Sastry V.S. Gollapudi

Enhanced phosphorylation of endogenous membrane proteins during induction of B lymphocyte proliferation.

Phosphorylation of endogenous proteins was assessed employing membrane preparations derived from splenocytes induced to proliferate in response to Sepharose linked anti-immunoglobulins. Time course studies revealed that enhanced protein phosphorylation was preceded by cell enlargement and was either followed by or closely related in time to the onset of DNA synthesis. Thus maximal enhancement of phosphorylation was initially observed at 24 h whereas cell enlargement was optimal at 16 h at a time when there was no enhancement in protein phosphorylation. Furthermore thymidine incorporation was maximal at 32 h and low at 24 h when phosphoprotein synthesis was maximally enhanced. Taken together, these results suggest that phosphorylation of endogenous membrane proteins may be involved in signalling entry of cells into S phase of the cell cycle.

Nonresponsiveness of immature B lymphocytes to anti-immunoglobulin is reversed by pronase

Splenic B cells are induced to proliferate upon culture with antibody having specificity for surface membrane immunoglobulins. Cells treated with pronase, washed and then cultured with antibody, exhibited a greater than 5-fold enhancement of DNA synthesis whereas pronase treatment, per se, was not mitogenic. The pronase effect exhibited specificity in that the induction of proliferation with either lipopolysaccharide or dextran sulfate was not enhanced by prior enzyme treatment. Cells from mice at two weeks of age which essentially do not show a proliferative response to antibody become responsive subsequent to pronase treatment. These results are interpreted to suggest a possible growth regulatory role for the pronase sensitive surface membrane component.

Anti-immunoglobulin-induced proliferation of B cells: parallelism in the inhibition by chloroquine, monensin and immunoglobulin

Proliferation of rabbit lymphocytes was induced with goat anti-rabbit immunoglobulin. Chloroquine and monensin, known to inhibit internalization-related events, yielded inhibition of proliferation that paralleled the inhibition by a specific competitive ligand, rabbit immunoglobulin (IgG), whereas inhibition by puromycin did not. Moreover, virtually all of the cells that can be activated in freshly isolated populations adhered to anti-immunoglobulin-coated Petri plates, whereas all of the activatable population was recovered in the non-adherent fraction after a brief incubation of the cells with anti-immunoglobulin to induce internalization of surface membrane immunoglobulin. Using immunofluorescence it was further observed that monensin and chloroquine inhibit the reappearance of surface immunoglobulins on the cell surface to some extent subsequent to their removal induced by anti-immunoglobulin.

Novel differences in the characteristics of spleen and peripheral blood lymphocytes activated by anti-immunoglobulin

A comparison of splenocytes and peripheral blood lymphocytes with regard to activation by anti-immunoglobulin revealed distinctions in the behavior of the populations. Thus, a three- to fourfold greater concentration of anti-immunoglobulin was required to achieve one-half maximal activation of splenocytes than that required for peripheral blood lymphocytes. This difference cannot be accounted for on the basis of binding data. That is, although the binding of 125I-labeled anti-immunoglobulin was found to be slightly greater for peripheral blood lymphocytes than for spleen cells, the concentration required for one-half maximal binding was essentially the same for the two populations. Other differences between the populations were also observed. For example, splenocytes but not peripheral blood lymphocytes were substantially inhibited in terms of activation by anti-immunoglobulin subsequent to the absorption and elution of cells from anti-immunoglobulin-coated petri plates at 4 degrees C, despite the fact that the recovery of cells in general and B cells in particular following absorption and elution from anti-immunoglobulin coated plates at 4 degrees C was essentially the same for both populations.

Non-identity of the triggering of activation of lymphocytes by anti-immunoglobulin and by antigen

Abstract Goat anti-rabbit immunoglobulins trigger DNA synthesis in lymph node cell populations. Premature removal of anti-immunoglobulin at three hours or even as late as the 31st hour of a 48 hour culture period resulted in a markedly diminished response. In contrast, within three hours after addition of antigen to cells its removal was without effect on the extent of activation. Similar results were obtained by using the delayed addition of competing ligands. More importantly, removal of anti-immunoglobulin from purified B cells at three hours resulted in virtually complete inhibition of activation whereas removal of antigen at three hours was without effect. Possible mechanisms to account for the difference in triggering of B cells are discussed.

Mechanism for signaling initiation and termination of B lymphocyte proliferation induced by anti-immunoglobulin

SummarySeveral lines of evidence were explored which taken together indicate that both the initiation and the termination signal for activation of rabbit lymphocytes to synthesize DNA in response to anti-rabbit immunoglobulin occurs at an immunoglobulin receptor on the surface membrane of B cells. Thus, the premature removal of anti-rabbit immunoglobulin by simply washing the cells at the 31st hour of a 48-h incubation period caused a 60% decrease in the induction of DNA synthesis. The addition of rabbit immunoglobulin to compete with B cell surface immunoglobulin for the combining sites on anti-rabbit immunoglobulin yielded a markedly diminished activation. Addition of rabbit immunoglobulin even during the latter part of a pulse label period with [3H]-thymidine was sufficient to result in reduced activation. Finally, insoluble anti-rabbit immunoglobulin at the same nominal concentration as soluble anti-rabbit immunoglobulin also was effective in inducing cells to DNA synthesis. However, it is noteworthy that under the incubation conditions used it was not possible to derive a soluble component from insoluble anti-rabbit immunoglobulin which stimulated DNA synthesis. These data have been interpreted to indicate a need for a continuous surface presence of anti-rabbit immunoglobulin to stimulate activation in a process that is not dependent upon internalization of anti-rabbit immunoglobulin.

A novel early effect of concanavalin A on thymocytes

Abstract Concanavalin A, added to freshly isolated rabbit thymocytes, markedly enhanced the extracellular appearance of non-immunoglobulin proteins. Time course studies revealed that the onset of enhancement occurred virtually without delay. The effect appeared to be restricted only to certain of the thymus-derived cells because thymocytes obtained from rabbits treated with hydrocortisone, as well as splenocytes derived from untreated rabbits essentially did not exhibit the enhancement. Stimulation by concanavalin A was specific in that pokeweed mitogen and lipopolysaccharide were without effect and also in that α-methyl-mannoside, but not galactose, abrogated the concanavalin A-mediated enhancement. Experiments with mouse thymocytes demonstrated that the cells which responded to concanavalin A were primarily cells that bear the θ-antigen on their surface (T-cells).

T cell induction of precursor B cells: temporal separation of helper T cell functional activities.

SummaryThe temporal relationships involved in T cell induction of immunoglobulin-secreting B cells have been studied by employing a pulse label technique, in vitro. It was shown that addition of rabbit thymocytes or splenic T cells to B cell-enriched splenocyte populations at the time of initiation of cultures resulted in a marked enhancement in induction of immunoglobulin-secreting cells. However, even a two-hour delay in the addition of thymus cells was sufficient to reduce substantially the extent of induction when measured 70 hours later. Besides this early requirement for thymocytes, a late requirement was also detectable. Thus, thymus cells and splenocyte populations upon being mixed, subsequent to being cultured separately for 72 hours, yielded a several-fold enhancement in [3H]-immunoglobulin secreted during the course of a 90-minute labeling period with [3H]-leucine. Moreover, both the early and late thymocyte effects were lost after treatment with anti-thymocyte serum and complement.The thymocyte-mediated enhancement of immunoglobulin secretion by splenocytes that occurs late in the induction process was detected with spleen cells cultured for two or three days but not with freshly-isolated splenocytes. Although the rate of appearance of extracellular immunoglobulins was markedly enhanced by fresh thymus cells, the rate of appearance of intracellular immunoglobulins in such spleen cells was unchanged. The secretion-stimulating (secretagogue) activity of thymocytes appeared to be specific in that thymus cells were without effect on the rate of secretion of serum albumin by liver cells.In regard to the induction of immunoglobulin-secreting cells, both B and T cell-enriched populations were sensitive to mitomycin C treatment performed before initiation of cell culture, indicating that not only B cells but also T cells undergo some form of differentiation or maturation prior to functioning in the induction of immunoglobulin-producing cells. It should be noted in this context that the late T cell requirement was unaffected by prior mitomycin C treatment of thymocytes. On the other hand, thymocytes heated at 60°C for 5 minutes did not enhance immunoglobulin secretion when added at any time and the late thymocyte requirement could not be replaced with medium in which thymocytes had been previously cultured.