Mitogen-activated Lymphocytes Research Articles

Synergistic action of phorbol esters in mitogen-activated bovine lymphocytes

Phorbol esters act synergistically with phytohemagglutinin (PHA) and Concanavalin A to promote DNA synthesis in bovine lymphocytes. Studies of this response indicate that phorbol esters are useful tools for elucidating the cellular processes that are related to the action of mitogens.

Maturation of mitogen-activated bone marrow-derived lymphocytes in the absence of proliferation.

AbstractMouse B lymphocytes respond by increased rates of DNA synthesis 14–24 hours after stimulation with the mitogen lipopolysaccharide. If, at this time, stimulated cells are treated for 12 hours with a “hot pulse” of thymidine, subsequent mitogen‐induced maturation to high rate IgM‐producing plaque‐forming cells is abolished. Thus, B cells stimulated by mitogen to mature also incorporate thymidine into DNA and proliferate.DNA synthesis is inhibited to 99 % in 48‐hour mitogen‐stimulated B lymphocytes by 10−2 M hydroxyurea or 10−3 M cytosine arabinoside, while protein and IgM synthesis and secretion and the plaque‐forming capacity of those cells are unaffected. When hydroxyurea is added to small, resting B cells at the time of initiation of mitogenic stimulation, these cells mature to 19 S IgM‐ secreting, plaque‐forming cells in the absence of DNA synthesis and proliferation. Maturation in the absence of DNA synthesis is also manifested by an increase in ratios of rates of synthesis and secretion of IgM over those of all proteins in the cell and by the attachment of the “branch” sugars, galactoses and fucoses, to 19 S IgM secreted by the cells. This maturation of B cells in the absence of DNA synthesis occurs within 12 to 30 hours after stimulation and is mitogen dose dependent. The inhibition of B cells to synthesize DNA and to develop into clones of plaque‐forming cells can be reversed by the removal of hydroxyurea for as long as 36 hours after mitogenic stimulation in the presence of the inhibitor.In the presence of hydroxyurea, B cells are stimulated to immature plasmablast‐like cells containing surface‐bound and little intracytoplasmic Ig within 16 to 24 hours of stimulation. Mature plasma cells containing no detectable surface‐bound Ig, but abundant intracytoplasmic Ig are only developed in the uninhibited, but not in the hydroxyurea‐inhibited mitogen‐stimulated cells later in the response. Thus, two stages of B cell maturation and differentiation can be distinguished: the first stage of an immature plasmablast develops in the absence of DNA synthesis, while the later stage of the development of the mature plasma cell requires DNA synthesis.

Role of Cyclic Adenosine 3′,5′-Monophosphate in Lymphocyte Mitogenesis

Abstract For assessment of the role of cyclic adenosine 3′,5′-monophosphate (cAMP) in lymphocyte mitogenesis, adenylate cyclase activity, cAMP content, 3H-thymidine (3H-THY) incorporation, and blast transformation were examined in lymphocytes incubated with mitogenic agents, and with prostaglandins, cholera toxin, or theophylline. When tested over a wide dose range with mouse spleen cells, the mitogens phytohemagglutinin-P (PHA-P), staphylococcal enterotoxin B (SEB), concanavalin-A (CON-A), and endotoxin did not stimulate adenylate cyclase activity or increase cellular cAMP content. Similarly, in human lymphocytes, neither SEB, CON-A, nor mitogenic doses of PHA-P altered cellular cAMP levels. A rapid increase in human lymphocyte cAMP was noted in response to high doses of PHA-P, but such doses resulted in little induction of 3H-THY incorporation compared to lower doses that were ineffective in elevating cAMP. Further, PGE1, cholera toxin, and theophylline, agents which all significantly increased lymphocyte cAMP content, uniformly suppressed both basal and mitogen-induced 3H-THY incorporation and blast transformation in a dose-related manner. The inhibitory effects of PGE1, cholera toxin, and theophylline on mitogen induced 3H-THY incorporation were time-dependent. After a 36-hr exposure of mouse spleen cells to CON-A increases in cellular cAMP no longer inhibited subsequent DNA synthesis, perhaps owing to the fact that those metabolic events influenced by cAMP were already completed in the majority of mitogen-activated cells by 36 hr. Moreover, washing mouse spleen cells which had been concomitantly incubated with CON-A and with PGE1 or theophylline for periods up to 36 hr abolished the inhibitory actions of the latter agents on 3H-THY incorporation. It seems possible from these results that elevated cellular cAMP levels may only delay, by a reversible process, the flow of mitogen-activated lymphocytes through their growth cycle and that lymphocytes remain susceptible to such inhibition for only a finite period after stimulation with mitogens.