Polyclonal activation of guinea pig spleen lymphocytes

The incorporation of 3H-thymidine in newly formed DNA was studied in guinea pig spleen cells stimulated by phytohemagglutinin (PHA) and/or concanavalin A (Con A). In spleen cells stimulated by PHA, two peaks of thymidine uptake were observed for two different doses of lectin whatever the number of cultured cells. Furthermore, thymidine uptake is proportional to the number of the cultured cells. During Con A-induced mitogenesis, two peaks were also observed but only when using a great concentration of cells. Maximal thymidine incorporation depended on both the cell number and the concentration of Con A. When cells were stimulated by both PHA and Con A, thymidine uptake was strongly depressed as compared to the one observed using PHA or Con A alone. On the other hand, supernatants from unstimulated spleen cells had opposite effects on blastogenesis induced by Con A or by PHA; they depressed PHA-induced blastogenesis while enhancing the one induced by Con A. These results suggest that, in guinea pigs, both PHA and Con A induce thymidine incorporation in at least two lymphocyte populations through different mechanisms. This heterogeneity of lectin-induced T cell mitogenesis has to be taken in account when studying the mechanisms by which the immunomodulators are active at the T cell level.     

Inhibition of lectin-stimulated lymphocyte agglutination and mitogenesis by hydroquinone: Reactivity with intracellular sulfhydryl groups

Benzene-induced lymphocytopenia and hypoplasia in bone marrow, spleen, and thymus correlate with concentrations of polyhydroxy metabolites of benzene in these target organs. Pretreatment of Ficoll-purified rat spleen lymphocytes with hydroquinone (HQ) enhanced in vitro phytohemagglutinin (PHA)-stimulated mitogenesis at lower concentrations but inhibited lectin-stimulated blast transformation at higher concentrations. Following preincubation with HQ, viable lymphocytes failed to agglutinate or undergo blast transformation in the presence of PHA. This effect occurred in the absence of depressed energy production which suggested an impairment of cytoskeletal function. Other polyhydroxy metabolites of benzene, including p-benzoquinone, 1,2,4-benzenetriol, and catechol produced similar effects on mitogen response. N-Ethylmaleimide (NEM), a membrane penetrating alkylating agent with specificity for sulfhydryl (SH) groups, inhibited lymphocyte mitogenesis and agglutination at the same concentrations as HQ. 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB), a poorly penetrating SH reagent, had no effect on lymphocyte function. The concurrent inhibition of mitogenesis and agglutination by HQ and NEM was prevented by the addition of dithiothreitol (DTT), a SH compound, suggesting an important interaction of the metabolite (or its products) with intracellular SH groups critical to early events in blastogenesis. We suggest that interaction of the polyhydroxy metabolites of benzene with particularly reactive SH groups on microtubules may explain their sublethal effects of lymphocyte function.     

Stimulation of murine lymphocyte blastogenesis by mitogens in heat-killed Histoplasma capsulatum yeast cells.

In vitro blastogenesis by normal murine splenocytes from several mouse strains has been detected after exposure to heat-killed Histoplasma capsulatum yeast cells. Maximal lymphocyte stimulation induced by 10(4) heat-killed cells resulted in 20- to 45-fold increases in [3H]thymidine uptake by splenocytes when compared with responses by normal unstimulated lymphocytes. The kinetics for this response to heat-killed H. capsulatum cells has shown peak mitogenesis 3 days after culture. Examination of the mitogenic potential of soluble antigen preparations from H. capsulatum has revealed stimulation of lymphocyte blastogenesis with yeast cell sonicates and autolysates but not substances from autoclaved yeast cells. The levels of lymphocyte blastogenesis induced by sonicates or autolysates were comparable to mitogen responses stimulated by heat-killed cells. Preliminary biochemical characterization of the mitogenic factor(s) associated with yeast cell sonicates show two peaks of activity, at 178,000 and less than 12,000 Mr, which have a protein or glycoprotein nature. Finally, analysis of lymphocyte blastogenesis in cultures enriched for selected lymphocyte subpopulations has shown that T lymphocytes are preferentially stimulated by yeast cell mitogens.     

Silybin inhibition of human T-lymphocyte activation

Silybin, a 3-oxyflavone occurring in the thistle Silybum marianum, displays a dose-dependent inhibition of in-vitro lymphocyte blastogenesis induced by lectins (phytohaemagglutinin, Concanavalin A and pokeweed) and by anti-CD3 monoclonal antibody. The drug has no effect on cell viability and spontaneous 3H-thymidine incorporation, suggesting that the inhibitory activity is not due to aspecific toxicity. Since all the T-cell responses investigated require cell-membrane-associated events, the effect of silybin is probably at the level of the cell membrane, as for other flavonoids. Addition of CuSO4 prevents the inhibitory activity of silybin on PHA-induced proliferative response, indicating that the drug could exert its activity also by virtue of a chelation mechanism.     

Modulation of Murine Lymphocyte Mitogen Responses by Glycerol-Teichoic Acid

Glycerol-teichoic acid (GTA) showed a modulatory effect on the in vitro response of murine splenocytes to the mitogens concanavalin A (Con A) and lipopolysaccharide (LPS) as measured by incorporation of ³H-thymidine. GTA inhibited the response to Con A when added prior to addition of the mitogen, while addition 24 hr after had no significant effect on the response. The degree of suppression was dose dependent in a range from 0.1-5μg GTA/culture. The spleen cell response to LPS was enhanced by GTA when added prior to the mitogen. Peak enhancement occurred at 1-2 μgGTA/culture, depending on the time of addition. GTA added 24 hr after LPS produced no significant effect on mitogenesis. Addition of GTA alone to spleen cell cultures produced a slight suppression of DNA synthesis and was toxic at 10 μg/culture if incubated at least 66 hr. GTA is bound to murine spleen cells as indicated by decreased passive hemagglutination inhibition activity of culture supernates.     

Gastrointestinal Regulatory Peptides Modulate Mouse Lymphocyte Functions Under Serum-Free Conditions In Vitro

Conditions are described for performing mitogen (Concanavalin A, Con A; lipopolysaccharide, LPS) and mixed lymphocyte reaction (MLR) cultures using serum-free medium. The effects of exogenously adding several gastrointestinal regulatory peptides (β-endorphin, substance P, met-enkephalin, vasoactive intestinal peptide, bombesin and somatostatin) on the incorporation of ³H-methyl-thymidine was determined. It was observed that mitogen stimulation of lymph node cells with Con A was inhibited (70% of control) by vasoactive intestinal peptide (VIP) but spleen cells stimulated by LPS were insensitive to immunomodulation (98% of control). The ability of VIP to inhibit Con A induced thymidine incorporation was concentration dependent (10^-6 to 10^-18 M) and was not attributable to kinetic shifts or cell toxicity. None of the other tested neuropeptides affected Con A or LPS induced blastogenesis. MLR cultures were inhibited by VIP, β-endorphin and somatostatin in a biphasic manner with maximal inhibition observed at 10^-8 to 10^-12 M. Both substance P and bombesin exhibited slight immunoenhancing properties at 10^-14 to 10^-18 M. Met-enkephalin was ineffective as an immunomodulator of MLR cultures. The utility of using serum-free medium in identifying neuropeptides with immunomodulatory properties are discussed.     

Phorbol ester circumvents the need for macrophages as well as for mitogenic lectins in the stimulation of lymphocytes with wheat germ agglutinin or the calcium ionophores A23187 or ionomycin

Numerous biochemical events precede the proliferation of primary lymphocytes stimulated by mitogenic lectins in the presence of macrophages. Various compounds can activate parts of this response. Specifically the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, (TPA), can replace the requirement for macrophages, apparently by mimicking the macrophage product interleukin 1 (IL1). Wheat germ agglutinin (WGA), itself a non-mitogenic lectin, is reported to cause a calcium flux, phosphatidylinositol turnover, and enhance interleukin 2 (IL2) synthesis. In spite of these positive responses, WGA inhibits DNA synthesis caused by mitogenic lectins. Nevertheless, in this study, we tested the possibility that together TPA and WGA could complement and bring about DNA synthesis. This prediction turned out to be true. The combination of two non-mitogenic compounds resulted in lymphocyte proliferation. The TPA overcame the inhibitory effects of WGA. Moreover, macrophages were not required. The TPA also synergized with the calcium ionophores A23187 or ionomycin to cause lymphocyte proliferation in the absence of macrophages. WGA and the ionophores together did not cause proliferation, a finding which suggested that they fulfill the same roles. These observations led us to conclude that at least two signals were required for lymphocyte stimulation. One signal caused the mobilization of calcium and the other signal circumvented the need for macrophages or macrophage products possibly by mimicking diacylglycerol, the activator of protein kinase C.     

T lymphocyte-mediated cytolysis. III. Delineation of mechanisms whereby mitogenic and non-mitogenic lectins mediate lymphocyte-target interaction

We have investigated the mechanism(s) by which mitogenic (concanavalin A [Con A], phytohaemagglutinin [PHA] and Lens culinaris agglutinin [LCA]) and non-mitogenic (soybean agglutinin, peanut agglutinin, wheat-germ agglutinin and pokeweed mitogen) lectins mediate, non-specifically, lectin-dependent lymphocytotoxicity (LDCC). We show that non-mitogenic lectins are ineffective mediators of LDCC, due to their inability to mediate effective binding of effector cytotoxic T cells (EC) and target cells (TC), and not to their failure to 'activate' TC-bound EC, as proposed before. Evidence is presented that in LDCC Con A and PHA exert their primary effect(s) by affecting the TC rather than the EC. Although the lectin LCA, unlike Con A and PHA, is equally reactive with either EC or TC, a direct comparison is difficult since the presence of LCA, but not Con A or PHA, during the entire assay is required for optimal kill. Furthermore, all three LDCC-supportive lectins (PHA, Con A and LCA) show a similar TC preference when tested in a EC-TC conjugation assay. Taken together, these results are inconsistent with the theory that lectins mediate LDCC by 'bridging' EC and TC through lectin-binding receptors followed by 'activation' of the TC-bound EC. We would like to suggest that the potential of mitogenic lectins to mediate EC-TC interaction is related to their modification of TC-surface constituents, possibly major histocompatibility complex determinants, rendering them recognizable, non-specifically by EC.     

The effect of particle size on the immunodepressive properties of silica

Five silica preparations were tested in the mouse for their effects on the in vivo humoral immune response to SRBC, the in vitro macrophage phagocytosis of SRBC and the in vitro blastogenic response of non-adherent spleen cells to Con A and LPS¹. One silica preparation was composed exclusively of small particles and produced the greatest depression of all functions tested with the least variability in results. Four of the silica preparations were heterogeneous with respect to particle size, and the heterogeneous preparations containing the highest percentage of small particles had the most depressive effect on the in vivo humoral immune response to SRBC and on in vitro macrophage phagocytosis of SRBC but not on the in vitro blastogenic response to mitogens. All 4 heterogeneous preparations produced great variability in each of the functions tested.Small sized particles from the 4 heterogeneous preparations were prepared by differential sedimentation, and were more depressive in the in vitro assays for macrophage phagocytosis and lymphocyte blastogenesis than the corresponding unfractionated heterogeneous preparation. The large sized particles either had no effect or enhanced both functions. All unfractionated silica preparations were toxic for non-adherent spleen cells, but the homogeneous small particle preparation and the small particles from the heterogeneous preparations were more cytotoxic. These data indicate that silica has a direct depressive effect on both macrophage and lymphocyte functions and that the depressive effects are inversely related to particle size.     

Inhibitory effects of tunicamycin on the mixed lymphocyte reaction and mitogen-induced lymphocyte blastogenesis

The role of asparagine(Asn)-linked saccharides on the surface of lymphocytes in cellular interactions was examined by performing studies on the effects of tunicamycin (TM), which inhibits the glycosylation of proteins N-glycosylated at asparagine residues, on the mixed lymphocyte reaction (MLR) and mitogen-induced lymphocyte blastogenesis of human lymphocytes by measuring 3H-thymidine (TdR) incorporation. Responses were expressed as percentages of that of the control (MLR without TM). The lymphocyte blastogenesis in the one- and two-way MLR were, respectively, 43.1% and 48.0% of the control at 0.1 microgram/ml of TM, and 5.5% and 7.2% at 1 microgram/ml of TM. The inhibitory effect of TM on the one-way MLR was shown using TM-pretreated stimulator cells, TM-pretreated responder cells or both. TM blocked lymphocyte blastogenesis in the secondary as well as in the primary MLR. The inhibitory effect of TM on the two-way MLR was observed when TM was added on day 0 to day 2, but not on day 4 of incubation. TM blocked mitogen-induced lymphocyte blastogenesis by phytohemagglutinin, concanavalin A or by pokeweed mitogen. As TM had no cytotoxic effect on cultured cells, these inhibitory effects of TM were thought to be due to the loss of Asn-linked saccharides from glycoprotein of the surface of lymphocytes. These findings indicated that Asn-linked saccharides of glycoprotein on the surface of lymphocytes were important in cellular interactions that are necessary for the cellular immune response.     

Inhibition of in vitro lymphocyte blastogenesis by inhibitor produced by cultured human lymphoblasts

Normal human lymphoblastoid cell lines, growing in continuous suspension culture, produce inhibitors of in vitro lymphocyte blastogenesis. The inhibitor reduces human lymphocyte blastogenic responses to phytohaemagglutinin, streptolysin-O and the mixed lymphocyte culture 90–99%, is non-cytotoxic and can inhibit both newly initiated and on-going responses. The inhibitor is heat-stable at 80°C but labile at 100°C, non-dialysable and degraded by pronase but not DNase or RNase. It is species- and tissue-specific and does not inhibit the proliferation of mouse lymphocytes, human melanoma cells or human bone marrow in vitro colony-forming cells. Inhibitor was produced only under very specific conditions of crowding. Thus, maximal inhibitor production occurred at 5 × 10⁶ lymphocytes per cm² culture surface area while only 0–5% of the maximal amount was produced at 10⁶ or 5 × 10⁷ lymphoblasts per cm². This data is relevant to the nature of feedback control of immunological reactions and may guide the development of new classes of immunosuppressants.     

驱虫斑鸠菊对正常小鼠免疫功能的影响

目的探讨驱虫斑鸠菊对小鼠免疫功能的影响,揭示其免疫作用机理.方法利用[3H]-TdR掺入法分别测定了驱虫斑鸠菊对环胞菌素A(Con A)诱导的小鼠脾脏T淋巴细胞和细菌脂多糖(LPS)诱导的B淋巴细胞增殖活性以及脾脏T淋巴细胞分泌白细胞介素-2(IL-2)活性的影响,运用酶联免疫吸附试验测定了驱虫斑鸠菊对小鼠血清中抗体活性的影响;采用流式细胞仪测定脾脏B淋巴细胞表面抗原CD19的表达水平.结果驱虫斑鸠菊的低、中、高3个剂量体内对脾脏T、B淋巴细胞的增殖活性、血清总抗体和针对人A375黑素瘤细胞抗原特异性抗体含量、B细胞表面抗原CD19的表达以及对脾脏T淋巴细胞分泌IL-2活性都具有明显抑制作用.结论驱虫斑鸠菊对机体体液免疫和细胞免疫功能都具有明显抑制作用.     

Mitogenic activity of staphylococcal peptidoglycan.

Staphylococcus aureus peptidoglycan displayed a marked dose-dependent mitogenic activity for mouse splenocytes and human peripheral blood lymphocytes in vitro, as measured by increased [3H]thymidine incorporation. Similarly it was mitogenic for athymic nude mouse spleen cells, whereas no blastogenic effect was observed in T cell-enriched and B cell-depleted mouse lymphocyte cultures. These data demonstrate that peptidoglycan-responding cells in mouse spleen cell cultures are B lymphocytes.     

Suppression of human lymphocyte mitogenesis mediated by phagocyte-released reactive oxygen species: comparative activities in normals and in chronic granulomatous disease

Human blood monocytes and neutrophils stimulated in vitro with phorbol myristate acetate, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, activated zymosan, or heat aggregated gamma-globulin were found to suppress lymphocyte mitogenic responses. In activated phagocyte-lymphocyte cocultures, both blast transformation and [3H]-thymidine incorporation were reduced while numbers of dead cells were increased, thus suggesting a cytolethal suppressive mechanism. Suppression was prevented by catalase but not by other oxygen radical scavengers nor by cyclooxygenase inhibitors, thus implicating H2O2 as the suppressive mediator. Activated monocytes and neutrophils but not lymphocytes released measurable quantities of H2O2 into cell supernatants. However, transfer of an inhibitory effect with these supernatants was not routinely achieved. Finally, as opposed to normals, lymphocyte blastogenesis in chronic granulomatous disease patients was not inhibited by their activated phagocytes. However, catalase -reversible suppression could be restored in cocultures of normal phagocytes and patient lymphocytes. In conclusion, these studies demonstrate a potentially important mechanism whereby activated phagocytes may alter lymphocyte reactivity.     

The suppressor factor NBxFO is not species-specific

The NBxFO factor was obtained from the supernatant liquids of neonatal spleen: myeloma fusions. Previously it had been shown that this factor could inhibit the proliferative response of alloreactive T cell lines. In this study the factor was found to inhibit the MLR of the parent species (mouse) as well as the MLRs of humans and rats. Thus, the NBxFO factor has activity that is not species-specific. Furthermore, the factor was found to inhibit the lectin-induced mitogenesis of these 3 species. Gel chromatography revealed that the moleclar weight of the molecule responsible for suppressing human lymphocyte mitogenesis is the same as previously determined for suppression of mouse proliferative responses.     

Monoclonal antibodies against a rat leucocyte antigen block antigen-induced T-cell responses via an effect on accessory cells.

The MRC OX-45 and OX-46 mouse monoclonal antibodies recognize a rat cell surface glycoprotein of 45,000 MW that is present on a wide variety of haematopoietic cells and on endothelial cells. MRC OX-45 IgG or F(ab')2 blocked the primary mixed lymphocyte response (MLR) and the secondary response of T lymphocytes to the soluble antigen DNP-BGG. In contrast, the antibodies had no effect on the cytotoxic activity of specific (CTL) or non-specific (NK) killer cells or on proliferative responses stimulated by lectins or oxidative mitogenesis. The inhibitory effect was at the level of stimulator cells rather than responders since mouse anti-rat xenogeneic MLRs were inhibited but rat anti-mouse responses were unaffected. However, the effect was not a direct one because inhibition was seen when irradiated spleen cells were used as stimulators but not when cell populations highly enriched for dendritic cells were used. In the latter case, inhibition potentiated by antibody could be restored if a peritoneal cell population enriched for macrophages was added back to the cultures. The inhibitory effects of these monoclonal antibodies seem most likely to be due to potentiation of nonspecific suppression by macrophages.     

Studies on mitogen activity of the dextrans

The influence of dextrans of various molecule sizes on blastic transformation induced by Con A and PHA lectins was studied. It was found that particularly dextran of smaller molecule inhibits blastogenesis induced by Con A. The dextran of greater molecule is less so inhibitory. On the other hand, in the effect of dextrans on the induction provoked by PHA their stimulating component is rather pronounced. Discussing the obtained results it was pointed out that the dextrans depending on the molecule exhibit the affinity to lymphocyte receptors for lectins and both blocking and stimulating effects may be involved.     

Differential Effect of Mixed D1/D2 and Selective D2 Dopaminergic Antagonists on Mouse T and B Lymphocyte Proliferation and Interleukin Production In Vitro

The effects of some dopaminergic antagonists were investigated on mouse lymphocyte proliferative responses in vitro. The mixed D₁/D₂ dopaminergic antagonists chlorpromazine, haloperidol and fIupentixol inhibited ³H-Thymidine incorporation into adult BALB/c mouse spleen cells stimulated by concanavalin A, lipopolysaccharide from Escherichia coli, and allogenic cells in a mixed lymphocyte reaction. The inhibition was achieved at concentrations greater than 10^-6M. It was not accounted for by decreased cell viability and it was no longer demonstrable when the compound was added 24h or 48h after the mitogenic stimulus. Conversely selective D₂ dopaminergic antagonists sulpiride, metoclopramide and domperidone had no inhibitory effect at concentrations ranging from 10^-9 to 10^-5 or 10^-4M. The three mixed D_l/D₂ antagonists inhibited the mitogenic effect of interleukin-1 on concanavalin A-stimulated thymocytes, but not the activity of interleukin-2 on the proliferaiton of the CTLL-2 cell line. The mixed D_l/D₂ antagonists interfered with the production of interleukin-2 but not with that of interleukin-1. These results indicate that dopaminergic antagonists may differerentially affect lymphocyte proliferative responses to T or B cell mitogens or alloantigens. The mechanisms involved in terms of receptor specific or non specific phenomenons are discussed.     

Studies on the mechanism of action of cyclosporin A

The mechanism of cyclosporin A (CS-A), a compound known to act selectively on the immunocompetent lymphocyte, was investigated in a series of in vitro studies. CS-A, provided it was added simultaneously with mitogen, inhibited the incorporation of tritiated uridine and thymidine into mouse spleen cells and human peripheral blood lymphocytes. Addition of CS-A 48 h after onset of culture did not affect cell division, indicating that it acted at an early stage of lymphocyte stimulation, exerted no inhibitory effect on lymphoblasts and did not possess anti-mitotic activity. It was further shown that CS-A was not lymphotoxic and that its effect was reversible, because suppressed spleen cells recovered their proliferative capacity after a 24 h elution period. Inhibition of PFC in the Mishell-Dutton assay provides evidence of an anti-T helper cell action of CS-A.     

Induction of lymphocyte proliferation and membrane changes by lipopeptide derivatives of the lipoprotein from the outer membrane of Escherichia coli.

Lipoprotein from the outer membrane of E. coli, a potent novel mitogen, was digested by pronase treatment resulting in lipopeptide fragments containing 2-5 amino acids bound to diacylglyceryl-N-acylcysteinthioether. The lipopeptides were characterized by amino acid analysis and gas chromatography and were checked for mitogenicity. We found that all lipopeptide fragments were able to stimulate the uptake of 3H-uridine into RNA and 3H-thymidine into DNA in mouse spleen cells of several strains. The response of splenocytes of congenitally athymic mice was comparable to that of normal animals. A weak stimulation of DNA synthesis was also observed in thymocytes. The mitogenicity of the products was abolished by mild alkali hydrolysis which removes the ester-bound fatty acids. We conclude that the N-terminal lipopeptide region of lipoprotein is responsible for the mitogenic activity of the molecule. Lipopeptide as well as lipoprotein were found to cause early membrane changes in lymphocyte plasma membranes. After 4 hours we found an increased incorporation of 14C-oleate and 14C-acetate into lecithin. The membrane changes observed are similar to those brought about by mitogenic lectins, which suggests a similar mechanism for the induction of lymphocyte activation for both types of mitogens.