Effect of polyamine depletion in vivo by DL-alpha-difluoromethylornithine on functionally distinct populations of tumoricidal effector cells in normal and tumor-bearing mice.

The objective of the present investigation was to examine the effect of in vivo polyamine depletion by DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on cell-mediated tumoricidal activity in normal and tumor-bearing (B16 melanoma) mice. DFMO treatment in vivo for 6 days reduced splenic leukocyte polyamine levels and the induction of cytotoxic T-lymphocytes (greater than 50%) in both normal and tumor-bearing mice. However, substantially less inhibition was observed in the ability to generate cytotoxic T-lymphocytes following 18 days of DFMO treatment. In contrast, DFMO treatment for 6 or 18 days did not impair splenic natural cell-mediated cytotoxicity, assessed against natural killer sensitive YAC-1 target cells and natural cytotoxic sensitive WEHI-164 target cells, in normal or tumor-bearing mice. Natural cell-mediated cytotoxicity was not observed against fresh B16 melanoma cells. However, macrophage-mediated tumoricidal activity directed against B16 melanoma cells was augmented 79% following 6 but not 18 days of DFMO treatment. These results demonstrate that DFMO can exert very selective effects on functionally distinct populations of antitumor effector cells in vivo depending upon the schedule of DFMO administration.

Potentiation of human lymphokine-activated killer cell activity by swainsonine, an inhibitor of glycoprotein processing.

Interleukin 2 (IL-2) is a secreted glycoprotein which acts as an activation and proliferative signal for lymphocytes expressing membrane-bound glycoprotein IL-2 receptors. We have recently established that swainsonine (SW), an inhibitor of mannosidase II during N-linked glycoprotein processing, augmented mitogen-induced mononuclear leukocyte IL-2 receptor expression and IL-2-induced proliferation. The objective of the present investigation was to examine the effect of SW on lymphokine-activated killer (LAK) cell induction. Human mononuclear leukocytes were treated with various concentrations of SW (0.1-10 micrograms/ml) and IL-2 (1-100 units/ml) for up to 72 h. SW augmented IL-2-induced LAK activity directed against human lung carcinoma, melanoma, and leukemia cells 2-3-fold. LAK activity generated in the presence of SW at suboptimal doses of IL-2 (10 units/ml) was similar to that observed with higher concentrations of IL-2 (100 units/ml) alone. SW treatment alone or in combination with IL-2 increased the percentage of IL-2 receptor-positive cells. Furthermore, pretreatment with SW subsequently enhanced IL-2-induced lymphocyte proliferation. SW-treated mononuclear leukocytes exhibited an increase in high-mannose type glycoproteins based upon [3H]mannose labeling, susceptibility to alpha-mannosidase, and binding to concanavalin A-Sepharose. These results indicate that modulators of glycoprotein processing may be useful in lowering the concentrations of IL-2 required for LAK induction and maintenance.

Ornithine decarboxylase induction and polyamine biosynthesis are required for the growth of interleukin-2- and interleukin-3-dependent cell lines.

The objective of this study was to evaluate induction of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, and subsequent polyamine accumulation in interleukin-2 (IL-2)- and interleukin-3 (IL-3)-dependent growth. The CTLL-20 and FDC-P1 cell lines, which have been shown to be absolutely dependent on IL-2 and IL-3, respectively, were used in these studies. The CTLL-20 and FDC-P1 cells each had different temporal patterns of ODC induction following lymphokine stimulation. ODC levels increased rapidly in the FDC-P1 cells, peaking 4 hr after stimulation with IL-3. In contrast, peak ODC activity in the CTLL-20 cells occurred 18 hr following stimulation with IL-2 and reached eightfold higher levels than those observed in the FDC-P1 cells. Treatment with D,L-alpha-difluoromethylornithine X HCl X H2O (DFMO), a specific irreversible inhibitor of ODC activity, completely abrogated lymphokine-dependent ODC induction in both the CTLL-20 and FDC-P1 cell lines. Similarly, intracellular levels of the polyamines putrescine and spermidine were reduced in both cell lines following DFMO treatment. DFMO treatment reduced both IL-2- and IL-3-dependent proliferation in a dose-dependent manner. However, this inhibition could be reversed by the addition of exogenous putrescine. DFMO treatment had no effect on cell viability. Polyamine-depleted CTLL-20 and FDC-P1 cells showed decreased absorption of IL-2 and IL-3 activity, respectively. However, the addition of exogenous putrescine restored the ability of the cells to absorb the appropriate lymphokine. These data are the first to demonstrate that ODC induction and polyamine biosynthesis are required in lymphokine dependent growth.

Increased ornithine decarboxylase activity and polyamine biosynthesis are required for optimal cytolytic T lymphocyte induction.

The objective of the present investigation was to evaluate the requirement for increased ornithine decarboxylase (ODC) activity and polyamine biosynthesis in the induction of cytolytic T lymphocytes (CTL). In this regard, we have utilized alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO treatment completely abrogated Con A-induced NW T-cell ODC activity. Similarly, DFMO treatment reduced putrescine and spermidine biosynthesis 100 and 87% respectively by the end of a 48-hr incubation period. Polyamine depletion reduced the Con A-mediated polyclonal induction of CTL by 52 and 81% at 24 and 48 hr of culture, respectively. The effect of DFMO on CTL induction could be reversed by the addition of exogenous putrescine. These data indicate that the observed effects of DFMO on CTL induction were mediated through inhibition of polyamine biosynthesis. Therefore, increased ODC activity and polyamine biosynthesis are required for optimal CTL induction. Furthermore, polyamine depletion did not impair IL-2 production; however, IL-2-dependent proliferation was reduced. These data are the first to discriminate between the requirement for polyamines with regard to IL-2 responsiveness, rather than IL-2 production, during a primary T-cell mitogenic response.

Alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, augments cyclosporin A inhibition of cytolytic T lymphocyte induction.

The objective of the present investigation was to examine the effect of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, in combination with the immunosuppressant cyclosporin A (CsA) on cytolytic T lymphocytes (CTL) induction in vitro and in vivo. Treatment with DFMO (0.2 mg/ml) or CsA (10 ng/ml) alone in vitro inhibited mitogen-induced CTL generation by 56% and 51%, respectively. Similarly, DFMO or CsA treatment alone inhibited alloantigen-induced CTL generation by 50% and 62%, respectively. Combination treatment with DFMO and CsA reduced mitogen- and alloantigen-mediated CTL induction by 79% and 90%, respectively. In vivo, DFMO treatment alone did not inhibit alloantigen induced CTL generation. However, DFMO potentiated the immunosuppressive effects of CsA in vivo on CTL induction. DFMO treatment reduced activated lymphocyte putrescine and spermidine levels by 81% and 91%, respectively. Combination treatment with DFMO and CsA, at concentrations that effectively inhibited CTL induction, did not further deplete polyamine levels beyond those levels observed with DFMO alone. CsA treatment with or without DFMO did reduce detectable levels of interleukin 2 (IL-2) activity. DFMO treatment alone did not impair IL-2 production. These results indicate that CsA and DFMO may inhibit different processes required for CTL induction, IL-2 production and polyamine biosynthesis. Therefore, inhibitors of polyamine biosynthesis may be useful in lowering the doses of CsA required to inhibit CTL induction.

Decreased interleukin-2 responsiveness in leukocytes from Trypanosoma brucei-infected mice.

Immunosuppression with respect to those inducible processes which regulate lymphocyte mitogenesis was examined in experimental trypanosomiasis. Splenic leukocytes from Trypanosoma brucei-infected mice showed a decreased proliferative response to the T-cell mitogen concanavalin A. Activated cells secreted normal levels of the endogenous T-cell proliferative signal interleukin-2 (IL-2) and expressed high-affinity IL-2 receptors. However, the ability of activated cells to proliferate in response to exogenous IL-2 was inhibited. These results indicate an impairment of processes which occur after IL-2-receptor binding during lymphocyte mitogenesis. Furthermore, these data indicate that expression of high-affinity IL-2 receptors does not necessarily correlate with an ability to respond to IL-2.

Inhibition of alloantigen-induced cytolytic T lymphocytes in vitro with (2R,5R)-6-heptyne-2,5-diamine, an irreversible inhibitor of ornithine decarboxylase.

The objective of the present investigation was to examine the effect of a new potent irreversible inhibitor of ornithine decarboxylase, (2R,5R)-6-heptyne-2,5-diamine (MAP) (MDL 72,175), on the induction of functionally reactive T-cell populations in vitro. We examined alloantigen-activated cytolytic T lymphocytes (CTL) and T-helper (TH) lymphocytes generated during a one-way mixed-leukocyte culture (MLC). The addition of MAP (1 mM) at the initiation of cell culture reduced intracellular putrescine, spermidine, and spermine levels by 81.9, 82.4, and 55.8% respectively. MAP reduced CTL induction 93.8, 78.4, and 37.5% when added at 0, 24, or 48 hr of culture, respectively. A dose-dependent inhibition of CTL induction and polyamine levels was observed following MAP treatment. In direct comparison with another ODC inhibitor, alpha-difluoromethylornithine (DFMO), MAP was five- to sixfold more potent in reducing CTL induction. CTL generation is dependent upon the endogenous production of the TH-cell product interleukin 2 (IL-2). MAP treatment reduced detectable IL-2 activity in a MLC by 54.8%. These results indicate that MAP is a potent inhibitor of alloantigen-activated CTL in vitro and deserves further investigation as a potential immunosuppressive agent.

Methyl-acetylenicputrescine (MAP), an inhibitor of polyamine biosynthesis, reduces the frequency and cytolytic activity of alloantigen-induced LyT 2.2 positive lymphocytes in vivo.

The objective of the present investigation was to examine the effect of (2R,5R)-6-heptyne-2,5-diamine (methyl-acetylenicputrescine; MAP), an irreversible inhibitor of ODC, on the induction of alloreactivity in vivo. Treatment of mice with MAP (0.5-0.01% in drinking water) inhibited CTL induction in a dose-dependent manner with an IC50 of approximately 144 mg/kg/day. MAP treatment reduced the frequency of LyT2+ (cytolytic/suppressor) splenic lymphocytes by greater than 75%. In contrast, MAP did not alter the number of L3T4+ (helper/inducer) lymphocytes. MAP treatment reduced lymphocyte putrescine and spermidine levels by 61 and 40%, respectively. The inhibitory effect of MAP on CTL induction could be reversed by simultaneous administration of putrescine (500 mg/kg). These data indicate that the observed inhibitory effect of MAP on CTL induction is mediated through inhibition of polyamine biosynthesis. Furthermore, results of the present investigation suggest that inhibition of polyamine biosynthesis may provide a unique target for immunosuppression.

The effect of alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, on mitogen-induced interleukin 2 production.

The objective of the present investigation was to examine the effect of DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, on mitogen-induced interleukin 2 production. Treatment with DFMO reduced nylon wool T cell polyamine levels. In contrast, DFMO treatment enhanced, greater than two-fold, detectable levels of concanavalin A-induced interleukin 2 activity. This observed augmentation was not limited to in vitro DFMO treatment, since oral administration of DFMO to C57BL/6 mice also enhanced concanavalin A-induced interleukin 2 levels in vitro. Treatment with exogenous putrescine reversed the effect of DFMO on interleukin 2 levels. These results suggest that the effect of DFMO on interleukin 2 levels is mediated through polyamines. Therefore, polyamine biosynthesis may play a role in the intracellular regulation of interleukin 2 production.

Enhancement of murine mixed lymphocyte response by 1,1-dimethylhydrazine: characterization and possible mechanism.

Treatment of mice with 1,1-dimethylhydrazine (UDMH) resulted in enhancement of the one-way mixed lymphocyte response (MLR); this effect was seen when both responder and stimulator mice were treated as well as when just the stimulator or just the responder mice were treated. Experiments in which splenocytes were exposed to UDMH in vitro indicated that exposure of the stimulator cells alone resulted in an enhanced MLR; exposure of the responder cells alone had no effect; and addition of UDMH to the assay (exposure of both populations) resulted in suppression of the response at higher concentrations. A possible mechanism for the enhancement of the MLR by UDMH was suggested by further experiments showing that UDMH inhibited prostaglandin E2 production by adherent splenocytes.

Intracellular polyamine biosynthesis is required for interleukin 2 responsiveness during lymphocyte mitogenesis.

The objective of the present investigation was to define a more precise role for intracellular polyamine biosynthesis with respect to specific inducible events which regulate lymphocyte mitogenesis. In this regard, we have examined the effect of polyamine depletion on interleukin 2 (IL-2) production, receptor expression, and responsiveness in Con A stimulated mononuclear leukocytes (MNL). Polyamine depletion was achieved utilizing the specific irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO). Polyamine depletion of MNL augmented detectable levels of Con A-induced IL-2 activity. In contrast, the ability of polyamine depleted MNL to respond to saturating levels of IL-2 (100 U/ml) following 72 or 96 hr of Con A stimulation was reduced 100 and 81%, respectively. Nonetheless, polyamine depletion did not impair the induction of IL-2 receptor expression. High-affinity IL-2 receptor density in the polyamine depleted population was greater than control cells late in culture (96 hr). The expression of high-affinity IL-2 receptors did not correlate with an ability to respond to IL-2 in the polyamine depleted population. The results of this study demonstrate for the first time that intracellular polyamine biosynthesis is required for IL-2 responsiveness during a primary mitogenic lymphocyte response.

The effect of alpha-difluoromethylornithine on natural killer cell and tumoricidal macrophage induction by interferon in vivo.

The objective of the present investigation was to evaluate the effect of DFMO (DL-alpha-difluoromethylornithine HCl H2O) administration on tumoricidal effector cell generation by IFN or IFN inducers in vivo. DFMO administration reduces both splenic leukocyte and peritoneal macrophage polyamine levels. In tumor bearing (B16 melanoma) mice, DFMO administration did not impair splenic natural killer (NK) cell augmentation, assessed against NK sensitive YAC-1 target cells, by IFN alpha/beta or the IFN inducers tilorone and polyriboinosinic: polyribocytidilic acid (poly I:C). Tumoricidal macrophage activation by IFN alpha/beta was similarly uninhibited by DFMO. However, only tumoricidal macrophage not NK cell activity was observed which could kill the B16 melanoma target cells. These results indicate that DFMO is not immunosuppressive regarding antitumor cytolytic cell induction in vivo.