Eur. J. Immunol. 1991. 21: 1933-1937 Regulation of IL 2 production and glutathione levels by lactate 1933

Steffen Roth and Wulf Droge

Regulation of interleukin 2 production, interleukin 2 mRNA expression and intracellular

Institute of Immunology and

Center, Heidelberg lymphocytes by lactate glutathione levels in ex ;ivo derived T Genetics, German Cancer Research

The concentration of L-lactate in the blood plasma of higher vertebrates is about 1 mM but can be as high as 30 mM under certain physiological and pathological conditions or in the vicinity of glycolytically active cells including macrophages. Here we report that high but physiologically relevant concentrations of lactate increase the expression of interleukin 2 (IL 2)-specific mRNA and the production of IL, 2 activity in cultures of mitogenically stimulated Tcells. Lactate supports IL 2 production most effectively if added 0-8 h after Tcell stimulation and only in cultures of CD4+ but not of CD8+ Tcells. In contrast to the DNA synthesis activity in these cell cultures, IL 2 production is not augmented but rather inhibited by exogenous glutathione (GSH). Lactate causes a reduction of intracellular GSH levels, and lactate-containing cultures require accordingly higher extracellular cysteine concentrations than control cultures to achieve similar intracellular GSH levels. In view of the strong variations of extracellular lactate concentrations in vivo, our experiments suggest that lactate may be part of a previously unknown mechanism by which the metabolic microenvironment modulates gene expression in T cells.

1 Introduction 2 Materials and methods

The contribution of the metabolic microenvironment to the regulation of immune responses is still largely unknown. In this report we describe the regulation of T lymphocyte functions by variations of the extracellular lactate concen- tration. Lactate occurs in the blood plasma at a concentra- tion of about 1-3 mM [l]; but plasma lactate levels can increase up to 20 -30m in healthy individuals after physical exercise and up to 30-40 m in pathological lactic acidosis [ l , 21. It is reasonable to assume that even higher concentrations occur in the vicinity of glycolytically active cells such as MQ, [3] or tumor cells [4,5]. MQ, can generate up to 60 rn lactate in culture SN [3]. Injection of TNF was also found to raise plasma lactate concentrations in mice [61*

It has been shown previously that variations of the extracellular lactate concentration in the physiologically relevant range between 1-30 m modulate Tcell functions and that lactate can have strong immunopotentiating activity in viva [3,7].This may be related to the observation that moderate physical exercise causes immune enhance- ment [8-121, whereas regular intense training programs can have damaging effects on the immune system [8]. The experiments in this report were designed to analyze the regulatory effect of the extracellular lactate concentration on the induction of IL2 production and IL2 mRNA expression.

[I 92961

Correspondence: Wulf Droge, German Cancer Research Center, Institute of Immunology and Genetics, Im Neuenheimer Feld 280, D-6900 Heidelberg, FRG

2.1 Animals and chemical materials

C3H/He (H-2k) and C57BL/6 (H-2b) were purchased from Charles River Wiga GmbH (Sulzfeld, FRG). Nine- to twelve-week-old mice were used in the experiments. Sodi- um L-lactate, L-glutathione (GSH; reduced form) and L-cysteine were purchased from Serva (Heidelberg, FRG). All substances were dissolved in the corresponding culture medium and adjusted to pH 7.3 before they were added to cell cultures.

2.2 Cell culture conditions and accessory cell-depleted splenic T cell preparations

Cell culture conditions and culture media have been described previously [13]. Spleen cells were incubated in two consecutive cycles in nylon wool columns and subse- quently incubated in RBC lysing buffer as described [3, 141.

2.3 Assay for IL 2

The amount of IL 2 in cell culture SN was determined by their ability to stimulate DNA synthesis of the IL2- dependent T cell clone W-2 as described [15] and is expressed as U/ml using an IL 2 standard as described by Farrar et al. [16]. Units were determined by calculating the values from the differences between the linear parts of the IL 2 standard regression plot and the sample regression plots. Control experiments have established that the rele- vant concentrations of lactate or GSH have no effect on the

Abbreviation: GSH: Glutathione IL 2 assay.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 0014-2980/91/0808-1933$3 S O + .25/0

1934

2.4 C-mediated lysis of CD4+ and CD8+ T cells

Splenic T cells (5 x lo6) were incubated with 100 pl cell culture SN of the hybridomas RL 172.4 (anti-CD4 anti- body) or 3.168.8.1 (anti-CD8 antibody) for 20 min on ice. The cells were subjected to centrifugation and the cell pellet was resuspended in 500 p1 of a 1 : 20 diluted Low- Tox-M rabbit C (Cedarlane Laboratories Ltd., Hornby, Canada, Cat. Nr. 4399) solution in ice-cold standard cell culture medium. The cells were then incubated at 37 "C for another 30 min. Finally, the cells were washed twice, counted and used in the experiments.

S. Roth and W. Droge Eur. J. Immunol. 1991.21: 1933-1937

2.5 Determination of intracellular GSH and analysis of IL 2-specSc mRNA expression by Northern blot and dot blot analysis

GSH was determined as described in [13]. Analysis of IL 2-specific mRNA has been performed as described in ~ 7 1 .

3 Results

3.1 Regulation of IL 2-mRNA expression and IL 2 production by lactate

To analyze the mechanistic details of the immunoregulatory effect of lactate [3], we determined in cultures of rnitogen- ically stimulated splenic T cells the expression of IL 2- specific mRNA by Northern blot analysis and the I L 2 activity in the SN after various time intervals. These experiments (Fig. 1) revealed that already within 16 h lactate causes a detectable increase of the IL 2 production and IL 2 mRNA expression. This increase was even more striking after longer time intervals, when IL 2 mRNA in control cultures had disappeared almost completely. A profound augmentation of IL 2 production was obtained with 30 mM lactate, but substantial effects were seen already with 1-10 mM (Fig. 2, left panel). Lactate had to be added within the first 8 h after culture initiation and Tcell stimulation in order to be optimally effective (Fig. 2, right panel). Control experiments revealed that the sodium salts of several other carboxylic acids including butyrate, ace- tate, dichloroacetate, citrate and malate did not augment IL 2 production under similar conditions (data not shown).

These effects of lactate on IL 2 production suggested the possibility that lactate may recruit T cell subsets into lymphokine production that normally do not produce IL 2. Our experiments (Fig. 3) showed, however, that lactate augments strongly the production of IL 2 by CD4+ cells but cannot induce IL 2 production in CD8+ Tcells. Cultures of CD4+ Tcells with lactate showed steadily increasing levels of IL 2 for at least 48 h after stimulation (Fig. 3)., whereas cultures of unfractionated T cells showed decreasing IL 2 levels after 40 h of incubation (Fig. l), indicating that the CD8+ Tcells in the unfractionated population of Fig. 1 may have consumed most of the IL 2 in the late phase of the culture period.

culture period (h)

o h - 4h - 4h + 8h - 8 h +

12h - la7 + 18h - 1M + u)h - 28h + *Oh - u H + 5 o h - 5 o h +

Figure 1. Effect of L-lactate on IL 2 production and IL 2 mRNA expression. (A) IL2 activity in the SN. Splenic T cells were incubated with Con A in standard RPMI 1640 culture medium with 2-ME. Some of the cultures contained 30 mM L-lactate. The SN were harvested at the indicated time points and assayed for IL 2 activity. (B) IL 2-specific mEWA expression. Splenic Tcells (7 X 107/14 ml) were incubated with Con A in standard culture medium with 2-ME. Some of the cultures contained 30 mM L-lactate. At the indicated time points the cells were counted, lysed, and subjected to Northern blot analysis with a 32P-labeled IL 2-specific DNA probe. The figure shows the expression of IL Zspecific mRNA (lower left panel) and the corresponding amount of 28 S and 18 S rRNA as stained by methylene blue (lower right panel). This control shows that equal quantities of RNA have been applied to the gel.

3.2 Lactate causes a reduction of intracellular GSH levels

Since cysteine and the cysteine derivative GSH are known to have profound effects on lymphocyte functions including proliferative responses and cytotoxic T cell activation [13], we analyzed among others the effects of lactate on the intracellular GSH level in a culture medium with approxi- mately physiological amino acid concentrations (i. e. mod- ified NCTC 135 culture medium). The results of these experiments confirmed earlier findings [13] that the intra- cellular GSH level in mitogenically stimulated splenic T cells is strongly influenced by the extracellular concentra- tion of cysteine (Fig. 4). Lactate was found to cause a

Eur. J. Immunol. 1991. 21: 1933-1937 Regulation of IL 2 production and glutathione levels by lactate 1935

i (A) 50 t

104

(A)

: : I 50

40 - 2 z30 2

20

10 ,'*

0 , 0 1.1 3.3 10 30

lactate ( mmol I L )

0 ' 1

0 1.1 3.3 10 30

lactate ( mmol I L )

0 4 8 12 16 20 24 28 32 36

time 01 administration of L- lactate ( h )

Figure 2. Augmentation of IL 2 production by lactate at graded doses and at various time points after Tcell stimulation. SplenicT cells were incubated with Con A in standard RPMI 1640 culture medium with 2-ME. (A) The indicated amounts of L-lactate were added at the start of the culture. (B) L-lactate (30 mM) was added at the indicated time points after Con A stimulation. Forty-eight hours after the start of the culture, SN were harvested and assayed for IL 2 activity.

substantial decrease of intracellular GSH levels at any given extracellular cysteine concentration. Accordingly, higher extracellular cysteine levels are required to obtain similar GSH levels as in control cultures without lactate (Fig. 4). It was, therefore, of interest to determine the role of gluta- thione in the lactate-mediated augmentation of IL 2 mRNA expression and IL 2 production.

3.3 The expression of IL 2 mRNA and IL 2 production is inhibited by GSH

The effect of GSH was analyzed with a cell culture system similar to that used in Fig. 4. IL 2 mRNA expression was determined by dot blot analysis. The results of these experiments showed that the lactate-mediated augmenta- tion of IL2 mRNA expression and IL2 production was virtually completely abrogated by millimolar concentra- tions of GSH (Fig. 5 ) . It is important to note in this context that concentrations of 2-5 m~ GSH are not generally toxic to mitogenically stimulated splenic T cells under these culture conditions. These concentrations of GSH were found, in fact, to augment strongly the rate of DNA synsthesis of mitogenically stimulated splenic Tcells under essentially identical conditions [ 171.

4 Discussion

Our experiments demonstrate that physiologically relevant variations of extracellular lactate concentrations exert strong regulatory effects on the induction of IL 2 produc- tion and IL2 mRNA expression. Lactate was found to augment the induction of IL 2 production and IL 2 mRNA expression and caused concomitantly a decrease of intra- cellular GSH levels. The mechanism for these effects of lactate is presently not known. However, the possibility that the stimulatory effect of lactate may be caused by an

0

i CLM+ Tcells + 0.0 mM Lactate o

3.3 mM Laclate A 10 mM Lactate v 30 mM Laaate 0

CD8+ Tcelk + 0.0 mM Lactate 3.3 mM Lactate A 10 mM Lactate (I 30 mM Lactate 8 a

0 16 32 48

time of incubation (hrs)

Figure 3. Failure of lactate to induce IL 2 activity in cultures of CD8+ T cells. CD8+ and CD4+ T cells have been prepared by treating splenicTcells (5 x 106/ml) with culture SN of hybridomas containing antLCD4 or anti-CD8 antibody, respectively, plus C. After washing, the cells were incubated with Con A (5 pg/ml) and graded doses of L-lactate in standard RPMI 1640 culture medium with 2-ME. The culture SN were harvested at the indicated time points and assayed for IL 2 activity.

alteration of the cytoplasmic pH seems unlikely, since exposure of T lymphocytes to 30 mM lactate was found to cause a brief and very moderate transient decrease of the cytoplasmic pH level (S. Roth, unpublished observa- tion).

The inhibition of the lactate-mediated IL 2 mRNA expres- sion and IL 2 production by GSH (see Fig. 5) in conjunc- tion with the fact that hydrogen peroxide augments IL 2 production in the presence of lactate but not in its absence [18] supports the hypothesis that the lactate-mediated augmentation of IL 2 production may involve the intracel- lular conversion of lactate into pyruvate. Reduced GSH is expected to lower the ratios of NAD+/NADH and of pyruvateflactate. Hydrogen peroxide, in contrast, raises NAD+/NADH ratios via the glutathione peroxidase and glutathione reductase reactions and favors thereby high pyruvateAactate ratios. Since a single activated MQ, can generate hydrogen peroxide at a rate of 2 x 1014-6 x 1014 moleculesh and up to a local extracellular concentration of

S. Roth and W. Drijge Eur. J. Imuno l . 1991. 21: 1933-1937

o without lactate with lactate

o / o - o

I I 1 I I

7.5 15 30 60 120 cysteine ( p mol I L )

Figure 4. Effect of lactate and cysteine on intracellular GSH levels of stimulated splenic T cells. Splenic T cells were incubated with Con A in modified NCTC 135 medium without 2-ME and with or without 30 mM L-lactate. The cultures received graded amounts of cysteine four times per day.The abscissa indicates the arriount per culture volume that was added each time. Protein and intracellular glutathione levels were determined after 48 h.

10-100 p~ [19, 201, it is possible that hydrogen peroxide may serve in conjunction with lactate as another immuno- regulatory small macrophage product. Pyruvate, in turn, is known to form covalent complexes with cysteine (i.e. hemimercaptal and thiazolidine derivatives) [21-23], and this may explain the effect of lactate on the intracellular GSH level (Fig. 4).

The effect of lactate on the intracellular GSH level and the observation that exogenous GSH abrogates the lactate- mediated augmentation of IL 2 mRNA expression and IL 2 production raise the question of whether lactate may augment IL 2 production by decreasing the intracellular concentration of potentially inhibitory GSH. This simple interpretation is unlikely, however, in view of the fact that depletion of intracellular GSH by the biosynthetic inhibitor buthionine sulfoximine does not substitute for lactate and does not cause by itself a substantial augmentation of IL 2 production [ 171.

Other immunologically relevant functions, including IL 2- dependent DNA synthesis and the activation of CTL are known to require high intracellular GSH levels and can be augmented by exogenous GSH [13]. Expectedly, these strongly GSH-dependent functions are negatively affected by lactate [14]. The fact that lactate augments certain lymphocyte functions but inhibits others may explain why the immunological reactivity is strengthened by moderate physical exercise and impaired by intensive training pro- grams [S-121. A detailed knowledge of the effects of lactate on the metabolism of lymphoid cells may enable 11s to use lactate as an immunopotentiating substance under defined

o 0 mM Lactate 030 mM L.eule >(\ 080 mM Laclate

0-

0 0.0s 0.31 1 .25 Y, glutathione ( m d L )

0.0 mM GSH 0.5 mM GSH 2.0 mM GSH 0.0 mM GSH + 30 mM lac 0.5 mM GSH+30 mM lac 2.0 mM GSH + 30 mM lac

Figure 5. Inhibition of IL 2 production and IL 2 mRNA expres- sion by GSH. (A) IL 2 activity in culture SN. Splenic T cells were incubated with Con A and graded doses of L-lactate in modified NCTC 135 medium without 2-ME and with the indicated concen- trations of GSH. The culture SN were harvested after 48 h and assayed for TCGF activity. (B) IL 2-specific mRNA expression. Splenic T cells were incubated with Con A and the indicated concentrations of GSH in modified NCTC 135 medium without 2-ME. Some of the cultures contained 30 mM L-lactate. After 24 h the cells were counted and subjected to dot-blot analysis with a 32P-labeled IL 2-specific DNA probe.

and controlled conditions. The immunosuppressive effect of glucocorticoids [24-261 may be explained at least partly by their effects on the glycolytic metabolism.

The assistance of Mrs. I. Fryson in the preparation of this manuscript is gratefully acknowledged.

Received January 24, 1991; in revised form May 13, 1991.

Eur. J. Immunol. 1991. 21: 1933-1937 Regulation of IL 2 production and glutathione levels by lactate 1937

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