Differential effect of DHEA on mitogen-induced proliferation of T and B lymphocytes

Dehydroepiandrosterone (DHEA) is the predominant steroid hormone secreted by adrenal gland, and it has been proposed in recent years that DHEA has significant effects on immune function. We investigated the effect of DHEA (1 x 10(-5) - 1 x 10(-8)M) on proliferation of human T cells and B cells and on immunoglobulin production, a representative function of B cells. High doses of DHEA (1 x 10(-5)) significantly inhibited proliferation of peripheral blood mononuclear cells (PBMCs) and T cells induced by T cell mitogens hemagglutinin (PHA) and concanavalin A (Con A). Proliferation of PBMCs induced by B cell mitogens pokeweed mitogen (PWM) was increased by 1 x 10(-7) - 1 x 10(-6)M DHEA. Proliferation of PBMCs and B cells induced by Staphylococcus aureus Cowan strain I (SAC) was not significantly changed at any concentrations of DHEA. However, a concentration of 1 x 10(-7)M DHEA tended to potentiate their proliferation. This study suggested that DHEA acted on T and B lymphocytes differentially in immune system.     

IgG subclass expression by human B lymphocytes and plasma cells: B lymphocytes precommitted to IgG subclass can be preferentially induced by polyclonal mitogens with T cell help

The human IgG subclasses expressed by circulating B lymphocytes, tissue plasma cells, and plasma cells generated from B cell precursors in response to the polyclonal mitogens LPS and PWM were examined by immunofluorescence using subclass-specific monoclonal antibodies. The subclass distribution observed for circulating B lymphocytes was IgG2 (48%) greater than IgG1 (40%) greater than IgG3 (8%) greater than IgG4 (1%), while the distribution among IgG plasma cells in bone marrow, blood, spleen, and tonsils was IgG1 (64%) greater than IgG2 (26%) greater than IgG3 (8%) greater than IgG4 (1%). Multiple IgG isotypes were not observed on B cells or in plasma cells. Although IgG plasma cell responses to both LPS and PWM were T cell dependent, the distributions of IgG subclasses elicited were strikingly different. In control and LPS-stimulated cultures of blood mononuclear cells, the induced plasma cells expressed the IgG subclass distribution: IgG2 greater than 80%, IgG1 less than 20%, IgG3 less than 1%, IgG4 less than 1%. In PWM-stimulated cultures, the subclass distribution, IgG1 approximately 65%, IgG2 approximately 25%, IgG3 approximately 7%, IgG4 approximately 1%, was in perfect concordance with the in vivo subclass distribution of IgG plasma cells. Selective inhibition of suppressor T cell activity by x-irradiation and mitomycin C treatment did not alter the IgG subclass distribution pattern induced by LPS and PWM. Monoclonal antibodies were used to deplete selectively the B cell precursors bearing IgG1, IgG2, or IgG3 before PWM stimulation of blood mononuclear cells. In each instance, a reduction was observed only in the subpopulation of plasma cells producing the homologous IgG subclass. The results indicate that T cells can preferentially influence the terminal differentiation of B cells that are precommitted to different IgG subclasses.     

Cross-linking of T cell mitogens: effects on B and T cell proliferation and immunoglobulin synthesis.

The T cell mitogens Pa-2, concanavalin A (con A) and its dimeric derivative succinyl-con A, were each cross-linked with the bifunctional reagent dimethyl suberimidate. Although the dose-response curves of these insoluble aggregated products were markedly changed from those of the soluble mitogens, each aggregate continued to stimulate DNA synthesis by murine thymus and T cells. Both aggregated Pa-2 and aggregated succinyl con A stimulated DNA synthesis by B cells from athymic (Nu/Nu) mice. Aggregated con A did not stimulate these cells and, like soluble con A, depressed the background incorporation of 3H-thymidine. Unlike soluble Pa-2, aggregated Pa-2 also greatly increased Ig production by both the B cell cultures and B + T cell cultures from normal (BALB/c) mice.     

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.     

Synthesis of sterol and phospholipid induced by the interaction of phytohemagglutinin and other mitogens with human lymphocytes and their relation to blastogenesis and DNA synthesis

Human peripheral blood lymphocytes (PBL) responded to phytohemagglutinin (PHA) and a variety of other mitogens by increased synthesis of sterol and phospholipid. This activity was established within 4–7 hr of the addition of mitogen and was dependent upon the binding of the ligand to the cell membrane. Sterol and phospholipid synthesis reached a peak at approximately 24 hr in association with blastogenic expansion of the lymphocyte membrane and initiation of DNA synthesis. Lipid synthesis and blast transformation occurred independently of replication of the genome since inhibition of DNA synthesis did not reduce the degree of blast transformation and lipid synthesis observed. However, inhibition of sterol synthesis using 20α-hydroxycholesterol resulted in decreased blastogenesis and DNA synthesis, demonstrating that early synthesis of lipid is important for these subsequent events. Human thymocytes responded to T-cell mitogens in the conventional manner as regards synthesis of lipid and blast transformation; however, they did not synthesize DNA. Possible reasons for this incomplete response are discussed. Several nonmitogenic agents which agglutinate lymphocytes were also found to initiate early increases in sterol and phospholipid synthesis, and the possible significance of this observation is considered.     

Selective responses of mouse T lymphocytes to different T mitogens and in mixed lymphocyte culture induced cytolysis reaction.

CBA spleen T lymphocytes were stimulated by the T mitogens concanavalin-A (Con-A), phytohemagglutinin (PHA), and leukoagglutinin (LA). On the 2nd to 3rd culture day the activated cells (blasts) were separated from the nonactivated cells (lymphocytes) by 1g velocity sedimentation. The lymphocytes which were not activated during the primary culture (lymphocyte fraction from the velocity sedimentation) were then stimulated by the same mitogens or in one-way MLC to DBA/2 m, and tested for relevant target lysis after MLC stimulation. Primary stimulation with Con-A abolished the responses to Con-A, to PHA, and to LA, whereas primary stimulation with PHA or with LA abolished the responses to these mitogens but left behind a considerable Con-A response. Stimulation with any one of the listed T mitogens did not significantly affect the MLC responses. While primary stimulation with Con-A abolished the relevant target cell lysis after MLC stimulation, primary stimulation with PHA or with LA reduced it only slightly. Assuming that the various mitogens stimulate separate subpopulations of T cells, the results seem to indicate that the Con-A-responsive population includes the PHA- and LA-responsive populations but not the MLC-responsive population. It also appears that the T cells generated to killer cells during MLC are mainly confined to the concanavalin-responsive population.     

Uridine labeling of human lymphocytes: Differential uptake by T and B cells

Human peripheral blood lymphocytes were labeled with [³H]uridine and separated into immunoglobulin (Ig)-positive-enriched and Ig-negative populations by rosetting and Ficoll sedimentation. The Ig-negative (T cell-rich) fraction was found to be more heavily labeled than the B cell-enriched population, in agreement with previous results in rats. Combined autoradiography and rosetting confirmed the differential uridine labeling of T and B cells. Incorporation of cytidine and adenosine by T and B cell-enriched populations showed similar but less dramatic differential labeling.     

Optimal conditions for blastogenic response of peripheral blood lymphocytes to T and B cell mitogens in cynomolgus monkeys

Optimal conditions for the mitogen-induced proliferation of T and B lymphocytes of cynomolgus monkeys were determined. The T cell mitogens concanavalin A and phytohemagglutinin, at concentrations of 1.25–10 μg/ml and 1.25–10 μg/ml, respectively, and the T and B cell mitogen pokeweed mitogen, at concentrations of 0.2–10 μg/ml, induced high lymphoproliferative responses, the average stimulation index (SI) being 34–93. Since suitable mitogens have not been reported for monkey B cells, we tested three types of lipopolysaccharide (LPS): two derived from Escherichia coli—one extracted with phenol and one extracted with trichloroacetic acid (TCA); and one derived from Salmonella typhimurium, extracted with phenol. All three LPS had a high mitogenic effect for monkey lymphocytes, with SI of 2.3–6.4. The highest response was observed for 25 μg/ml of Salmonella LPS, and the addition of trypsin to the culture augmented the proliferative response of low or non-responder lymphocytes. © 1994 Wiley-Liss, Inc.     

Effect of alpha-thrombin on DNA synthesis and excretion by lymphocytes

Thrombin-activated spleen lymphocytes of mice synthesize 1.5 times more DNA than unstimulated cells. The proportions of palindromic sequences in the newly-synthesized nuclear DNA of control and thrombin-stimulated lymphocytes are 1.1% and 0.5%, respectively. Both the control and thrombin-stimulated lymphocytes release about 10-12% of the newly-synthesized cellular DNA into the culture medium. It is shown that the newly-synthesized DNA of viable, rather than non-viable cells, is the source of extracellular DNA.     

Unscheduled synthesis induced by thiophosphamide in human lymphocytes

An unscheduled DNA synthesis in human nonreplicative lymphocytes is shown to follow cell exposure to thiophosphamide at a dose level of 1-10 microgram/ml. Incorporation of 3H-thymidine in the absence of the mutagen is probably due to spontaneous reparative processes. Significant variations are found of spontaneous and thio-phosphamide-induced levels of a reparative DNA synthesis in normal individuals.     

Lymphocyte receptors for polyclonal T mitogens: I. Concanavalin A binding to lymphocytes inhibits mitogenesis induced by galactose oxidase

Mitogenic stimulation of mouse lymphocytes by the enzyme galactose oxidase (GO) is inhibited if Con A is present during the enzymatic oxidation. The mechanism of this inhibition appears to involve steric hindrance of GO action at cell surface sites which bind Con A because (a) similar pulse exposure of unoxidized cells to Con A does not affect their subsequent ability to respond to GO stimulation; (b) Con A binding to fetuin interferes with GO oxidation of that glycoprotein substrate; and (c) sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cells labeled by $\text{GO}\text{NaB}{}^3\text{H}{}_4$ in the presence of Con A shows a selective inhibition of labeling of some high-molecular-weight glycoproteins compared to controls labeled in the absence of Con A.     

Differential susceptibility of murine T and B lymphocytes to freeze-thaw and hypotonic shock

Experiments were carried out to determine if thymic-derived lymphocytes (T-cells) could be differentially damaged by hypotonic and/or freeze-thaw stress. The uptake of ³H-thymidine after stimulation of murine spleen cells with phytohemagglutinin-P (PHA-P) or bacterial endotoxin (LPS) was used as an indicator of recovery. Optimal freezing and thawing techniques showed that 100% of LPS-responsive cells could be recovered, compared to 65% of PHA-responsive cells. These differences could be increased by treatment of spleen cells with 0.17 m NH₄Cl prior to freezing and thawing. This represented a recovery of 50% of LPS-responsive cells and less than 10% of PHA-responsive cells. A similar effect could be obtained by treating NH₄Cl-treated spleen cells with distilled water prior to culture. It is hypothesized that T-cells are more susceptible to osmotic damage than B-cells due to their differences in membrane characteristics.     

Differential effect of insulin and elevated glucose level on adenosine handling in rat T lymphocytes

Reduced proliferation potential is among other T cell functional defects long known feature of diabetes. However, the mechanism responsible for this impairment is still unknown. Our study was undertaken to investigate the effect of changes in glucose and insulin concentrations on adenosine metabolism, transport and receptor-mediated action in rat T lymphocytes. Presented results indicate that vulnerability of T cells to metabolic stress is determined by insulin but not by glucose concentration. However, glucose and insulin differentially affected the activities of adenosine metabolizing enzymes in resting and proliferating T cells. The Con A-induced proliferation of cultured T lymphocytes did not depended on expression level and functional state of nucleoside transporters. Inhibition of adenosine kinase (AK) with 5-iodotubercidin lowers the proliferation potential of T cells to the level observed for insulin-deprived cells. Moreover, insulin-deprived T lymphocytes but not cells cultured in the presence of insulin released significant quantities of adenosine. Under resting conditions, the cAMP level was fivefold higher in cells deprived of insulin comparing to cells cultured in the presence of insulin. Exposition of insulin-deprived T lymphocytes to specific antagonist (ZM241385) of A2a receptor but not to specific antagonist (Alloxazine) of A2b receptor suppressed cAMP elevation and completely restored the proliferation potential of T cells. Concluding, adenosine released by insulin-deprived T cells due to suppressed AK activity by acting on A2a receptors leads to increases in cAMP level and suppression of T cell proliferation. We assume that this mechanism may significantly contribute to immune impairment observed in diabetes.