for the first 12 hr in cells activated with either reagent. It declines to basal levels in Fab-GARIg stimulated cells, but a continuous increase occurs in cells stimulated with 5 and 50 micrograms/ml of complete antibody. Simultaneous addition of 50 micrograms/ml Fab-GARIg with 5 microgram/ml of GARIg causes greater RNA synthesis for 12 hr after stimulation than is caused by GARIg alone. After 12 hr the monovalent reagent has an inhibitory effect on RNA synthesis. Fluorescence-activated cell sorter analysis of acridine orange-stained cells shows that Fab anti-Ig-stimulated cells have higher RNA content than resting cells, but lower than GARIg-activated cells. These findings suggest that rabbit B cells can be activated from the G0 stage of cell cycle to G1 by monovalent anti-Ig reagents but further cell cycle progression requires maintenance signals provided by receptor crosslinking. The implications of these results for B cell activation signalling are discussed in the context of the floating receptor model.
Lagoo
A
A
Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, Houston 77025.
Sell
S
S
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D002453:Cell Cycle; D004261:DNA Replication; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D008213:Lymphocyte Activation; D008297:Male; D011485:Protein Binding; D011493:Protein Kinase C / Q000032:analysis; D012313:RNA / Q000096:biosynthesis; D011817:Rabbits; D011947:Receptors, Antigen, B-Cell / Q000276:immunology*; D011961:Receptors, Fc / Q000276:immunology*; D015398:Signal Transduction
D000888:Antibodies, Anti-Idiotypic; D007140:Immunoglobulin Fab Fragments; D011947:Receptors, Antigen, B-Cell; D011961:Receptors, Fc; D012313:RNA; D011493:Protein Kinase C