Genetic modification of the manganese superoxide dismutase/glutathione peroxidase 1 pathway influences intracellular ROS generation in quiescent, but not contracting, skeletal muscle cells

Increased amounts of reactive oxygen species (ROS) are generated by skeletal muscle during contractile activity, but their intracellular source is unclear. The oxidation of 2',7'-dichlorodihydrofluorescein (DCFH) was examined as an intracellular probe for reactive oxygen species in skeletal muscle myotubes derived from muscles of wild-type mice and mice that were heterozygous knockout for manganese superoxide dismutase (Sod2(+/-)), homozygous knockout for glutathione peroxidase 1 (GPx1(-/-)), or MnSOD transgenic overexpressors (Sod2-Tg). Myoblasts were stimulated to fuse and loaded with DCFH 5-7 days later. Intracellular DCF epifluorescence was measured and myotubes were electrically stimulated to contract for 15 min. Quiescent myotubes with decreased MnSOD or GPx1 showed a significant increase in the rate of DCFH oxidation whereas those with increased MnSOD did not differ from wild type. Following contractions, myotubes from all groups showed an equivalent increase in DCF fluorescence. Thus the oxidation of DCFH in quiescent skeletal muscle myotubes is influenced by the content of enzymes that regulate mitochondrial superoxide and hydrogen peroxide content. In contrast, the increase in DCFH oxidation following contractions was unaffected by reduced or enhanced MnSOD or absent GPx1, indicating that reactive oxygen species produced by contractions were predominantly generated by nonmitochondrial sources.     

Murine delayed hypersensitivity (DHS): modulation of in vitro antigen-induced responsiveness of lymph node cells from mice expressing DHS to human gamma-globulin by lymphocyte populations from normal syngeneic animals

Using a chemically defined, protein-free medium, the modulatory effect of normal (N) lymphocytes on in vitro antigen-induced proliferation by lymph node cells (LNC) from mice immunized to express delayed hypersensitivity (DHS) to human γ-glogulin (HGG) was quantitated in coculture. LNC from normal syngeneic animals exerted little if any effect on immune-LNC proliferation. Compared with immune-LNC plus N-LNC coculture response. N thymus cells (TC) were consistently suppressive while N spleen cells (SC) varied in their effect from a marginal to a marked potentiation of radiolabeled thymidine incorporation. Inactivation of N-SC suspensions by X irradiation prior to coculture with immune LNC abrogated the increased responsiveness. It therefore appeared that interaction of immune LNC and antigen resulted in recruitment of N-SC to proliferate. Separation of N-SC suspensions to provide enriched populations of thymic-independent (B) and thymic-dependent (T) lymphocytes showed that B cells augmented and T cells suppressed HGG-induced incorporation of [3H] thymidine when cocultured with immune LNC.     

In vitro immune blastogenesis during contact sensitivity in tumor-bearing mice: I. Description of progressive impairment and demonstration of splenic suppressor cells

T-cell responsiveness was measured by the DNA response of disassociated spleen and lymph node cells when exposed to antigen in vitro. Sensitized splenic lymphocytes from fibrosarcoma-bearing mice immunized with 2,4-dinitro-1,5-difluorobenzene (DN2FB) demonstrated a progressive decrease in T-cell responsiveness to the haptenprotein conjugate DNP-BSA. Hyporesponsiveness to the dinitrophenylated-protein conjugate appeared in the spleens but not lymph nodes of tumorous animals. Normal host lymph node cells (LNC) responded strongly 24 to 48 h after sensitization and subsequently declined with a corresponding increase in responsiveness in the spleen. Tumor-bearing hosts (TBH) had similar LNC kinetics during immunization, however, spleen cells were significantly suppressed when compared to normal BALB/c mice sensitization kinetics. Spleen cells from TBH were also capable of suppressing the in vitro response of normal primed lymphocytes to DNP-BSA when admixed. Results from these experiments suggest that in vitro measurement of contact sensitivity was affected by suppressor cells/products existing in the spleens but not lymph nodes of fibrosarcoma-bearing mice.     

Generation of human autologous melanoma-specific cytotoxic T cells from tumor-involved lymph nodes

Cytotoxic T lymphocytes (CTL) specific for autologous human melanoma have been successfully generated in vitro from tumor bearing lymph nodes without any stimulation by the autologous tumor. Tumor-involved lymph node cells (LNC) were cultured in serum free medium (AIM-V) containing 1,000 U/ml of recombinant interleukin-2. The best expansion and specific cytotoxicity of CTL were achieved in 4 to 6 weeks of culture. The predominant populations in cultured LNC-derived CTL were CD2+, CD3+, CD4-, CD8+, CD56-, and HLA-DR+ T cells. These data suggested that tumor-involved LNC may provide an alternative source for the generation of tumor-specific CTL in adoptive immunotherapy.     

Effects of antithymocyte serum on lymph node cells participating in the graft-vs-host reaction.

BALB/c mice were injected with 0.1 ml of antithymocyte serum (ATS) and tested at various times thereafter for graft-versus-host (GVH) reactivity of lymph node cells (LNC) and spleen cells (SC). Splenomegaly produced by LNC or SC injected alone was used as a measure of precursor T cell function and the capacity of such cells to produce synergy when combined with normal thymocytes was used to evaluate amplifier T cell activity. In lymph node, both precursor and amplifier function were strikingly depressed 2 days after ATS administration; by day 11, precursor function showed slight recovery while amplifier activity had recovered to supranormal levels. In spleen, precursor activity recovered two fold between 2 and 11 days after ATS inoculation but no amplifier activity could be detected at day 11. Since donors had not been deliberately stimulated with alloantigens prior to testing of LN and SC GVH acivity, these studies demonstrate (a) that precursors and amplifiers are disinct T cell populations that are committed to express unique functional activities before antigen exposure and (b) that, following depletion with ATS, these two populations recover independently at different rates in separate lymphoid compartments.     

Intratracheal priming with ovalbumin- and ovalbumin 323-339 peptide-pulsed dendritic cells induces airway hyperresponsiveness, lung eosinophilia, goblet cell hyperplasia, and inflammation

Dendritic cells (DC) are the primary APC responsible for the capture of allergens in the airways and the shuttling of processed allergens to the draining lymph nodes where Ag presentation and T cell activation take place. The mechanism of this Ag handling and presentation in asthma is poorly understood. In addition, the feasibility of asthma induction by DC priming has not been directly tested. In this report an asthma model using intratracheally (i.t.) injected splenic DC was used to address these issues. DC pulsed with a model Ag OVA or the major MHC class II-restricted OVA T epitope peptide OVA(323-339) and instilled i.t. primed mice to exhibit asthma-like diseases. With OVA as the Ag, mice exhibit airway hyperresponsiveness (AHR), lung eosinophilia and inflammation, and pulmonary goblet cell hyperplasia. In OVA(323-339)-immunized mice, AHR and goblet cell hyperplasia were noted, with little eosinophilia and parenchymal inflammation. The latter finding provides evidence for dissociating AHR from eosinophilia. In both cases mediastinal node hypertrophy occurred, and high levels of Th2 cytokines were produced by the lung and mediastinal lymph node cells (LNC). Interestingly, mediastinal LNC also produced high levels of Th1 cytokines. Lung cells produced much less Th1 cytokines than these LNC. These results demonstrate that DC when introduced i.t. are potent in inducing asthma-like diseases by recruiting lymphocytes to the lung-draining lymph nodes and by stimulating Th2 responses and also suggest that the lung environment strongly biases T cell responses to Th2.     

Electrokinetic behaviour of subpopulations of T lymphocytes in allografted mice.

In AKR(H-2k) mice transplanted with DBA/2(H-2d) skin grafts, the mean electrophoretic mobilities (EPM) of total lymph node cells (LNC) and T cells were significantly reduced. Subpopulations of T lymphocytes, viz. CD4- (CD8- (CD4+) T cells were obtained by depletion treatment of T cells with monoclonal antibodies specific for these surface antigens and complement. Determination of EPM of these two subpopulations revealed that the electrokinetic change following immunostimulation equally afflicted these two subpopulations. These data thus confirmed that CD4+ as well as CD8+ T cells were activated in MHC unmatched allograft rejection.     

Gammadelta T cells enhance the expression of experimental autoimmune encephalomyelitis by promoting antigen presentation and IL-12 production

Using an adoptive transfer model of experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP)-reactive lymph node cells (LNC), we have shown that depletion of gammadelta T cells from LNC resulted in diminished severity of EAE in recipient mice, both clinically and histopathologically. The reduced potency of gammadelta T cell-depleted LNC to induce EAE correlated with decreased cell proliferation in response to MBP. The gammadelta T cell effect upon the threshold of MBP-induced LNC proliferation and EAE transfer was restored by reconstitution of gammadelta T cells derived from either MBP-immunized or naive mice, indicating that this effect was not Ag specific. The enhancing effect of gammadelta T cells on MBP-induced proliferation and EAE transfer required direct cell-to-cell contact with LNC. The gammadelta T cell effect upon the LNC response to MBP did not involve a change in expression of the costimulatory molecules CD28, CD40L, and CTLA-4 on TCRalphabeta(+) cells, and CD40, CD80, and CD86 on CD19(+) and CD11b(+) cells. However, depletion of gammadelta T cells resulted in significant reduction in IL-12 production by LNC. That gammadelta T cells enhanced the MBP response and severity of adoptive EAE by stimulating IL-12 production was supported by experiments showing that reconstitution of the gammadelta T cell population restored IL-12 production, and that gammadelta T cell depletion-induced effects were reversed by the addition of IL-12. These results suggest a role for gammadelta T cells in the early effector phase of the immune response in EAE.     

Effect of chlorophyllin against oxidative stress in splenic lymphocytes in vitro and in vivo

Chlorophyllin (CHL) has been examined as an antioxidant/radioprotector in splenic lymphocytes from BALB/c mice. CHL inhibited lipid peroxidation induced by 2,2'-azobis(2-propionimidinedihydrochloride) (AAPH) in lymphocytes in vitro. It also partially prevented radiation-induced suppression of mitogenic stimulation of lymphocytes in vitro. Generation of intracellular reactive oxygen species (ROS) by radiation or AAPH was measured as oxidation of dichlorodihydrofluorescein diacetate (H(2)DCF-DA) using flow cytometry. Addition of CHL to lymphocytes in vitro significantly inhibited the increase in intracellular ROS. Further, lymphocytes from mice treated with CHL (100-400 microg/gbw i. p.) showed varying levels of ROS depending on the dose and the time (24 to 72 h) after injection. The extent of radiation-induced apoptosis and suppression of concanavalin A (con A)-induced mitogenesis ex vivo corresponded with changes in ROS levels in CHL-administered mice. Antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) were also estimated in lymphocytes from CHL-treated mice. CHL offered protection against whole body irradiation (WBI)-induced lipid peroxidation and apoptosis in lymphocytes at all the time points studied. These results demonstrate antioxidant effect of CHL in vivo.     

Neonatal infection with mouse thymic virus. Differential effects on T cells mediating the graft-versus-host reaction.

Neonatal infection with mouse thymic virus (TA), a murine herpes virus, produced extensive but temporary necrosis of the thymus which was maximal at 10 to 14 days of age. Studies of precursor and amplifier cells mediating graft-vs-host (GVH) reactivity of thymocytes, spleen cells (SC), and lymph node cells (LNC) of normal and TA-infected mice were made at 4 and 8 weeks of age. Infection with TA resulting in a profound reduction (70 to 80%) in the direct GVH reactivity of thymocytes at both ages; by comparison, the capacity of thymocytes to produce synergy when combined with normal LNC was normal at 8 weeks. Direct GVH reactivity of SC was depressed 90% 4 weeks after infection with TA but returned to near normal at 8 weeks. Direct GVH reactivity of LNC from TA-infected mice was normal at 4 and 8 weeks of age, but amplifier T cell activity in LNC was markedly depressed at 8 wekks. These results demonstrate that TA has highly selective effects upon subpopulations of T cells in thymus and lymph node.     

Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.     

Kinetic analysis of phagosomal production of reactive oxygen species

Phagocytes produce large quantities of reactive oxygen species for pathogen killing; however, the kinetics and amplitude of ROS production on the level of individual phagosomes are poorly understood. This is mainly due to the lack of appropriate methods for quantitative ROS detection with microscopic resolution. We covalently attached the ROS-sensitive dye dichlorodihydrofluorescein (DCFH(2)) to yeast particles and investigated their fluorescence due to oxidation in vitro and in live phagocytes. In vitro, the dye was oxidized by H(2)O(2) plus horseradish peroxidase but also by HOCl. The latter produced a previously unrecognized oxidation product with red-shifted excitation and emission spectra and a characteristic difference in the shape of the excitation spectrum near 480 nm. Millimolar HOCl bleached the DCFH(2) oxidation products. Inside phagosomes, DCFH(2)-labeled yeast were oxidized for several minutes in a strictly NADPH oxidase-dependent manner as shown by video microscopy. Inhibition of the NADPH oxidase rapidly stopped the fluorescence increase of the particles. At least two characteristic kinetics of oxidation were distinguished and the variability of DCFH(2) oxidation in phagosomes was much larger than the variability upon oxidation in vitro. We conclude that DCFH(2)-yeast is a valuable tool to investigate the kinetics and amplitude of ROS production in individual phagosomes.     

A novel CD8 T cell-restricted CD45RB epitope shared by CD43 is differentially affected by glycosylation.

The mAb 1B11 has been characterized as recognizing the activation-associated glycoform of murine CD43, a heavily O-glycosylated protein implicated in leukocyte homing. When hemopoietic cells from CD43-/- mice were stained with 1B11, CD43-independent binding of 1B11 was observed on peripheral CD8 T cells and at low levels on thymocytes, while no binding was detected on CD4 T cells, B cells, or bone marrow cells. Levels of 1B11 staining were comparable in lymph node CD8+ T cells from both CD43-/- mice and CD43+/+ mice. We sought to identify the CD43-independent target of 1B11 expressed on CD8 T cells. Previous work had demonstrated that neuraminidase treatment of lymph node cells (LNC) enhanced 1B11 binding on CD43+/+ LNC; this enhancement was also observed in CD43-/- LNC. We show that neuraminidase-enhanced 1B11 binding in CD43-/- LNC and EL4 thymoma cells is CD43 independent and that 1B11 detects a novel target of apparent mass of approximately 200 kDa identified as a hyposialylated form of CD45RB preferentially expressed on peripheral CD8, but not CD4, T cells. Our data also show that the recognition of CD43 and CD45RB by 1B11 is differentially affected by O-linked glycosylation and sialic acid. Whereas 1B11 recognition of CD43 on activated T cells required both core 2 O-glycan branching and sialic acid, 1B11 recognition of CD45 only occurred in the absence of both core 2 glycosylation and sialic acid.     

Decreased IL-12 production underlies the decreased ability of male lymph node cells to induce experimental autoimmune encephalomyelitis

Myelin basic protein (MBP)-specific T lymphocytes from male SJL mice were shown to be less encephalitogenic than MBP-specific T lymphocytes from females. Mechanisms underlying this gender difference in the induction phase of EAE were examined. Following immunization with MBP, draining lymph nodes contained fewer cells, and Ag-specific proliferative responses were decreased in males as compared with females. These gender differences in the proliferative response were not unique to MBP-specific responses since they were also observed after immunization with hen eggwhite lysozyme. Short-term MBP-specific T cell lines derived from females and males mapped with identical specificity, indicating no defect in the ability of male APCs to process Ag. Interestingly, IL-12 and IFN-gamma production was decreased following Ag-specific stimulation of draining lymph node cells (LNC) from males as compared with females, but IL-10 and IL-4 were no different. While male-derived LNCs were less encephalitogenic than female derived LNCs, cotransfer and coculture of male LNCs with female LNCs demonstrated that male LNCs were not immunosuppressive. Administration of IL-12 to LNCs from male mice enhanced encephalitogenicity. These data indicate that deficient endogenous IL-12 production within draining LNCs of male SJL mice is central to gender differences in the induction phase of experimental autoimmune encephalomyelitis.     

Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide

Free radicals are produced continuously by skeletal muscle fibers. Extracellular release of reactive oxygen species (ROS) and nitric oxide (NO) derivatives has been demonstrated, but little is known about intracellular oxidant regulation. We used a fluorescent oxidant probe, 2',7'-dichlorofluorescin (DCFH), to assess net oxidant activity in passive muscle fiber bundles isolated from mouse diaphragm and studied in vitro. We tested the following three hypotheses. 1) Net oxidant activity is decreased by muscle cooling. 2) CO(2) exposure depresses intracellular oxidant activity. 3) Muscle-derived ROS and NO both contribute to overall oxidant activity. Our results indicate that DCFH oxidation was diminished by cooling muscle fibers from 37 degrees C to 23 degrees C (P < 0.001). The rate of DCFH oxidation correlated positively with CO(2) exposure (0-10%; P < 0.05) and negatively with concurrent changes in pH (7.0-8.5; P < 0.05). Separate exposures to anti-ROS enzymes (superoxide dismutase, 1 kU/ml; catalase, 1 kU/ml), a glutathione peroxidase mimetic (ebselen, 30 microM), NO synthase inhibitors (N(omega)-nitro-l-arginine methyl ester, 1 mM; N(omega)-monomethyl-l-arginine, 1 mM), or an NO scavenger (hemoglobin, 1 microM) each inhibited DCFH oxidation (P < 0.05). Oxidation was increased by hydrogen peroxide, 100 microM, an NO donor (NOC-22, 400 microM), or the substrate for NO synthase (l-arginine, 5 mM). We conclude that net oxidant activity in resting muscle fibers is 1) decreased at subphysiological temperatures, 2) increased by CO(2) exposure, and 3) influenced by muscle-derived ROS and NO derivatives to similar degrees.     

The excitotoxic effect of NMDA on human lymphocyte immune function

N-Methyl-d-aspartate (NMDA)-activated glutamate receptors are expressed in lymphocytes, but their roles have not yet been defined. We show that incubation of human peripheral blood lymphocytes with NMDA resulted in increased intracellular calcium and reactive oxygen species (ROS) levels through effects on NMDA-activated glutamate receptors. In terms of ROS production, T cells were most affected, followed by NK cells, whereas B cell ROS levels were not increased. In unstimulated T and NK cells, interferon-gamma (IFN-gamma) production was unaffected by NMDA, whereas interleukin-2 stimulation of IFN-gamma production was significantly suppressed by NMDA. Simultaneous incubation of the cells with NMDA and IL-2 resulted in a dramatic increase in the amount of cells expressing the NR1 subunit of the NMDA-activated receptors. We conclude that NMDA-activated glutamate receptor activation, accompanied by the changes in intracellular calcium and ROS levels, may be involved in the modification of immune functions of human T and NK cells.     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.     

Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions

We examined intra- and extracellular H(2)O(2) and NO formation during contractions in primary rat skeletal muscle cell culture. The fluorescent probes DCFH-DA/DCFH (2,7-dichlorofluorescein-diacetate/2,7-dichlorofluorescein) and DAF-2-DA/DAF-2 (4,5-diaminofluorescein-diacetate/4,5-diaminofluorescein) were used to detect H(2)O(2) and NO, respectively. Intense electrical stimulation of muscle cells increased the intra- and extracellular DCF fluorescence by 171% and 105%, respectively, compared with control nonstimulated cells (p <.05). The addition of glutathione (GSH) or Tiron prior to electrical stimulation inhibited the intracellular DCFH oxidation (p <.05), whereas the addition of GSH-PX + GSH inhibited the extracellular DCFH oxidation (p <.05). Intense electrical stimulation also increased (p <.05) the intra- and extracellular DAF-2 fluorescence signal by 56% and 20%, respectively. The addition of N(G)-nitro-L-arginine (L-NA) completely removed the intra- and extracellular DAF-2 fluorescent signal. Our results show that H(2)O(2) and NO are formed in skeletal muscle cells during contractions and suggest that a rapid release of H(2)O(2) and NO may constitute an important defense mechanism against the formation of intracellular (*)OH and (*)ONOO. Furthermore, our data show that DCFH and DAF-2 are suitable probes for the detection of ROS and NO both intra- and extracellularly in skeletal muscle cell cultures.     

Preparation, characterization, and functions of rabbit lymph node populations. III. Antigen-induced uptake of thymidine and dibutyryl cyclic AMP: inhibition of thymidine uptake by cholera toxin and dibutyryl cyclic AMP.

Keyhole limpet hemocyanin (KLH)-primed lymph node cell (LNC) populations were incubated with various amounts of KLH and the cellular incorporation of tritiated thymidine ([³H]TdR) or tritiated N⁶, O^2′ dibutyryl cyclic AMP ([³H]DbcAMP) was determined. T LNC responded more vigorously than did complement receptor lymphocytes (CRL), i.e., B cells, at all KLH concentrations, during all time intervals examined, and in the presence or absence of normal rabbit serum (NRS). The depletion of adherent cells from KLH-primed LNC resulted in no significant decrease in KLH-induced incorporation of either [³H]TdR or [³H]DbcAMP in any of the LNC populations. Thus it appeared that variation among LNC populations in the incidence of macrophages did not account for the marked variation in their responses. Cultures containing equal numbers of T and CRL were induced to incorporate more [³H]TdR or [³H]DbcAMP than either population cultured separately or the sum of their individual responses. It was concluded that KLH-induced incorporation of these substances into primed, isolated LNC, was primarily manifested in the T-cell population. The synergism seen in cultures containing mixtures of T and CRL suggested that B cells are induced to incorporate [³H]TdR or [³H]DbcAMP in the presence of antigen and T-cell product(s). KLH-induced incorporation of [³H]TdR into KLH-primed LNC was inhibited by cholera enterotoxin (CT) and DbcAMP as previously reported. However, CT or DbcAMP inhibited this incorporation into T LNC to a greater extent than into CRL or unfractionated LNC.     

The generation of oxygen radicals: a positive signal for lymphocyte activation

The induction of ornithine decarboxylase (ODC) activity in lymphocytes is associated with activation and the initiation of cellular proliferation. ODC is also an essential component in tumor promotion. Phorbol myristic acetate (PMA) is a mitogen for lymphocytes, but can also promote tumor formation. Tumor promotion is linked to the generation of free radicals induced by PMA. Modulation of intracellular glutathione is associated lymphocyte activation and in protection of cells from damage due to oxygen radicals. We examined the interaction between ODC activity and intracellular glutathione concentrations in EL4 murine lymphoblastoid cells. The intracellular glutathione concentration could be augmented in EL4 cells when cultured with the cysteine delivery agents 2-oxothiazolidine 4-carboxylate (OTC) and 2-mercaptoethanol (2-ME) and suppressed with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO). OTC and 2-ME suppressed ODC activity in fresh serum and PMA-activated EL4 cells. BSO had no effect on ODC activity of EL4 cells cultured in the presence of PMA. While both OTC and 2-ME augmented the total intracellular glutathione concentration, PMA enhanced only the level of oxidized glutathione. To determine if the mechanism by which PMA or fresh serum altered intracellular glutathione and ODC activity was through the generation of oxygen radicals, EL4 cells were cultured with free radical scavengers. The nonpermeant electron acceptor potassium ferricyanide, and the H2O2 scavenger catalase, lowered ODC activity in both serum-stimulated and PMA-activated EL4 cells. Similarly, incubation of EL4 cells with either potassium ferricyanide or catalase elevated intracellular glutathione concentrations. These data suggest that (a) modulation of intracellular glutathione in the EL4 lymphoblastoid cell line alters ODC activity induced by fresh serum and by the mitogen PMA; (b) activation of EL4 cells by PMA alone alters intracellular glutathione metabolism, which may be associated with its role as a mitogen in lymphocyte activation; and (c) the generation of free radicals in EL4 cells may play a positive role in cellular activation.