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Previous studies of secretion from basophils have demonstrated the phenomenon called nonspecific desensitization, the ability of one IgE-mediated stimulus to alter the cell's response to other non-cross-reacting IgE-mediated stimuli, and a process that would modify phosphatidylinositol 3,4,5-phosphate levels was speculated to be responsible for nonspecific desensitization. The current studies examined the changes and characteristics of SHIP1 phosphorylation as a measure of SHIP1 participation in the reaction. Based on the earlier studies, two predictions were made that were not observed. First, the kinetics of SHIP1 phosphorylation were similar to reaction kinetics of other early signals and returned to resting levels while nonspecific desensitization remained. Second, in contrast to an expected exaggerated SHIP phosphorylation, cells in a state of nonspecific desensitization showed reduced SHIP phosphorylation (compared with cells not previously exposed to a non-cross-reacting Ag). Discordant with expectations concerning partial recovery from nonspecific desensitization, treatment of cells with DNP-lysine to dissociate bound DNP-HSA, either enhanced or had no effect on SHIP phosphorylation following a second Ag. These experiments also showed a form of desensitization that persisted despite dissociation of the desensitizing Ag. Recent studies and the results of these studies suggest that loss of early signaling components like syk kinase may account for some of the effects of nonspecific desensitization and result in a form of immunological memory of prior stimulation. Taken together, the various characteristics of SHIP phosphorylation were not consistent with expectations for a signaling element involved in nonspecific desensitization, but instead one which itself undergoes nonspecific desensitization.

Multivalent DNP-BSA is commonly used to cross-link anti-DNP IgE bound to Fc epsilon RI to stimulate cellular responses, although key features of the binding process are unknown. Fluorescence quenching can be used to study the kinetics of DNP-BSA binding to FITC-IgE. We observe that DNP-BSA binds more slowly to IgE than does an equimolar amount of a monovalent DNP ligand, suggesting that the average effective number of DNP groups per BSA is less than one. The binding data are well described by a transient hapten exposure model in which most of the DNP groups are unavailable for binding but have some probability of becoming exposed and available for binding during the time of the binding measurement. Additional experiments indicate that, for suboptimal to optimal concentrations of DNP-BSA, most of the FITC fluorescence quenching on the cell surface is due to cross-linking events. With these concentrations at 15 degrees C, the kinetics of FITC fluorescence quenching by DNP-BSA correlates with the kinetics of DNP-BSA-stimulated tyrosine phosphorylation of Fc epsilon RI. At 35 degrees C, the phosphorylation kinetics are biphasic during the time period in which cross-linking continues to increase. Our results establish a quantitative relationship between the time-course for cross-linking by multivalent Ag and Fc epsilon RI-mediated signaling, and they provide the means to predict the kinetics of cross-linking under a wide variety of conditions.

The shift from IgM anti-hapten antibody production to IgG anti-hapten antibody production, subsequent to challenge with a T-dependent conjugate, is inhibited in mice by preimmunization with the T-independent DNP-LE conjugate. The results suggest that DNP on levan triggers off 19A T-independent anti-hapten precursors and paralyzes 7S T-dependent anti-hapten precursors.

A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases of antigen-presenting cells causes a profound inhibition of both the proteolytic degradation and the presentation of the synthetic antigen dinitrophenyl-poly-L-lysine. In contrast, the presentation of another synthetic antigen, the copolymer of L-glutamic acid and L-alanine, was enhanced by the same inhibitor. Another inhibitor, pepstatin A, which selectively blocks aspartic proteinases, did not block the presentation of dinitrophenyl-poly-L-lysine. The results identify cysteine proteinases, probably lysosomal, as one of the groups of enzymes involved in antigen processing.

In this study we have investigated the role played by the mode of attachment of the hapten on autologous IgG in the induction of carrier-determined tolerance. Three conjugates of the dinitrophenyl hapten and mouse gamma-globulin were prepared: 1) DNP-epsilon-lysine-MGG, 2) DNP-azo-MGG (which binds to histidine and tyrosine), 3) DNP-must-MGG (which binds to the carboxyl groups of glutamic or aspartic acid). DNP was also bound to the immunogenic carrier keyhole limpet hemocyanin (KLH) by the three different linkages. The three DNP mouse-gamma-globulin conjugates were used to induce tolerance and immediately thereafter the animals were immunized with various DNP-KLH conjugates. Antibody response was tested by a modiciation of the Jerne plaque assay. It was found that DNP-epsilon-lysine-MGG induced tolerance to both DNP-epsilon-lysine-KLH and to DNP-azo-KLH. In contrast, neither DNP-azo-MGG nor DNP-must-MGG induced tolerance to DNP-epsilon-lysine-KLH or to DNP-azo-KLH and DNP-must-KLH, respectively. Clearance studies showed no differences in the catabolic rate of the various hapten mouse gamma-globulin conjugates as compared to unconjugated MGG. However, although DNP-must-MGG and DNP-azo-MGG were not tolerogenic, they were immunogenic. In conclusion, this study demonstrated that not only the nature of the carrier but the mode of hapten binding to his carrier are critical for the induction of hapten specific tolerance.