David A. Hart

Potentiation of phytohemagglutinin stimulation of lymphoid cells by lithium.

Stimulation of hamster lymph node cells, splenocytes and thymocytes by the mitogen phytohemagglutinin-P (PHA) was found to be greatly enhanced by addition of 1–10 mM LiCl to the cultures. Lithium enhanced stimulation, as determined by [3H]TdR incorporation, only if added within the first 24 h of culture. The enhancing effect of lithium was specific for this monovalent cation since equivalent concentrations of KCl or NaCl did not induce a similar effect on [3H]TdR incorporation. The divalent cations Mg2+ (1–10 mM) and Ca2+ (1-1.6 mM), also had an enhancing effect on PHA stimulation. However, addition of Li+ to cultures enhanced with Mg2+ and/or Ca2+ led to an additional potentiation of the response to PHA. These results suggest that Li+ modifies a unique early event during stimulation of lymphoid cells by this mitogen.

Effect of protease inhibitors on mitogen stimulation of hamster lymphoid cells

Abstract Incorporation of [ 3 H]thymidine ([ 3 H]TdR) into normal and mitogen-stimulated MHA hamster lymphoid cells was suppressed by addition of certain protease inhibitors to the cultures. Phenylmethylsulfonyl fluoride (PMSF) and e-aminocaproic acid (EACA) were not effective in suppressing stimulation. N -α-tosyl- l -phenylalanyl-chloromethane (TPCK) inhibited both concanavalin A (ConA) stimulation and lipopolysaccharide (LPS) stimulation when added at the initiation of cultures and after 24 h. However, N -α-tosyl- l -lysyl- l -chloromethane (TLCK) differentially inhibited LPS stimulation. At high concentrations, TLCK inhibited ConA stimulation but this inhibition, unlike the inhibition of LPS stimulation, was reversible by adding reduced glutathione or l -systeine. In addition, similar concentrations of TLCK were not effective in inhibiting either ConA or LPS stimulation when added 24 h after stimulation, as assayed by l -leucine and [ 3 H]TdR incorporation. These results add to the evidence that a proteolytic event may be involved in an early activation event in B-cells.

Stimulation of a subpopulation of hamster lymphoid cells by trypsin and chymotrypsin

Abstract Hamster lymph node and spleen cells can be stimulated to incorporate tritiated thymidine ([ 3 H]TdR) in vitro under serum-free conditions by the proteases trypsin and chymotrypsin. Under similar conditions, thymocytes could be stimulated by concanavalin A (ConA) but not lipopolysaccharide (LPS) or the proteases. The subpopulation of cells responding to the proteases correlated with the cells responding to LPS on fractionation of spleen and lymph node cells on discontinuous bovine serum albumin (BSA) gradients or on nylon-wool columns. The stimulation induced by trypsin was completely blocked by soybean trypsin inhibitor (SBTI) while that induced by chymotrypsin was only partially blocked. The inhibition by SBTI of protease activation was not effective when added 24 h after initiation of stimulation. On the other hand, addition of clarified isologous serum to protease activated cultures after 24 h still lead to greater than 50% inhibition of [ 3 H]TdR incorporation.

Modulation of concanavalin A stimulation of hamster lymphoid cells by lithium chloride

Abstract Addition of LiCl (1–25 m M ) to serum-free cultures of MHA hamster thymocytes, lymph node cells, or splenocytes stimulated with concanavalin A had a biphasic effect on [ 3 H]thymidine incorporation. These concentrations of LiCl enhanced stimulation of [ 3 H]thymidine incorporation by suboptimal levels of concanavalin A but inhibited stimulation of optimal and supraoptimal concentrations of concanavalin A. This effect was specific for Li + since it was not observed when similar concentrations of Na + , K + , or Mg 2+ were added to cultures stimulated by concanavalin A. The inhibitory effect of LiCl on concanavalin A stimulation was not reversed by addition of Na + , Ca 2+ , Mg 2+ , or Ca 2+ + Mg 2+ to the cultures. Significant reversal of LiCl inhibition of stimulation was observed when KCl was added to the cultures. However none of the ions tested blocked the Li-induced enhancement of [ 3 H]thymidine incorporation in the presence of suboptimal concentrations of concanavalin A.

Evidence that manganese inhibits an early event during stimulation of lymphocytes by mitogens

Abstract Addition of low concentrations of Mn 2+ , 1–10 μM, to mitogen-stimulated hamster lymphoid cells enhanced [ 3 H]thymidine ([ 3 H]TdR) incorporation. Enhanced [ 3 H]thymidine incorporation was observed for stimulation of both B- and T-lymphocytes. Higher concentrations of Mn 2+ , 100 μM, differentially inhibited the responses to ConA and phytohemagglutinin (PHA) while the responses to lipopolysaccharide and dextran sulfate was unaffected. This effect was specific for Mn 2+ since it was not observed with Zn 2+ , Co 2+ , or Fe 2+ . Addition of 100 μM Mn 2+ to T cell mitogen-stimulated cultures at varying times after initiation of stimulation, indicated that Mn 2+ was inhibitory only if added within the first 4–16 h after stimulation. The inhibition of stimulation of splenocytes, and to a lesser extent thymocytes, induced by 100 μM Mn 2+ could be reversed by adding 1.6 mM CaCl 2 or SrCl 2 to the cultures. However, Mn 2+ inhibition of stimulation of lymph node cells could not be reversed by additional CaCl 2 . Studies with [ 3 H]acetyl ConA indicated that 100 μM Mn 2+ did not inhibit binding of ConA to the cells. Therefore, the inhibitory effect of Mn 2+ is most likely a post-binding effect on the cells themselves. Addition of 100 μM Mn 2+ to ConA and PHA-stimulated guinea pig lymphoid cells yielded results very similar to those obtained with hamster lymphoid cells. These results, obtained with lymphoid cells from two experimental animals indicate that the biochemical event inhibited may be a widespread cellular mechanism which could extend to non-lymphoid cells.

Hamster immune responses in infectious and oncologic diseases

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Augmentation of zinc ion stimulation of lymphoid cells by calcium and lithium

Abstract Stimulation of hamster lymph node cells by optimal concentrations of ZnCl 2 (10 μM) was found to be enhanced by addition of 1–25 mM LiCl to the serum-free cultures. Maximal enhancement occurred at 10 mM Li + . Similar concentrations of either KCl or NaCl did not potentiate stimulation. Addition of 1 mM CaCl 2 , but not 1–25 mM MgCl 2 , also potentiated Zn 2+ stimulation of lymph node cells. When the cultures were supplemented with 1 mM Ca 2+ + 10 mM Li + , a synergistic potentiation of Zn 2+ stimulation occurred. In addition, the dose response curve for Zn 2+ was shifted such that maximal stimulation occurred at 100–250 μM Zn 2+ , a concentration of Zn 2+ which was toxic for the unsupplemented cultures. In Ca 2+ + Li + supplemented cultures, Zn 2+ stimulated [ 3 H]thymidine incorporation to levels comparable to those obtained when hamster lymphoid cells were stimulated with lectins. In addition to Zn 2+ stimulation, Ca 2+ + Li + supplemented medium also enhanced Hg 2+ stimulation of hamster lymph node cells but did not change the dose response curve for Hg 2+ . Therefore, the observed ionic effects on Zn 2+ stimulation of lymphocytes were unique to this mitogen, when compared to either Hg 2+ stimulation or previously reported lectin stimulation of hamster lymphoid cells.

The effect of soybean trypsin inhibitor on concanavalin A and phytohemmagglutinin stimulation of hamster, guinea pig, rat, and mouse lymphoid cells☆

Abstract Lymphoid cells from MHA hamsters, Hartley guinea pigs, DA and Fischer rats, and BDF1 mice can be stimulated to incorporate tritiated thymidine by the mitogens concanavalin A and phytohemmagglutinin P. Addition of soybean trypsin inhibitor to cultures of hamster thymocytes or spleen cells had no effect on stimulation. Similar concentrations inhibited stimulation of a subpopulation of hamster lymph node cells. The inhibitable population was concentrated in the peripheral lymph nodes. Concentrations of soybean trypsin inhibitor up to 1 mg/ml did not inhibit mitogen stimulation of guinea pig lymph node cells and partially inhibited the response of spleen cells. The inhibition of splenocyte stimulation was consistently 30–40% which may indicate a specific subpopulation of cells is inhibited. Mitogen stimulation of rat splenocytes was completely inhibited by addition of soybean trypsin inhibitor. On the other hand stimulation of lymph node cells was relatively resistant to inhibition by the protease inhibitor. Mitogen stimulation of BDF1 splenocytes and lymph node cell cultures was inhibited 60–80% by the addition of soybean trypsin inhibitor.

Evidence that lithium ions can modulate lectin stimulation of lymphoid cells by multiple mechanisms

Abstract Inhibition of PHA stimulation of hamster lymph node cells by theophylline, DBcAMP, or indomethacin or PHA stimulation of thymocytes by theophylline or DBcAMP was partially reversed by addition of 10 m M LiCl to the cultures. Addition of LiCl to Con A-stimulated lymphoid cells treated with the same reagents did not alter the inhibition. In contrast, addition of 10 m M LiCl to Con A-stimulated cultures enhanced the inhibition induced by the Na,K ATPase inhibitor, ouabain. Like LiCl, this latter inhibitor was found to be effective in modulating stimulation only if added early in the culture. These data support the hypothesis that LiCl can modulate lymphocyte responsiveness at the level of cyclic nucleotide metabolism, as exemplified by PHA stimulation, or at the level of the Na,K ATPase, exemplified by Con A stimulation. The site of involvement of Li + ion would appear to be dependent on the biochemical nature of the stimulating signal.

Differential effect of trypsin and trypsin inhibitors on lipopolysaccharide stimulation of hamster lymphoid cells

Abstract Activation of hamster lymphoid cells by the mitogen lipopolysaccharide was shown to be greatly potentiated by addition of the protease trypsin. However, activation of cells by the polyanion, dextran sulfate, was not altered by this enzyme. Addition of the soybean trypsin inhibitor inhibited lipopolysaccharide stimulation of lymph node cells from animals 1–4 months of age but only partially inhibited stimulation of splenocytes from young animals (5–6 weeks) and did not inhibit stimulation of splenocytes from older animals. Dextran sulfate stimulation was not inhibited by soybean trypsin inhibitor.