Microtubules and lymphocyte responses: effect of colchicine and taxol on mitogen-induced human lymphocyte activation and proliferation

The role of microtubules in mitogen-induced human lymphocyte activation and proliferation was examined. The effect of colchicine, a microtubule-disrupting agent, was compared with taxol, a microtubule-stabilizing drug, and with isaxonine (N-isopropyl-amino-2-pyrimidine orthophosphate), a proposed microtubular-active drug. Lymphocyte proliferation, assessed by measuring the increase in the number of cells in mitogen-stimulated cultures, was completely suppressed by both colchicine and taxol (100 nM) whereas significant inhibition by isaxonine required much higher concentrations (5 mM). In order to characterize the inhibition, initial lymphocyte blast transformation and subsequent DNA synthesis were investigated. Neither colchicine nor taxol inhibited lymphocyte blast transformation assessed by quantitating the change in volume of the stimulated cells after a 24-hour incubation. In contrast, isaxonine (2-5 mM) suppressed blast transformation. Initial DNA synthesis, evaluated by measuring the cumulative incorporation of [3H]thymidine between 30 and 48 hours of culture, was inhibited in a concentration-dependent manner by both isaxonine and colchicine but not by taxol. Electron microscopic studies confirmed that both taxol and colchicine (10 nM) arrested the responding lymphocytes in mitosis, and that isaxonine inhibited initial activation. These results suggest that normal microtubule function is only necessary for cell division and that drug effects on blast transformation and initial DNA synthesis are unrelated to microtubules.     

Potentiation of lymphocyte activation by colchicine.

Proliferation of human lymphocytes induced by IO4- is potentiated by 30 min exposure to colchicine (10(-6)M), whereas the response to Con A is inhibited. Treatment with colchicine before or after IO4- modification has similar enhancing effects. Lumicolchicine does not alter proliferative responses. In addition to the proliferation of IO4--oxidized cells, irradiated IO4- modified lymphocytes induce proliferation when mixed with untreated lymphocytes. Enhancement occurs in both these conditions only when IO4--modified cells are treated with colchicine. Preliminary data indicate that proliferation in mixed lymphocyte cultures is also potentiated when either stimulating or responding cells are pretreated with colchicine. These findings suggest a selective stimulatory effect of colchicine on lymphocyte responses induced by cell-cell contact. Agents that modify microtubular assemblies might regulate the induction of immune responses that involve cellular interactions.     

Inhibitory activity of medium-dialyzed transfer factor on lymphocyte blastogenesis.

Human transfer factor was prepared from normal donors with marked skin reactivity against common microbial antigens by dialysis of lymphocyte extracts versus tissue culture medium (TFmd). Several batches of TFmd were tested for their ability to specifically increase the DNA synthesis of unsensitized lymphocytes in vitro in the presence of the corresponding antigens (PPD, SKSD, Candida, Thistoplasmin). No transfer or immunologic reactivity by TFmd was observed. There was, however, a significant unspecific inhibition by TFmd of lymphocyte cultures stimulated by antigens, PHA or allogeneic cells.     

The effects of corticosteroid concentrations on lymphocyte blastogenesis

High concentrations of methylprednisolone added for prolonged periods in vitro to lymphocyte cell cultures inhibited allogeneic-cell-induced or phytohemagglutinin (PHA)-induced blastogenesis in contrast to lower concentrations which were inhibitory only if added before or within several hours of blastogenic induction.     

Glucocorticosteroid modification of lymphocyte blastogenesis in tumor-bearing hosts

The present study was done to evaluate glucocorticosteroid modulation of the individual cells contributing to tumor-induced dualistic (macrophage and suppressor T-cell) suppression in mitogen-induced lymphocyte proliferation. Steroid-sensitive T cells from normal mice showed a characteristic decrease in phytohemagglutinin responsiveness in the in vitro presence of 0.1 μg hydrocortisone sodium-21 succinate (HC), while tumor-bearing host (TBH) T cells demonstrated little or no decrease. This correlates well with kinetic study results demonstrating decreased HC-mediated suppression of mitogen-induced TBH T-cell DNA synthesis at 8 to 10 days posttumor cell inoculation. Hydrocortisone-treated cells from mice with advanced tumors had significantly higher degrees of blastogenesis than untreated TBH cells. In the optimal presence of both macrophages (Mφ) and HC, TBH cells had significantly greater blastogenesis than their normal counterparts. Alone, Mφ or HC depressed normal host PHA responsiveness. Experiments were carried out to determine if the increased blastogenesis was due to the additional Mφ presence in TBH. In vivo administration of methylprednisolone acetate demonstrated that steroids can inhibit tumor growth. A significant delay in tumor appearance occurred when the steroid was administered 4 to 7 days after tumor transplant perhaps due to the measurable lack of suppressor T cells. These studies indicated that steroid suppression was directed against a sensitive suppressor T cell and possibly acts by preventing its differentiation. The role of the Mφ in the afferent limb of the cell-mediated immune response seems less clear, but the results suggest nonspecific inhibition of TBH T-cell response to blastogenic stimulus.     

The effect of hyposensitization on alternaria-induced lymphocyte blastogenesis

Twenty-one atopic children were treated with alternaria hyposensitization therapy. Leukocytes from these children were cultured with and without alternaria and phytohemagglutinin prior to treatment, and after 6 and 12 months of treatment. Patients demonstrating positive immediate cutaneous hypersensitivity showed significant lymphocyte transformation prior to hyposensitization whereas the skin test-negative patients did not. The skin test-negative patients developed significant lymphocyte transformation after 6 months of hyposensitization. Twelve months after the initiation of therapy both skin test-negative and skin test-positive patients showed a decreased lymphocyte reactivity, and there was no longer any significant change in the concomitant phytohemagglutinin responses indicating that hyposensitization had a specific effect on alternaria-induced in vitro lymphocyte reactivity. Plasma factors were found to have modulating effects on the lymphocyte reactivity of hyposensitized patients.     

Inhibition of lymphocyte blastogenesis by C3c and C3d.

The C3 cleavage products C3c and C3d were tested for their ability to alter the immunoproliferative response of human peripheral mononuclear cells to the antigens SLO and SK-SD, and to the mitogens PHA and PWM. It was found that both C3c (30 to 120 micrograms/ml) and C3d (10 to 40 micrograms/ml) inhibited lymphocyte blastogenesis in the presence on antigens but not mitogens, when cells were cultured in either autologous plasma or FCS. Similarly, the response to antigens of cell populations enriched for T lymphocytes was inhibited, whereas the response to optimal or suboptimal doses of mitogens was unaffected. When nonadherent (NA) cells were reconstituted with increasing numbers of adherent (AD) cells to potentiate the proliferative response of NA cells to the antigen SLO, the addition of either C3c or C3d abolished the potentiation of the response at low levels of reconstitution. However, at given dose of C3c or C3d, addition of excess AD cells could restore the proliferative response. These results suggest that both C3c and C3d can inhibit T cell proliferation in response to antigen and that they may act at the level of the monocyte-T lymphocyte interaction to modulate cellular immune responses.     

Effect of cell-free murine liver extract on lymphocyte blastogenesis in vitro.

The effect of cell-free liver extract (LE) on the proliferation of spleen cells in vitro was examined using [³H]thymidine incorporation. LE inhibited the blastogenic response of murine lymphocytes stimulated with plant mitogens, phytohemagglutinin, and concanavalin A and in the mixed lymphocyte reaction (MLR). Suppression of cell proliferation occurred whether the LE was syngeneic or allogeneic to the responding cells. This effect was observed only when LE was present in cultures, as preincubation of cells with LE did not impair their capacity to respond to stimulation. Profound suppression of proliferation was achieved with the addition of LE to the culture up to 48 hr after the onset of stimulation. However, the inhibitory effect was readily reversible upon removal of LE from the culture. Furthermore, although LE was capable of suppressing the generation of cytotoxic lymphocytes, LE did not interfere with their capacity for cytolysis. These findings indicate the presence of a potent inhibitor of lymphocyte proliferation in a cell-free extract of murine liver.     

Functional analysis of macrophage hybridomas. III. Inhibition of lymphocyte blastogenesis and tumor proliferation

Four cloned macrophage hybridoma cells prepared by fusion of splenic adherent cells with a P388D1 macrophage tumor markedly inhibited the growth of lymphocytes and tumor cells. Macrophage clones 5, 8, 63, and 64 are strong inhibitors of B-cell blastogenesis, T-cell blastogenesis, and tumor proliferation, while the P388D1, tumor line and clones 13, 59, and 67 demonstrated little inhibitory activity in all three systems. The inhibitory effect of macrophage hybridomas can be detected within 8 hr, although greater inhibition was noted following longer incubation. The correlations among these three assay systems suggest that similar mechanisms may be involved. The data indicate that the inhibition of cell proliferation was not due to cell lysis. Furthermore, the inhibition of lymphocyte proliferation in Con A-stimulated cultures was not accompanied by inhibition of lymphokine production in the same cultures. Neither prostaglandins nor hydrogen peroxide appear to be primarily responsible for growth inhibition. The inhibitory properties of these macrophage hybridoma lines represent a stable phenotype and provide a homogeneous source of cells for further analysis of the phenomenon.     

Antigen-induced in vitro lymphocyte blastogenesis in the rabbit: a T-cell response.

In vitro lymphocyte stimulation of sensitized rabbit lymphocytes to specific antigen (ovalbumin) was found to depend on thymic-dependent lymphocytes. This conclusion is based on an enhanced response upon enrichment with T lymphocytes by passage of lymphocytes through nylon wool, and on the elimination of the response after treatment of lymphocytes with complement and an antiserum to rabbit thymus cells prepared in a goat. Specificity of the antiserum was demonstrated by elimination of in vitro T-cell function and retention of in vitro B-cell functions.     

Suppression of phytohemagglutinin-stimulated lymphocyte blastogenesis by ovine uterine milk protein

Two basic glycoproteins (UTM-P) with molecular weights of 57,000 and 59,000 were purified from ovine uterine milk collected on Days 125 and 130 of pregnancy. The UTM-P were evaluated for immunosuppressive activity in phytohemagglutinin (PHA)-treated, mixed lymphocyte (MLC) and resting lymphocyte (RLC) cultures. For PHA and RLC cultures, UTM-P (2.5 to 800 micrograms UTM-P/ml) were added to 1 X 10(6) lymphocytes and 0.8 micrograms of PHA (for PHA cultures only), while for the MLC, UTM-P (50 to 1600 micrograms UTM-P/ml) were added to 5 X 10(5) lymphocytes combined from each of two ewes. Following [3H] thymidine addition, cells were later harvested for determination of thymidine incorporation. Lymphocyte blastogenesis was suppressed by UTM-P in PHA (R2 = 0.32 to 0.92, P less than 0.01 to 0.001), MLC (R2 = 0.8, P less than 0.001) and RLC (R2 = 0.65, P les than 0.01) experiments. To determine reversibility, PHA-treated lymphocytes were incubated with UTM-P for 6, 12 or 24 h, then washed to remove surface UTM-P. Incubation was continued in the presence of PHA as with other experiments. Exposure of lymphocytes to UTM-P for 6 or 12 h did not result in suppression of blastogenesis, whereas exposure for 24 h was sufficient for suppression (P less than 0.01). In an additional experiment, UTM-P were added to PHA-treated cultures at 0, 6, 12 or 24 h. Suppression (P less than 0.01) of blastogenesis was observed for each time period. Immunosuppressive activity was not mediated by overall cytotoxicity and was not affected by routine handling and storage of UTM-P. Data from these experiments provide one explanation for tolerance of the conceptus allograft during defined stages of ovine pregnancy.     

Relation of 8-ketotrichothecene and zearalenone analog structure to inhibition of mitogen-induced human lymphocyte blastogenesis.

The 50% effective doses of fusarenon X, nivalenol, deoxynivalenol, and 15-acetyldeoxynivalenol required to reduce [3H]thymidine uptake in mitogen-stimulated human lymphocytes by 50% were 18, 72, 140, and 240 ng/ml, respectively. These results indicated that lymphotoxicity of 8-ketotrichothecenes decreased according to the C-4 substituent order acetyl greater than hydroxyl greater than hydrogen, whereas acetylation of position C-15 of deoxynivalenol caused a slight decrease in in vitro toxicity. The 50% effective doses for zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were 3,500, 6,300, 36,000, 3,750, and 33,000 ng/ml, respectively, suggesting that a keto group or alpha-hydroxyl at the position C-6' contributed to the lymphotoxicity of the parent molecule. The inhibitory effects of zearalenone analogs observed in the blastogenesis assay did not correlate with the estrogenic potencies of these compounds. All 8-ketotrichothecenes and zearalenone analogs tested were capable of inhibiting B- and T-cell subsets stimulated by a mitogen panel of leukoagglutinin, concanavalin A, and pokeweed mitogen.     

Mechanisms of C-type viral leukemogenesis. I. Correlation of in vitro lymphocyte blastogenesis to viremia and leukemia.

Various inbred strains of mice respond immunologically to genetically transmitted ecotropic C-type viruses. Part of this response is T cell blastogenesis with type specificity for the viral envelope glycoprotein gp71. Of those nonviremic, nonleukemic strains, and F1 crosses examined, in which virus expression occurs early in life, gp71-specific blastogenic T cells were detected within the first 2 months of age and temporally preceded the development of a humoral immune response. However, in the viremic, highly leukemic strain of AKR mice, gp71-specific T cell blastogenesis in vitro was readily detectable throughout the preleukemic phase, the first 5 months of age. In appropriate F1 crosses and backcrosses, the persistent in vitro blastogenic response segregated with viremia and leukemia. These data suggest that in vivo T cell stimulation by endogenous viral gp71, caused by viremia, may contribute to virus-induced leukemogenesis in mice.