Reversal of immune dysfunction in osteopetrotic rats by interferon-gamma: augmentation of macrophage Ia expression and lymphocyte interleukin-2 production and proliferation

Lymphocytes from osteopetrotic (op) rats, compared to their normal (n) littermates, exhibit defective immune functions associated with their inability to resorb bone. Among these immune defects are the failure of their spleen cells to proliferate normally to mitogens and to generate IL-2. Addition of exogenous IL-2 failed to reverse the suppressed proliferation in the op spleen cells, indicating that additional defects were involved in the suppression. Phenotypic analysis of cellular constituents of op and n spleens revealed that the percentages of T cells, macrophages, and IL-2 receptor positive cells were not different. Furthermore, there was no difference in CD4 (W3/25) and CD8 (OX8) cells. However, the Ia+ (OX3) cells in the op spleen represented less than 50% of those found in the n spleen, but the op had higher levels of transferrin receptor (OX26). On the basis of the ability of interferon-gamma (IFN-gamma) to increase Ia expression, this cytokine was added to op spleen cells (10-50 U/ml) and found to increase the number of Ia+ cells to the level found in n spleen cells. Moreover, pretreatment of op spleen cells with IFN-gamma restored their ability to proliferate to mitogens and their responsiveness to IL-2. Not only did IFN-gamma reverse the defective response to IL-2, but it also augmented the defective IL-2 production by op spleen cells. Taken together, these findings demonstrate that IFN-gamma can reverse many of the impaired immune functions characteristic of op spleen cells in vitro. Furthermore, these data suggest that IFN-gamma may provide an important avenue of treatment in these animals that may contribute to restoration of normal bone resorption.     

Characterization of a suppressor cell line which downgrades experimental autoimmune uveoretinitis in the rat

A Ts lymphocyte line was isolated from spleens of rats primed with the retinal soluble Ag (SAg) in the anterior chamber of the eye. This line could inhibit in vitro SAg-driven proliferation of uveitogenic Th lymphocytes, in a radioresistant, Ag-independent manner. Adoptively transferred Ts line cells were found to downgrade experimental autoimmune uveoretinitis in actively immunized syngeneic recipients. The initial surface phenotype (OX8+) of the Ts line was unstable in vitro, however, the cells expressed suppressor function irrespective of phenotype. The mechanism of suppression did not appear to involve consumption of IL-2 or direct cytolysis of uveitogenic Th lymphocytes, but rather the production of a soluble suppressor factor. These findings may suggest an in vivo role for suppressor lymphocytes, capable of inhibiting primed Th cells, in the regulation of experimental autoimmune uveoretinitis.     

Predominance of Th1 cells in ocular tissues during herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) appears to represent an immunopathologic response in the cornea of the eye to HSV-1. T cells of the CD4+ subset were shown to be involved in the mediation of HSK, but how they subserve an immunopathologic role is uncertain. In the present report, we have isolated cells from eyes in the active phase of HSK and studied their cytokine profile after culture in vitro or stimulation with Ag or nonspecific mitogens. Inflammatory cells recovered from eyes consist of polymorphonuclear leukocytes, macrophages, and lymphocytes. As reported before, all the lymphocyte recovered were of the CD4+ phenotype. After stimulation in vitro with Ag or mitogen the cytokines IL-2, IFN-gamma, and TNF-alpha/beta were produced, but not the cytokines IL-4 and IL-10. Thus, on the basis of cytokine profile, ocular lymphocytes were identified as Th1 cells. Ocular cells were also stimulated with PMA and shown to produce IL-1. The results were discussed in terms of the possible means by which the Th1 cells induce tissue damage in HSK as well as in terms of the possible means by which a preferential accumulation of Th1 cell occurs in the eye.     

Activation of rat B lymphocytes. I. Characterization of anti-immunoglobulin responses and isotype density of rat B cells

Spleen cells of two rat strains, Lewis and Brown Norway (BN), have been activated by lectins and by antibodies specific for immunoglobulin isotypes embedded in their cell membranes. Optimal concentrations of antibodies specific for mu, gamma, or delta-chains of rat augments in vitro incorporation of 3H-TdR 5 to 18-fold in Lewis B lymphocytes and 1.5 to 4-fold in BN B lymphocytes. In addition, F(ab')2 fragments of anti-Ig reagents induced Lewis splenic B cells but not BN B cells to incorporate 3H-TdR. Responses to LPS and dextran sulfate, B lymphocyte mitogens, measured by radioactive uptake, were five to 10 times greater in Lewis B cell populations than in BN B cell populations. Density of surface Ig isotypes and capping kinetics were similar in the two rat strains, although the percentage of T cells, T cell subsets, B cells, and Ia+ B cells differed in the spleens of these strains of rats. Both T lymphocytes and macrophages were needed in culture to effect an optimal response. IL-2 restored the response in B cell cultures depleted of T cells and macrophages, and enhanced 3H-TdR uptake in whole spleen cells of Lewis but not BN rats. The strain-dependent responsiveness of B cells to specific anti-Ig reagents or B cell mitogens appears to be associated with inherent (genetic) defects in T cells and B cells or defects in T cell to B cell cooperation in BN rats.     

Effects of Aluminum on Immune Functions of Cultured Splenic T and B Lymphocytes in Rats

The effects of Aluminum (Al) exposure on immune functions of cultured splenic T and B lymphocytes of rats were studied. The lymphocytes were isolated from spleen of healthy male Wistar rats weighing 110-120 g. The cultured cells in RPMI-1640 medium were exposed to 0 (control group), 0.035 (low-dose group), 0.07 (medial-dose group), and 0.14 (high-dose group) mg/mL Al(3+) as aluminum trichloride (AlCl(3)) in an incubator under 5% CO(2) at 37°C for 24 h. The T and B lymphocyte proliferation was measured with a tetrazolium dye colorimetric assay. The levels of interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α were determined by iodine [(125)I] IL-2, IL-6, and TNF-α radioimmunoassay kits, respectively. The proportions of CD3(+), CD4(+), and CD8(+) T lymphocytes were measured with a flow cytometer. The results showed that the T and B lymphocyte proliferation, the levels of IL-2, IL-6, TNF-α, the proportions of CD3(+) and CD4(+) T lymphocytes, and the ratio of CD4(+)/CD8(+) T lymphocytes were lowered by Al treatments, while the proportion of CD8(+) T lymphocytes was increased. These findings indicate that Al exposure can inhibit the immune functions of splenic T and B lymphocytes of rats in vitro.     

Novel mechanism for Trypanosoma cruzi-induced suppression of human lymphocytes. Inhibition of IL-2 receptor expression

Co-culture of blood forms of Trypanosoma cruzi, the causative agent of Chagas' disease, with human PBMC impaired the capacity of T lymphocytes to express surface receptors for IL-2. This effect was evidenced by marked reductions in both the proportion of Tac+ cells and the density of Tac Ag on the surface of the positive cells, determined by flow cytometry. The extent of the inhibition increased with parasite concentration. Under optimal or suboptimal conditions of stimulation with either PHA or monoclonal anti-CD3, specific for an epitope of the T3-Ti human T cell Ag receptor complex, the presence of T. cruzi curtailed the capacity of T lymphocytes to proliferate and express Il-2R but did not affect IL-2 production. Furthermore, the addition of exogenous IL-2 did not restore the responsiveness of suppressed human lymphocytes but did when mouse lymphocytes were used instead. Therefore, unlike mouse lymphocytes, human lymphocyte suppression by T. cruzi did not involve deficient IL-2 production and was accompanied by impaired IL-2 utilization. Co-culture of human monocytes/macrophages with suppressive concentrations of T. cruzi increased IL-1 production, and the parasite did not decrease IL-1 secretion stimulated by a bacterial LPS. Therefore, the suppression of IL-2R expression and lymphoproliferation is not likely to have been an indirect consequence of insufficient IL-1 production due to infection of monocytes or macrophages. We have shown that suppression of human lymphocyte proliferation by T. cruzi is not caused by nutrient consumption, absorption of IL-2, lymphocyte killing, or mitogen removal by the parasite. Therefore, these results uncover a novel suppressive mechanism induced by T. cruzi, involving inhibited expression of IL-2R after lymphocyte activation and rendering T cells unable to receive the IL-2 signal required for continuation of their cell cycle and mounting effective immune responses.     

Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.Copies of articles are available through ISI Document Delivery Services c/o The Genuine Article, 3501 Market Street, Philadelphia, PA 19104.     

Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production

Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8 alpha (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1 beta and promoted TNF and IL-1 beta secretion. Similarly, OX8 Ab (CD8 alpha) stimulated NR8383 cells to secrete TNF, IL-1 beta, and NO. Activation of CD8 beta (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1 beta and stimulated secretion of TNF, but not IL-1 beta. Interestingly, anti-CD8 Abs did not stimulate IFN-gamma or PGE2 production, or phagocytosis by macrophages. OX8 (CD8 alpha)-induced TNF and IL-1 beta production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1 beta, stimulation by CD8 alpha is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1 beta production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8 alpha (OX8)-induced TNF and IL-1 beta production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1 beta expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.     

Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms

Human mesenchymal stem cells (MSCs) have immuno-modulatory properties. They inhibit T-cell proliferation to mitogens and alloantigens in vitro and prolong skin graft survival in vivo. We found that MSCs inhibited the proliferation of peripheral blood lymphocytes (PBLs) to phorbol myristate acetate (PMA), suggesting that MSCs exert an inhibitory effect downstream of the receptor level. We analyzed cytokine profiles of PBLs co-cultured with MSCs. MSCs increased interleukin (IL)-2 and soluble IL-2 receptor in mixed lymphocyte cultures (MLCs), while IL-2 and IL-2R decreased in phytohemagglutinin (PHA)-stimulated PBL cultures. MSCs inhibited IL-2 induced proliferation, without absorbing IL-2. IL-10 levels increased in MLCs co-cultured with 10% MSCs, while the levels were not affected in PHA cultures. In MLCs inhibited by MSCs, antibodies against IL-10 further suppressed proliferation but had no effect in PHA cultures. Addition of indomethacin, an inhibitor of prostaglandin-synthesis, restored part of the inhibition by MSCs in PHA cultures. However, indomethacin did not affect MSC-induced inhibition in MLCs. To conclude, our data indicate that MSC-induced suppression is a complex mechanism affecting IL-2 and IL-10 signaling and may function differently, depending on T-cell stimuli. Prostaglandins are important in the inhibition by MSCs when the T cells were activated by PHA, but not alloantigens.     

Inhibition of proliferation without affecting the generation of cytotoxicity in the human mixed lymphocyte reaction

Phosphoramide mustard (PM) is considered to be the major tumoricidal metabolite of cyclophosphamide in vivo. The effects of this metabolite in vitro on several immune functions of human lymphocytes have been investigated. Very low concentrations (10(-7) to 10(-9) M) of PM added to lymphocyte cultures inhibited proliferation of the lymphocytes in response to mitogens and alloantigens. At these concentrations, inhibition of proliferation appeared to be due to a direct action of PM on the proliferative cells. Thus, concanavalin A-stimulated lymphocytes still acquired IL-2 receptors (Tac antigen) normally in the presence of PM (10(-6) to 10(-9) M). Only exceedingly high concentrations of PM (10(-5) M or greater) prevented the acquisition of Tac antigen. Similarly, the inhibition of proliferation was probably not related to endogenous IL-2 levels: addition of exogenous IL-2 to PM-containing cultures did not result in any restoration of proliferation. Further evidence that PM directly affected proliferative cells was that low concentrations of PM inhibited the proliferation of T cells continuously growing in IL-2. The exposure time to PM necessary for inhibition was essentially identical to those for lymphoproliferative responses to mitogens and alloantigens. Paradoxically, however, the generation of cytotoxic lymphocytes in mixed lymphocyte reactions (MLRs) and mixed lymphocyte tumor cell cultures (MLTCs) was very resistant to PM. In parallel MLRs and MLTCs the cytotoxic responses were resistant to approximately 1000-fold more PM than were the proliferative responses. Only at 10(-5) M PM were these inhibited. These data suggest that clonal expansion of cytotoxic lymphocytes or their precursors by proliferation is not an absolute requirement for the generation of cytolytic activity.     

The requirement for macrophage-lymphocyte interaction in T lymphocyte proliferation induced by generation of aldehydes on cell membranes.

Guinea pig T lymphocyte proliferation induced by sodium periodate (NaIO4) or neuraminidase-galactose oxidase (NG) occurs when lymphocytes and macrophages are cultured together after treatment of either purified T lymphocytes or macrophages with these agents. Regardless of which cell initially bears the modified surface carbohydrate, lymphocyte proliferation requires the presence of viable homologous macrophages and fails to occur when they are replaced with fibroblasts, erythrocytes, L2C leukemia cells, thymocytes, PMN, line I hepatoma cells, or murine macrophages. Lymphocyte proliferation resulting from NaIO4 or NG treatment of lymphocytes is diminished when these cells are treated with proteolytic enzymes or aged in in vitro culture for 48 hr. By contrast, proteolytic enzyme treatment or in vitro aging has no effect on the ability of NaIO4 or NG-treated macrophages to induce lymphocyte proliferation. The requirement for macrophage-lymphocyte interaction in NaIO4 or NG-induced lymphocyte proliferation is indicative of a central role for the macrophage in the initiation of T lymphocyte proliferation.     

Variable lymphocyte responses in rats after space flight.

Most studies of human blood lymphocyte function following space flight have indicated that microgravity suppresses T cell proliferation. However, several other postflight experiments with animals have shown no decrease in proliferation of lymphocytes from peripheral lymphatic tissues, suggesting that different tissues may be variably affected by microgravity. Therefore, we examined the proliferation of lymphocytes from both spleen and lymph nodes of rats following a 4-day flight aboard the Space Shuttle. The experiments were designed to investigate tissue variability as well as potential mechanisms involved in suppressing proliferation. We found that proliferation of lymph node lymphocytes (LNL) from flight (FLT) animals stimulated with the antigen receptor-dependent T cell mitogen concanavalin A was depressed and could not be restored by supplementing cultures with interleukin 1 or interleukin 2 (IL-2). Response to another receptor-dependent mitogen, phytohemagglutinin, was not decreased. However, proliferation of FLT LNL following stimulation with the receptor-independent, mitogenic combination of phorbol ester and ionomycin was depressed. LNL IL-2 activity, cell surface marker expression, and B cell responses to mitogen were normal. Thus, deficits in antigen receptor/ligand interactions, cell surface marker expression, or IL-2 did not account for the suppressed lymphocyte proliferation observed postflight. In contrast to LNL, FLT splenocyte proliferation was not depressed. Assayable IL-2, IL-2 receptor expression, and cell surface marker expression likewise were unaffected by space flight. The differences between lymph node and splenic responses demonstrate the tissue-specific nature of microgravity effects on individual lymphatic tissues.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Helper and suppressor functions of panned populations of immune peritoneal T cells with reactivity to the rat mammary adenocarcinoma 13762A

The phenotype of glass-adherence-depleted tumor-immune peritoneal exudate lymphocytes (PEL) which generated anti-tumor reactivity against the rat mammary adenocarcinoma 13762A in vitro was examined by indirect panning. Monoclonal antibodies W3/25 and OX8, directed against the CD4 and CD8 differentiation antigens, respectively, were used to separate immune PEL into subsets of functionally different T cells. The panned populations of immune PEL were examined for anti-tumor reactivity in three different in vitro assays. Tumor-specific proliferation, tumor-specific induction of the helper lymphokine interleukin 2 (IL-2), and tumor-specific induction of an antiproliferative tumor-induced suppressor lymphokine (TISL) were determined. Panning experiments together with indirect immunofluorescence analysis of unpanned immune PEL indirectly indicated that a proportion of cells (15-20%) coexpressed CD4 and CD8 antigens. Strong tumor-specific proliferation and IL-2 and TISL production were generated from these double-positive cells, as determined by double-panning experiments. Significant tumor-specific proliferation and IL-2 production were produced also from CD4+CD8- cells, but TISL production was minimal, and could be accounted for by contaminating CD4+CD8+ cells. CD4-CD8+ cells produced negligible responses against the tumors as measured by these three assays, and no synergistic responses were demonstrated when CD4-CD8+ cells were incubated together with CD4+CD8- cells. These data demonstrated that CD4+CD8+ T cells exist in primed populations of rat peripheral lymphocytes, and that both helper and suppressor functions were generated from these double-positive cells.     

Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.     

Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor).

IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation. Studies with purified subpopulations of PHA-activated lymphoblasts and with cloned lines of human T cells indicated that IL-12 caused the proliferation of activated T cells of both the CD4+ and CD8+ subsets. This effect of IL-12 was independent of IL-2 because it was not blocked by antibodies to either IL-2 or IL-2R. The maximum proliferation induced by IL-12 was 31 to 72% of the maximum caused by IL-2; however, IL-12 was active at a lower effective concentration (EC50 = 8.5 +/- 1.3 pM) than IL-2 (EC50 = 52 +/- 8 pM). Combination of suboptimal amounts of IL-12 and IL-2 resulted in additive proliferation, up to the maximum induced by IL-2 alone. IL-12 also caused the proliferation of lymphocytes activated by culture with IL-2 for 6 to 12 days. CD56+ NK cells were among the IL-12-responsive cells in the IL-2-activated lymphocyte population. Unlike IL-2 or IL-7, IL-12 caused little or no proliferation of resting peripheral blood mononuclear cells (PBMC). In this regard, IL-12 was similar to IL-4. However, IL-12 could enhance the proliferation of resting PBMC caused by suboptimal amounts of IL-2, whereas IL-4 inhibited IL-2-induced PBMC proliferation. Thus, IL-12 is a growth factor for activated human T cells and NK cells; however, its spectrum of lymphocyte growth-promoting properties is distinct from that of IL-2, IL-4, or IL-7.     

Effects of N-acyl-2-hydroxymethyl aziridines on in vitro proliferative responses of human lymphocytes stimulated by mitogens

Aziridines have been shown to possess marked immunotropic activity. The aim of this work was to study the in vitro effects of different concentrations of three novel aziridines, 2-hydroxy-methyl-1-(N-phtaloylglycyl) aziridine (aziridine 1), 2-hydroxy-methyl-1-(N-phtaloylalanyl) aziridine (aziridine 2) and 2-hydroxy-methyl-1-(N-phtaloylphenylalanyl) aziridine (aziridine 3), on the proliferative responses of human lymphocytes stimulated by mitogens (concanavalin A (Con A) and lipopolysaccharide (LPS)), and interleukin-2 (IL-2), interleukin-6 (IL-6) secretion. The results showed that aziridines 1 and 3 significantly stimulated the resting and Con A or LPS lymphocyte proliferation at concentrations between 1 micromol/l and 1 mmol/l, in a dose-dependent manner, the action of aziridine 3 being the highest. They also increased IL-2 and IL-6 secretion. However, aziridine 2 had no effect on the resting lymphocyte proliferation in the absence of mitogens, at any concentration used, reduced Con A-stimulated T lymphocyte proliferation and LPS- stimulated B lymphocyte proliferation in a dose dependent manner and diminished IL-2 and IL-6 production. None of the three aziridines affected cell viability. In conclusion, the three aziridines used in this study displayed immunomodulatory properties. Aziridines 1 and 3 are potentially immunostimulant while aziridine 2 is immunosuppressive and could be used to provide nonspecific cell-mediated immune responses.     

Glutathione modulates activation-dependent proliferation of human peripheral blood lymphocyte populations without regulating their activated function

L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.