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J C Mani

Publications and source records attributed to J C Mani.

At least 73 records · Page 4Linked to original sources
PubMed1989

Flow cytometric analysis of oxidative product formation in phytohemagglutinin-stimulated ethanol-treated immune mononuclear cells.

Oxidative products formed by immune mononuclear cells were studied by flow cytometry. JURKAT T cells and peripheral blood mononuclear cells were incubated with 2,7-dichlorofluorescin diacetate. This substance was hydrolysed in the cells, leading to a non-fluorescent product which was oxidized into highly fluorescent 2,7-dichlorofluorescein by oxygen reactive species. These latter products were analysed by flow cytometry in phytohemagglutinin (PHA)-stimulated ethanol (ETH)-treated mononuclear cells. The level of fluorescence intensity (FI) was found higher in stimulated cells than in non-stimulated cells. ETH displayed two different effects on the cells: either a decrease of FI associated with a decrease of the number of fluorescent cells (FC) or an increase in FI. Both effects were dose-dependent. ETH is an effective scavenger of .OH radicals, but it is also oxidized by the microsomal ETH oxidizing system with production of oxygen reactive species, which probably explains the opposite effects of ETH. In the presence of desferal, an iron-chelating agent, and nordihydroguaiaretic acid, an inhibitor of the lipooxygenase pathway, the cells showed a decrease of FI and FC. These results suggest that .OH and other oxygen reactive species are involved in stimulation by PHA of ETH-treated immune mononuclear cells.

Adult↗
PubMed1989

Effects of ethanol in vitro on some parameters of the immune response.

The action of ethanol (ETH) on murine lymphocyte subpopulations and on human peripheral blood mononuclear cells (PBMC) stimulated in vitro by mitogens was studied. ETH caused a concentration-dependent decrease in DNA synthesis in the different murine cell types. ETH was more immunosuppressive for T lymphocytes than for B lymphocytes. An enhancement of the blastogenic response was observed for B cells at 0.5% ETH. Interleukin 2 synthesis by murine splenocytes was inhibited by ETH in a concentration-related manner; the lowest concentrations of ETH caused an increase in interleukin 2 synthesis. The highest concentrations of ETH tested decreased the size of the cell clusters formed in cultures of mitogen-stimulated lymphocytes, whereas an increase in PBMC cluster size was observed in the presence of 0.5 and 1% ETH.

Animals↗
PubMed1989

Extracellular ATP induces a nonspecific permeability of thymocyte plasma membranes.

We have previously demonstrated that extracellular ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis, without mobilization of intracellular calcium. We describe here the effects of extracellular nucleotides on membrane permeability to monovalent and divalent cations in mouse thymocytes. Among all nucleotides tested, under physiological conditions, only ATP and, to a lesser extent, 2-methylthio-ATP, adenosine 5'-O-(3-thio-triphosphate), and ADP were able to depolarize thymocyte plasma membranes and to induce Na+ and Ca2+ influxes into thymocytes; other nonhydrolysable ATP analogs were only effective in the absence of Mg2+. The ATP-induced effects were inhibited in a dose-dependent manner by Mg2+ and greatly potentiated in its absence, which suggests that the tetrabasic ATP4 is probably the active species and that a phosphotransferase activity is not involved in its effects. There ATP-mediated changes in ion fluxes result from an increase in nonspecific permeability of thymocyte membranes, probably by pore formation. These ion flux changes might be responsible for the mitogenic induction of phorbol 12-myristate 13-acetate treated medullary thymocytes. The potency order for the adenine derivatives to affect these fluxes (ATP greater than ADP much greater than AMP greater than adenosine) suggests the presence of ATP specific receptors (P2 purinergic receptors) on thymocyte plasma membranes.

Adenosine Triphosphate↗
PubMed1988

Determination of mitogenic properties and lymphocyte target sites of Dolichos lablab lectin (DLA): comparative study with concanavalin A and galactose oxidase cell surface receptors.

A mannoside-directed lectin has been isolated and purified from the seeds of Dolichos lablab L. by affinity chromatography. We have established that this glycoprotein, which displays high erythroagglutinating activity without blood group specificity, highly activates murine T lymphocytes, and we have described for the first time its mitogenic properties. Although its main properties are close to those of concanavalin A (Con A), the well-known mannoside-directed mitogen devoid of sugar moiety, several differences were found in some of the early events triggered by the two lectins during lymphocyte mitogenic stimulation: higher level of interleukin-2 (IL-2) synthesis, optimal dose for IL-2 synthesis at suboptimal mitogenic concentration, lack of ecto-5' nucleotidase inhibition, and lack of mitogenic inhibition at high lectin concentration. Because the two lectins did not act on the cell surface in exactly the same way, we have compared their receptors involved in mitogenesis on the plasma membrane of murine lymphocytes. We had previously established that the polyclonal activation of these cells probably occurred through high-molecular-weight receptors (200-230 kDa). Since the mitogenic stimulation of lymphocyte by galactose oxidase (GO), like that of Con A, was inhibited by DLA, we analyzed the cell surface receptors that were common to these three polyclonal mitogens. After labeling the neuraminidase/GO-treated cell surface glycoproteins with NaB3H4, we immunoprecipitated the Con A and DLA receptors which are the target of GO mitogenic action. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the precipitates demonstrated that there exist on the lymphocyte cell surface receptors common to the polyclonal mitogens DLA, Con A, and GO. Because Con A and DLA sterically inhibit GO mitogenic stimulation, the common glycoproteins which represent the necessary sites of oxidative mitogenic action are probably those which are involved in DLA and Con A-triggered mitogenesis, despite the different properties of the two lectins. These differences could be explained by the lower molecular weight receptors of the two lectins which are not identical.

5'-Nucleotidase↗
PubMed1987

PCP and analogs prevent the proliferative response of T lymphocytes by lowering IL2 production. An effect related to the blockade of mitogen-triggered enhancement of free cytosolic calcium concentration.

The psychotomimetic drug PCP displays a vast array of known pharmacological effects, among them its capacity to affect cation transport in nervous and myocardiac tissues. Since increased movements of cations are essential for the immune responses, it has been mentioned that PCP could also depress immune functions by this mechanism. In order to check this hypothesis, we have investigated the effects of PCP and of many other structural derivatives on the blastogenic response of murine or human T lymphocytes. We find that all the drugs block an early event of T lymphocyte activation and prevent their further proliferation; conversely they do not affect primed lymphocytes. The compounds, which do not inhibit interleukin-1 (IL-1) production in stimulated macrophages, lower interleukin-2 (IL-2) synthesis in activated T helper cells. This negative action appears to be related to the inhibition of the rise of free cytosolic calcium concentration [Ca2+]i observed soon after the T receptor triggering and which is an essential message for IL-2 production. The lymphocyte membrane depolarization induced by the drugs could explain the blockade of the lectin-induced [Ca2+]i changes. The study of the structure-activity relationship shows that the PCP analogs which possess a quasi-rigid conformational structure express an inhibitory capacity of T lymphocyte proliferation higher than that of PCP (200 times for some products). Since these compounds interact poorly with the CNS tissues and have few behavioral effects, we suggest that PCP exerts its negative action on lymphocytes on cell components different from its receptor(s) in the CNS.

Animals↗
PubMed1987

Extracellular ATP increases cytosolic free calcium in thymocytes and initiates the blastogenesis of the phorbol 12-myristate 13-acetate-treated medullary population.

Exogeneous nucleotides or nucleosides may influence lymphocyte functions such as proliferation and cytotoxicity. We report that ATP, and to a lesser extent ADP, at concentrations as low as 0.3 mM, are highly mitogenic for medullary mature thymocytes, when added in combination with phorbol myristate acetate (PMA), which is only weakly mitogenic by itself. Under the same conditions, the other nucleotides (AMP; GTP, ITP, 2'd-deoxyATP), the non-hydrolysable ATP analogs (p[NH]ppA, pp[CH2]pA) and adenosine are unable to trigger thymocyte blastogenesis. p[NH]ppA, a potent inhibitor of ATP hydrolysis, potentiates the ATP mitogenic effect. In contrast, T-cell-enriched splenocytes do not proliferate in response to ATP + PMA. These data and measurements of interleukin 2 synthesis suggest that ATP may efficiently deliver in thymocytes the calcium signal necessary for the initiation of blastogenesis (in medullary cells). Indeed, among all nucleotides tested, only ATP or ADP were able to increase the intracellular free calcium level in thymocytes, but not in splenocytes. Our results led us to suggest that thymocytes express on their surface receptors specific for ATP, which might be P2 type nucleotide receptors and could be involved in the lymphocyte response through the regulation of intracellular free calcium levels.

Adenosine Triphosphate↗
PubMed1987

Lipoxygenase inhibitors suppress IL-2 synthesis: relationship with rise of [Ca++]i and the events dependent on protein kinase C activation.

The present study was performed in an attempt to understand the mechanism involved in the inhibition of interleukin 2 (IL-2) synthesis by lipoxygenase (LO) pathway inhibitors. Using the two IL-2-producing lymphoid cell lines, (Jurkat and EL4 cells), we showed first that the inhibitory effect of the phenolic compounds tested (NDGA, BHA and caffeic acid) acted on lymphoid cells themselves and not on eventual monocytic or granulocytic contaminant cells. Secondly, these inhibitors were demonstrated as exerting their effect on two levels: they affected the events controlled by both second messengers implicated in T cell activation, namely rise of intracellular free calcium concentration [( Ca++]i) and protein kinase C (PKC) activation. For this purpose, LO inhibitor effects have been compared: (a) on IL-2 production by the two different lines: Jurkat cells, which need both signals, and EL4 cells, which require only PKC activation for the induction of this production; and (b) on the events induced by the different ways of Jurkat cell activation: PHA (or anti-CD3 monoclonal antibody) versus calcium ionophore. These results are discussed with respect to an eventual involvement of arachidonic acid [AA] derivatives in IL-2 synthesis.

Animals↗
PubMed1987

Sensitivity of human lymphocytes to acetaldehyde: comparison between alcoholic and control subjects.

The action of acetaldehyde (ACH), the first metabolite of ethanol, was studied on human lymphocytes stimulated in vitro by phytohemagglutinin (PHA) or Concanavalin A (Con A). ACH caused a dose-dependent decrease of [3H]thymidine uptake in lymphocytes from both alcoholic and control subjects. The area under the curve of [3H]thymidine incorporation as a function of ACH concentration was determined for each subject and referred to as the lymphocyte sensitivity index. Indexes for alcoholic subjects were found to be higher than those for controls, indicating a lower sensitivity to ACH of lymphocytes from alcoholics. We also found a wide range of sensitivity indexes within the same group. These results are consistent with the current hypothesis that not everybody is at equal risk to develop alcohol related disorders.

Acetaldehyde↗
PubMed1987

Amiloride-induced suppression of lymphocyte proliferation: inhibition of IL 2 receptor expression after blockade of early sodium influx.

Increased rates of monovalent cation fluxes are implicated in the activation of lymphocytes by mitogens. Our report shows that amiloride and dimethylamiloride, two inhibitors of the Na+/H+ antiport, dose dependently prevent the proliferation of mitogen-activated human peripheral blood lymphocytes. The action of these drugs follows several mechanisms, since their inhibitory effect can be reversed by extensive washing when they are used at low concentrations (150 microM for amiloride, 40 microM for dimethylamiloride), while at higher non cytotoxic concentrations this reversibility is no longer observed. We have studied the mechanism whereby amiloride inhibits the blastogenesis by measuring their effect on: 1) IL 2 production, 2) acquisition of IL 2 responsiveness and induction of IL 2 receptors, 3) IL 2-induced proliferation. Unlike the expression of IL 2 receptors, IL 2 production and IL 2-dependent proliferation were not inhibited by the low drug concentrations which indeed prevent blastogenesis. Moreover under these conditions, an enhanced accumulation of IL 2 was observed in the supernatants of stimulated cells. These results show that the drugs interact differently with the different cell populations involved in T cell proliferation: increase of an amiloride-dependent sodium influx is an obligatory step required to induce the early increase of the ouabain-dependent potassium influx which is needed for the expression of IL 2 receptors. On the contrary, the influx of potassium necessary for the IL 2-dependent proliferation does not seem to be controlled by the amiloride-dependent sodium flux.

Amiloride↗
PubMed1986

Enhancement of cell-cell contact by a nonmitogenic lectin increases blastogenic response and IL-2 release by mitogen-stimulated mouse thymocytes.

We have examined the influence of peanut agglutinin (PNA), a lectin which agglutinates but does not stimulate mouse thymocytes, on the responsiveness of these cells to concanavalin A (Con A) or galactose oxidase stimulation. Binding low amounts of PNA on unseparated mouse thymocytes pretreated with neuraminidase highly enhances the mitogenic response and the level of interleukin 2 release in the culture medium upon Con A stimulation. We have shown that PNA present on the cell surface acts as a crosslinking agent which favors intercellular binding between accessory cells (macrophages) and thymocytes, leading through this enhanced cooperation by cell-cell contact to an enhanced blastogenic response.

Adjuvants, Immunologic↗
PubMed1986

Mechanism whereby ouabain inhibits human T lymphocyte activation: effect on the interleukin 2 pathway.

Through the blockade of the Na-K-ATPase, ouabain inhibits several biochemical and biological events leading to the proliferation of activated lymphocytes. Since we already found that interleukin 1 production was not prevented by ouabain, we investigated by which mechanism this drug inhibits mitogen-induced human T lymphocyte activation, with respect to the interleukin 2 (IL 2) pathway. Our data revealed that at concentrations lower than 0.2 microM, IL 2 accumulation was not reduced in ouabain-treated cultures, even when cell proliferation was completely inhibited (0.1-0.2 microM ouabain). Moreover, in this concentration range, ouabain stimulated in a dose-dependent manner the accumulation of IL 2 in the supernatant of Con A-stimulated lymphocytes (optimum for 0.05 microM corresponding to half inhibition of lymphocyte proliferation). Such an effect, which appears correlated to the inhibition of Na-K-ATPase, suggests a failure of the cell to utilize IL 2. At concentrations higher than 0.3 microM, ouabain inhibited both lymphocyte proliferation and IL 2 production. These observations show that the glycosteroid interacts differently with the different cell populations involved in the cascade of reactions leading to cell proliferation, and suggest that the mitogenic inhibition resulting from the blockade of Na-K-ATPase is not related to the blockade of IL 2 production. On the other hand, we observed that: ouabain inhibited the expression of the receptors for IL 2, an obligatory step in lymphocyte proliferation; ouabain blocked the proliferation of an IL 2 sensitive human T cell line; in both cases the inhibition paralleled that of lymphocyte proliferation. Our data suggest that the essential steps of lymphocyte proliferation in which Na-K-ATPase-dependent K+ fluxes play a critical role are the expression of IL 2 receptors and the IL 2-dependent proliferative step.

Cell Line↗
PubMed1985

[Purine metabolism and blastogenesis in lymphocytes of alcoholic subjects].

Immune deficiency is characteristic of alcoholic subjects. These subjects usually show altered lymphocyte function. We determined the activities of adenosine deaminase (ADA) and ecto-5'-nucleotidase (ecto-5'N) in lymphocytes from 54 subjects: 15 healthy controls, 28 non-cirrhotic alcoholics, 8 alcoholic cirrhotics and 3 non-alcoholic cirrhotics. Whereas ADA activity was the same for all 54 subjects, ecto-5'N activity was in general lower in alcoholic subjects after cessation of alcohol intake. Following alcohol intoxication, however, ecto-5'N activity increased. The decrease of ecto-5'N activity in alcoholic subjects might be explained by shedding of the ecto-enzyme and alteration of lymphocyte subpopulations. We observed decreased mitogenic-induced lymphoblastic transformation in 3 patients with cirrhosis. All other subjects (including healthy controls) had normal mitogenic-induced blastogenesis. Interestingly, following alcohol intake, non-stimulated lymphoblastic transformation increased, leading to an apparently decreased stimulation index.

5'-Nucleotidase↗
PubMed1984

The proposed 5'-nucleotidase inhibitor in human leukemic cells is an artefact.

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5'-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577-584) mentioned that this phenomenon resulted from the presence of a 5'-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5'-[3H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [3H]Adenosine derived from 5'-[3H]AMP hydrolysis was further transformed into [3H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [3H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5'-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5'-nucleotidase inhibitor. We did not observe 5'-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.

5'-Nucleotidase↗
PubMed1984

Theophylline-like properties of xanthine analogs.

Theophylline and other methylxanthines display a large number of biological effects, some of which are clinically important. The effects of these compounds are commonly ascribed to an inhibition of cyclic AMP breakdown. However, it becomes actually evident that another mechanism, namely adenosine receptor antagonism, could be responsible for certain methylxanthine effects. It could be of interest to find new compounds displaying only one of these mechanisms, either phosphodiesterase inhibition or adenosine receptor antagonism. We have studied several synthetic imidazol[1,2a]pyrazines, some of which display theophylline-like pharmacological properties at lower doses than theophylline. We showed that some of these compounds inhibited mitogen-induced [3H]-thymidine uptake by human lymphocytes, which is consistent with increases in cyclic AMP levels: the most efficient compounds were those which were better phosphodiesterase inhibitors than theophylline and poorer adenosine receptor antagonists.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Paradoxical production of mouse thymocyte activating factor by ouabain-treated human mononuclear cells.

At concentrations as low as 10(-7) M, the cardiotonic glycosteroid ouabain, a specific inhibitor of the membrane Na+, K+-ATPase, is known to inhibit in vitro human lymphocyte proliferation produced in mixed lymphocyte cultures or induced by various stimulating agents (PHA, Con A, PWM, soluble antigens), while mouse lymphocyte proliferation is unaffected at this concentration. Ouabain inhibits most of proliferative response parameters at all stages of the transformation. This observation prompted us to suggest that ouabain could also act through inhibition of interleukin production which is known to occur during the first hours after T-cell stimulation in the presence of monocytes. In order to check the possible influence of ouabain on interleukin production, conditioned media from stimulated human mononuclear cells, prepared in the presence or in the absence of inhibitor, were tested for their ability to promote a mouse thymocyte response to PHA. Instead of the expected inhibition, we found that ouabain, even at high concentrations (2 X 10(-6) M) enhanced the stimulatory effect and/or the production of murine thymocyte activating factor(s). Moreover conditioned media from serum-free cultures of unstimulated human mononuclear cells exposed for 24 hr to low ouabain concentrations (10(-8) to 10(-7) M) showed a high activating effect on the response of murine thymocytes to PHA. This soluble factor produced upon ouabain treatment is produced by adherent cells and appears to be functionally similar to interleukin 1.

Animals↗