Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

CELLULAR IMMUNOLOGY 108,4 17-424 ( 1987)

Regulation of T-Cell Activation and T-Cell Growth Factor (TCGF) Production by Hydrogen Peroxide

STEFFEN ROTH AND WULF DR~GE

Institut ftir Immunologic und Genetik, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg, West Germany

Received January 22,1987; accepted March 17.1987

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T- cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations alter stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (0;) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures ( 18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 1O-5 M) do not inhibit the incorporation of [‘H]thymidine; and higher concentrations (3 X 10m5-1 X lo-“ M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10e5 M Hz02. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IG2) in contrast, are not augmented by 1 X 10e5 A4 H202. The relatively high concentration of 1 X 10e4 M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indi- cates that CTL precursor cells may be relatively resistant against Hz02. o 1987 Academic press, hc.

INTRODUCTION

T cells of the helper variety require macrophage-like stimulator cells or accessory cells for optimal stimulation (l-4) and come into intimate contact with these cells (see, for example, Fig. 1 in (5)). Macrophage-like accessory cells are known to release various immunoregulatory substances including interleukin 1 (IL-l)’ (6-9), lactate, and hydrogen peroxide. Recent experiments in our laboratory revealed that lactate is capable of supporting the production of T-cell growth factor (TCGF) in murine accessory cell-depleted T-cell populations (10). However, the level of TCGF production that was obtained with optimal concentrations of splenic accessory cells was still

’ Abbreviations used: CTL, cytotoxic T lymphocytes; IL-l, interleukin 1; IL-2, interleukin 2; Hepes, 4- (2-hydroxyethyl)- I-piperazineethanesulfonic acid; MLR, mixed lymphocyte reaction; PFC, plaque-form- ing cell; TCGF, T-cell growth factor; TPA, tetradecanoylphorbol acetate.

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0008-8749187 $3.00 Copyright 0 1987 by Academic Pmss, Inc. All rights of reproduction in any form reserved.

418 ROTH AND DR6GE

an order of magnitude higher than the level obtained with optimal concentrations of L-lactate. This suggested that optimal TCGF production may require still other macrophage products in addition to lactate. We, therefore, analyzed the effects of superoxide anion and its biochemical derivative hydrogen peroxide on the production of TCGF. Hydrogen peroxide is generated by activated macrophages in response to various stimuli and is derived by dismutation from superoxide anion, which is formed by the enzyme NADPH oxidase ( 1 l-20).

MATERIALS AND METHODS

Animals. Mice were obtained from the breeding stock of the German Cancer Re- search Center or purchased from Bomholtgard (Ry, Denmark). Eight- to fourteen- week-old mice were used in the experiments.

Accessory cell-depleted splenic T-cell preparations. Spleen cells were incubated twice in two consecutive cycles in nylon wool columns and eluted each time as described (2 1). The T-cell-enriched nonadherent spleen cell fraction was subsequently incubated for 2 min at a density of 1 spleen equivalent per milliliter in a mixture of 9 parts of a 0.83% NH&l solution in water and 1 part 2% Tris buffer adjusted with HCl to pH 7.5. The cells were subsequently washed three times in culture medium.

Assaysfir TCGF activity. The TCGF titers were determined with the interleukin 2-responsive W-2 T-cell line as described previously (22, 10). Control experiments established that the IL-2-dependent DNA synthesis of the W-2 cells is not affected by lactate (up to 3 X 10e2 M) or by hydrogen peroxide up to 1 X 10T5 M. Higher concentrations of hydrogen peroxide were suppressive (data not shown).

Activation of cytotoxic T lymphocytes in mixed lymphocyte cultures. C3H spleen cells (8 X 106) were incubated with 2 X lo6 irradiated (1500 rad) allogeneic DBA/2 spleen cells in 2 ml culture medium for 5 days. The culture medium (RPM1 1640; GIBCO) was supplemented with L-glutamine (final concentration, 5 mM; GIBCO), streptomycin/penicillin (100 U/ml; GIBCO), 0.5% Hepes (GIBCO), 10% fetal calf serum (GIBCO), and 3 X 10V5 A4 2-mercaptoethanol if not indicated otherwise. The cytotoxic activity was determined 5 days later against 2 X lo4 concanavalin A-activated BALB/c spleen cell blasts per well as target cells.

Generation of superoxide anion (0;) in cell cultures. Cell cultures were supplemented with a final concentration of 1 X lo5 Mxanthine (Sigma) and graded concentrations of xanthine oxidase (Sigma) (23-25).

Thepreparation ofIL-2-containing supernatants. Supematants were obtained from an IL-Zproducing EL4 thymoma subline after induction with tetradecanoylphorbol acetate (TPA) as described (26). Briefly, lo6 EL4 cells/ml were incubated together with 10 ng TPA/ml (Sigma) for 48 hr. The supematant was collected and stored frozen at -20°C.

RESULTS

Regulation of TCGF Production and DNA Synthesis in Concanavalin A-Stimulated Accessory Cell-Depleted T-Cell Populations by Superoxide Anion and Hydrogen Peroxide

Accessory cell-depleted splenic T-cell preparations were obtained by incubating murine spleen cells twice with nylon wool and by subsequent treatment with ammo-

REGULATION OF T-CELL FUNCTIONS BY H202 419

o+-- I I I I

FIG. 1. Augmentation of TCGF production by an O;-generating system. Accessory cell-depleted splenic T cells (4 X 106) from C3H mice were incubated with 5 pg of concanavalin A and 3 X 1 O-* M lactate, 1 X 10-j M xanthine (Sigma), and graded concentrations of xanthine oxidase (Sigma) in 1 ml of culture medium without 2-mercaptoethanol. The supematants of these cultures were harvested after 48 hr and assayed for TCGF activity.

nium chloride solution for removal of erythrocytes. These accessory cell-depleted T- cell preparations produce by themselves only minute amounts of TCGF in response to concanavalin A, but TCGF production is reconstituted (at least partly) by lactate ( 10) (see also Fig. 2). Since macrophages produce not only lactate (10) but also superoxide anion (0;) ( 14- 17), we investigated the TCGF production in cultures which contained lactate in combination with a superoxide anion-generating system (i.e., xanthine plus xanthine oxidase (23-25)). The experiments revealed that the production of TCGF in these cultures is indeed markedly augmented by the O;-generating system (Fig. 1).

Since 0; is known to d&mutate in the presence of superoxide dismutase and to some extent even spontaneously into O2 and H202, we investigated then the possibil- ity that the acting principle in this system may be hydrogen peroxide. Our experiments showed (Fig. 2) that TCGF production in accessory cell-depleted T-cell cultures without lactate was not detectably reconstituted by hydrogen peroxide at concentrations between 4 X lop6 and 3 X 10e4 M(Fig. 2A). Cultures with 3 X lo-’ A4 L-lactate, however, supported a substantial production of TCGF and this was further augmented by the addition of hydrogen peroxide. Optimal TCGF production was observed at a concentration of about 1 X 1 O-’ M hydrogen peroxide (Fig. 2A).

Oppenheim and colleagues provided indirect evidence that hydrogen peroxide can inhibit the DNA synthesis in mitogenically stimulated lymphocyte cultures ( 17,27). Mitogenic responses were shown to be suppressed by a superoxide anion-generating system (17) and also by activated macrophages ( 17, 27) and this suppression was in

420 ROTH AND DR6GE

FIG. 2. Regulation of TCGF production and DNA synthesis in concanavalin A-stimulated accessory cell-depleted T-cell populations by hydrogen peroxide. (A) TCGF production: 5 X lo6 accessory cell-depleted splenic T cells from BALB/c mice were incubated in 1 ml of culture medium with 5 pg of concanavalin A and with graded concentrations of L-lactate and H202 in 1 ml ofculture medium without 2-mercaptoethanol. The supematants of these cultures were harvested after 48 hr and assayed for TCGF activity. (B) [3H]Thymidine incorporation: 7 X lo4 accessory cell-depleted splenic T cells from BALB/c mice were incubated with 1 pg of concanavalin A in 0.2 ml of culture medium without 2-mercaptoethanol. The cultures received 1 &i [3H]thymidine 18 hr later and were incubated for another 8 hr at 37’C. The incorporation of [3H]thymidine was finally determined with a Dunn cell harvester.

both cases at least partly abrogated by catalase thus pointing to hydrogen peroxide as the active principle. Our experiments confirmed this conclusion and showed that the DNA synthesis in mitogenically stimulated accessory cell-depleted splenic T-cell cultures is strongly inhibited by a concentration of 1 X 10e4 M hydrogen peroxide (Fig. 2B). A concentration of 1 X 10e5 M hydrogen peroxide, in contrast, caused a slight augmentation of the [3H]thymidine incorporation in cultures without lactate and practically no effect in cultures with 3 X 1 OP2 ML-lactate.

Farrar and his colleagues reported that the production of interleukin 2 is often induced or augmented by signals, which inhibit the DNA synthesis in the IG2-pro-


H202 ( moles / L )

FIG. 3. Regulation of the DNA synthesis (3day MLR) by hydrogen peroxide and catalase. C3H spleen cells (I X 105) were incubated with 1 X 10’ irradiated (1500 rad) DBA/Z spleen cells in 0.2 ml of culture medium with 3 X 1 O-’ M 2-mercaptoethanol and with the indicated final concentrations of Hz02 with or without 5000 U of catalase (Sigma). The cultures received 1 pCi of [3H]thymidine 3 days later and were incubated for another 8 hr. The incorporation of [‘H]thymidine was finally determined with a Dunn cell harvester.

ducing cells (26,28). In line with this rule, we observed that the administration of L- lactate not only induced the production of TCGF but also inhibited the incorporation of [3H]thymidine as tested after 18-26 hr (see Fig. 2). The addition of 3 X 1 Oe5- 1 X 1 Oe4 M hydrogen peroxide, in contrast, was found to inhibit not only the incorporation of [3H]thymidine but also the production of TCGF.

The Regulation of Mixed Lymphocyte Reactions by Hydrogen Peroxide

The allogeneic mixed lymphocyte reaction (MLR) was found to be inhibited by catalase and augmented by small concentrations of hydrogen peroxide on the order of 1 X 10P5 M(Fig. 3), indicating that hydrogen peroxide also plays a role in proliferative T-cell responses after antigenic stimulation. Higher concentrations of hydrogen peroxide ( 10e4 M) were again suppressive. The DNA synthesis and the activation of cytotoxic T lymphocytes in allogeneic mixed lymphocyte cultures with an external source of IL-2, in contrast, were not augmented by moderate concentrations of hydrogen peroxide (1 X lop5 M), indicating that hydrogen peroxide augments primarily the production of TCGF but not directly the TCGF-mediated DNA synthesis or the generation of cytotoxic activity (Fig. 4). The concentration of 1 X 10W4 A4 hydrogen peroxide in combination with external IL-2 again suppressed the DNA synthesis (Fig. 4A) but not the activation of cytotoxic T lymphocytes (Fig. 4B). This indicated that the CTL precursor cells are more resistant against this relatively high concentration of hydrogen peroxide than the TCGF-producing helper T cells and the cells which account for most of the DNA synthetic response.

DISCUSSION

The experiments in the accompanying paper (10) have shown that the production of T-cell growth factor by mitogenically activated accessory cell-depleted splenic T

422 ROTH AND DR6GE

A ) DNA synthesis ( 3 day MLR )

60

B ) CTL activity

Q3xlO%Z.ME

AlxlO%Z-ME

I 0 1.i I I I I

0 8X10-6 2.3 x 1O-5 7X10-5 2x10‘4

H202 ( moles I L )

FIG. 4. Regulation of DNA synthesis and cytotoxic T-cell activity by hydrogen peroxide in the presence of IG2. (A) DNA synthesis: The same cell preparations and culture conditions were used as in Fig. 3 (same experiment) except that 7 ~1 of an IL-2containing EL-4 cell supematant was added to the cultures. (B) CTL activity: 8 X lo6 C3H spleen cells were incubated with 2 X lo6 irradiated DBA/2 spleen cells in 2 ml ofculture medium. The cytotoxic activity against BALB/c target cells was assayed 5 days later by a standard “Cr release assay. Some of the cultures (0) contained 3 X lo-’ M 2-mercaptoethanol and others (A) contained 1 X 10m6 M 2-mercaptoethanol. The difference in the concentration of 2-mercaptoethanol had a strong effect on the proliferative activity in these cultures but had practically no effect on the relative degree of suppression by hydrogen peroxide.

lymphocytes is at least partly reconstituted by the macrophage product L-lactate. The experiments in this report now demonstrate that this effect of lactate is potentiated by superoxide anion and by its biochemical derivative hydrogen peroxide. Both substances were found to have only little effect on the TCGF production by accessory cell-depleted T lymphocytes in the absence of L-lactate.

Hydrogen peroxide and superoxide anion are formed by adequately activated macrophages during the oxidative burst (1 l-20), and lactate is released by macrophages as a product of the glycolytic metabolism ( 10). In a recent report we have shown that peritoneal macrophages produce lactate at a rate of 1.2 X lo-l3 mol/hr per cell and


can generate concentrations of lactate up to 6 X lo-* M in the extracellular space ( 10). Hydrogen peroxide is reportedly produced by activated macrophages at a rate of 2-6 X 1 O-r4 mol/hr per cell (12,20). Since the inhibitory effect of activated macrophages on mitogenic responses of lymphocytes was shown to be mediated at least partly by hydrogen peroxide (17, 27) and since the inhibition of the mitogenic response requires about low4 A4 hydrogen peroxide (see Fig. 2), it is reasonable to as- sume that concentrations of this order of magnitude do in fact occur at the surface of the activated macrophages.

Immunoregulatory effects of the macrophage product hydrogen peroxide have al- ready been the subject of earlier studies (17, 27, 29, 30). A concentration of 10w5- 1 Od4 M H202 has been reported to inhibit antibody formation (PFC responses (29)), and indirect evidence indicated that hydrogen peroxide also inhibits the mitogen- induced proliferation of T cells (17, 27) and the cytotoxic activity of natural killer cells (30). Our experiments confirmed and extended these studies and showed that a concentration of 3 X 1 O-5- 1 X 1 Op4 A4 H202 inhibits not only the DNA synthesis in mitogenically stimulated T-cell populations but also the production of TCGF (see Fig. 2). The role of hydrogen peroxide as a mediator of accessory cell function and especially the augmenting effect of hydrogen peroxide ( 1 X lop5 M) on the TCGF production in accessory cell-depleted T-cell populations has not been reported previously.

The inhibitory effects of hydrogen peroxide were previously proposed to result (i) from a depletion of nicotinamide adenine dinucleotide (NAD) and ATP as a conse- quence of the activation of poly(ADP-ribose) polymerase (25, 3 l), (ii) from a depletion of ATP as a result of the inhibition of glycolysis and stimulation of the hexose monophosphate shunt (31) (iii) from the loss of Ca*+ from intracellular stores (32, 33) and (iv) from hydrogen peroxide-induced lipid peroxidation (34). Whether the mechanism of the augmenting effect of hydrogen peroxide on TCGF production is also related to one of these biochemical events remains to be investigated.

ACKNOWLEDGMENTS

The expert technical assistance of Mrs. S. Nick and Mrs. H. Schmidt and the assistance of Mrs. I. Fryson in the preparation of this manuscript is gratefully acknowledged.

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Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide