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Regulation of T-cell functions by l -lactate
CELLULAR
IMMUNOLOGY
108,405-416
(1987)
Regulation of T-Cell Functions by L-Lactate WULF DR~GE, STEFFENROTH, ANNETTEALTMANN, AND SABINEMIHM Institut fir Immunologic und Genetik, DeutschesKrebsforschungszentruum, Im Neuenheimer Feld 280, D-6900 Heidelberg, West Germany Received January 22,1987; acceptedMarch 17, I987 Lactate is a product ofglycolytically active macrophages. After stimulation with concanavalin A accessory cell-depleted splenic T-cell populations were found to produce only minute amounts of T-cell growth factor (TCGF); but substantial amounts of TCGF were produced if the cultures were supplemented either with splenic adherent cells or with lactate but not with interleukin-1 (IL 1). IL- 1 was capable, however, of supporting TCGF production by the thymoma subline EL4-6.1. TCGF production in cultures of accessorycell-depleted splenic T-cell populations was demonstrable with lo-’ ML-lactate, and optimal responses(plateau level) were obtained with 4-6 X lo-* ML-lactate. Cultures of macrophages were found to accumulate up to 5 X lOA2M lactate. Our experiments indicate, therefore, that lactate serves as a regulatory signal by which macrophage-like accessorycells enhance helper-T-cell functions. Lactate is apparently not the only mediator of accessorycell function since plateau levels of TCGF production were markedly lower with lactate than with splenic accessorycells; but L-lactate was found also to determine the magnitude of T-cell-mediated immune responsesin vivo and in cultures of unfractionated lymphocyte populations. The production of interferon in accessory celldepleted and concanavalin A-treated T-cell cultures, however, was not significantly affected by lactate. Concanavalin A-stimulated splenic T-cell populations were found to consume glucose rapidly and to release lactate into the supematant. This indicates that the cells contain more lactate and pyruvate than they can utilize by their respiratory metabolism. The administration ofexternal lactate or pyruvate was found to inhibit the utilization ofglucose by the mitogenically stimulated T cells. o 1987 Academic FWSS, 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)). The molecular nature of the accessorycell function is not entirely clear. Experiments from several laboratories (6-9) suggestedthat interleukin- 1 (IL-l)’ is one of the mediators which are released by accessory cells and which support the activation of helper T cells. The experiments in this report demonstrate, however, that T-cell growth factor (TCGF) production in murine accessory cell-depleted splenic T-cell populations is not reconstituted by interleukin-1, but is reconstituted at least to some extent by the macrophage product L-lactate. The experiments indicate that the delivery of lactate is an important component of accessory ’ Abbreviations used: CTL, cytotoxic T lymphocytes; IL 1, interleukin-1 ; IL-2, interleukin-2; TCGF, T-cell growth factor; TPA, tetradecanoylphorbol acetate. 405 0008-8749187$3.00 Copyright Q 1987 by Academic F’Rs, Inc. All rights of reproduction in any form reserved.
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cell function. Lactate was found to determine the magnitude of immune responses in vivo and in cultures of unfractionated lymphoid cell populations in vitro. MATERIALS AND METHODS Animals. Mice were obtained from the breeding stock of the German Cancer Research Center or purchased from Bomholtgard (Ry, Denmark). Mice 8- 14 weeks old were used in the experiments. Materials. Lactate was obtained from Serva (Heidelberg, FRG), mitomycin C from Sigma, and concanavalin A from Pharmacia. 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 (10). 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. The preparation of splenic adherent cells (AC). The term “splenic adherent cells” was used in this report for a fraction of spleen cells prepared by a procedure that has been reported to enrich mainly for dendritic cells and some immature macrophages ( 11). Spleen cells from three mice (about 3 X lo* cells) were incubated in 10 ml of culture medium in plastic petri dishes with a IO-cm diameter (Greiner, Niirtingen, West Germany, type TC94/ 16) at 37°C. After 2 hr, the supernatant was discarded, the nonadherent cells were washed off carefully by extensive rinsing with warm culture medium or balanced salt solution (BSS), and the adherent cells wer again incubated with 10 ml of fresh culture medium for another 18 hr. The cells that detached spontaneously during this time period (usually 1 to 2 X lo6 cells) were then collected in the supematant and used as the “splenic adherent spleen cell population” in these studies. The IL-l l-dependent IL-2-producing EL-4 thymoma subline. EL4-6.1 has been described elsewhere ( 12) and was kindly provided by Dr. R. McDonald. Interleukin 1. Human IL-l was kindly provided by Dr. W. Falk. It was prepared by incubating human monocytes from huffy coat with pansorbin (Calbiochem) for 24 hr. Assay for TCGF activity. The amount of TCGF in supematants was measured by their ability to support the growth of the T-cell clone W-2 (originally M-2, cloned by Michael Stock, Hannover, West Germany) (13). The culture supematants were titrated by serial two- or threefold dilutions in flat-bottom tissue culture plates (Flow Laboratories) in a volume of 0.1 ml of culture medium without fetal calf serum (FCS). One hundred microliters of W-2 cell suspension (105/ml) in culture medium was added per well, and the plates were incubated at 37°C in moist air with 5% CO* for 20 hr. [3H]Thymidine (1 PCi) in 25 ~1 of RPM1 1640 was added per well, and the plates were incubated for another 4 to 6 hr. The cells were then harvested with a Dunn cell harvester, and the filters were counted in a liquid scintillation counter. Each point was assayedin duplicate. Data are given as TCGF units which are equivalents of the IL-2 activity units of an IL-2 standard, based on the method described by Farrar et al. (14). Units were calculated with an HP 9815 computer using linear regression analysis of the linear part of the titration curve. Control experiments (not shown) have established that lactate (up to a concentration of 2 X lo-’ M) has no
REGULATION
OF T-CELL FUNCTION BY LACTATE
0
o--o
0 /‘1-I no IL-1
407
loo0 reciprocal
titers
I
I
100
10
of IL-l
FIG. 1. TCGF production by accessory cell-depleted splenic T cells is augmented by lactate or splenic adherent cells but not by IL 1. Upper panel: The activity of the IL 1 preparation under test was demonstrated in the murine thymocyte proliferation assay with 1 X IO6C3H-HeJ thymocytes and 0.2 pg PHAM (Sigma) in 0.2-ml microcultures. Bottom panel: Nylon-wool-nonadherent and NH&l-treated C3H/Tif spleen cells (5 X 106)were incubated with 5 pg concanavalin A in 1 ml of culture medium for 58 hr. Some of the cultures also received 2 X lo-’ M L-lactate or 1 X 10’ splenic adherent cells and graded doses of IL-l at the start of the culture period. The supematants were subsequently assayed for TCGF activity. Corresponding concentrations of lactate (up to 2 X lo-* M) had no effect on the TCGF assay, i.e., they had no effect on the DNA synthesis of W-2 cells in cultures with or without TCGF (data not shown). 0, graded concentrations of IL1 only; 0; L-lactate + graded concentrations of IL-l; A, AC + graded concentrations of IL- 1.
effect on the TCGF assay.The assayhas been shown by W. Falk (personal communication) to detect not only interleukin-2 (TCGF-1) activity but also interleukin-4 (TCGF-2) which has been discovered recently ( 15,16) and shown to be identical with B-cell-stimulating factor- 1. Interferon was assayed as described (17). Briefly, 2 X lo4 L929 cells were cultured in 0.1 ml for 24 hr, then mixed with 0.05 ml of threefold serial dilutions of the sample and incubated for 24 hr. The supernatant was then removed and the cells were mixed with 0.1 ml of a vesicular stomatitis virus preparation and incubated for another hour. The supernatant was again removed, 0.2 ml of fresh culture medium was added, and the cultures were incubated for another 48 hr. The supernatants were removed again and the cells were stained with crystal violet. One interferon (IF) unit was defined as the minimal amount of interferon capable of conferring protection to 50% of the cells. All titers are expressedin laboratory units. Activation of cytotoxic T lymphocytes in mixed-lymphocyte cultures (microcultures). Spleen cells (3 X 105) or lymph node cells (1 X 105) were incubated with 1 X lo5 irradiated (1500 rad) allogeneic C57BL/6 spleen cells or 7 X IO4 irradiated
408
DRijGE ET AL. TABLE 1 IL- 1 Supports TCGF Production in Cultures of the Thymoma Line EL4-6.1 IL-2 (units/ml) IL 1 dilution
0.3 ng TPA/ml
1 ng TPA/ml
1:5 1:25 1:125 1:625 1:3125 No. IL- 1
4 15 11 4 0.1 0.0
19 68 53 15 4 0.1
Note. EL4-6.1 cells (2 X 10S/ml)were incubated with the indicated concentration of tetradecanoylphorbolacetate (TPA) and graded concentrations of IL- 1. The supematants were collected after 3 days and assayedfor TCGF.
lymph node cells, respectively, in 0.2 ml of culture medium for 5 days. The culture medium (RPM1 1640, GIBCO) was supplemented with r&ttamine (GIBCO, final concentration 5 mM), streptomycin/penicillin (GIBCO, 100 U/ml), 0.5% Hepes (GIBCO), 10% fetal calf serum (GIBCO), and 3 X 10e5M 2-mercaptoethanol. The cytotoxic activity was determined 5 days later against 2 X lo4 concanavalin A-activated C57BL/6 spleen cell blasts per well as target cells in a 4-hr ‘lCr-release assayas described previously ( 18). The activation of secondary cytotoxic T-cell responses in vitro (macrocultures). Spleen cells (2 X 10’) from immunized mice were incubated as responder cells with 1 X lo6 mitomycin C-treated (Sigma) (80 pg/ml, 45 min) tumor cells and 5 X lo6 irradiated ( 1500 rad) allogeneic stimulator cells in a total volume of 5 ml of culture medium for 5 days at 37°C in 5% C02. The cultures were tested after 5 days for cytotoxic activity against tumor cells and concanavalin A-activated allogeneic spleen cell blasts in a 4-hr “Cr-release assayas described previously ( 18). The tumor line ESb-D. The tumor line was derived from the methylcholanthreneinduced T-cell lymphoma L 5 187Y (DBA/2 origin). The history and properties of this tumor line have been described recently in detail ( 19). Enzymic determination of L-lactate. Cell culture supernatants were incubated with perchloric acid (0.5 M) for 15 min at 4°C. Precipitated protein was removed by highspeed centrifugation and the resulting supematants were subjected to the following procedure: 1.4 ml of glycine buffer (37.5 g glycine (Merck) plus 84 ml of 24% hydrazine hydrate (Merck) dissolved in 1 liter and adjusted to pH 9.0 with NaOH) was mixed with 0.1 ml of @-nicotinamide adenine dinucleotide (Sigma, 30 mg/ml = 40 mM) and 0.02 ml of supematant sample. The extinction of this mixture at 334 nm (E,) was determined in a Gilford 250 spectrophotometer. Finally, 0.0 1 ml of L-lactate dehydrogenase (Serva, from rabbit muscle, 10 mg/ml; 600 U/mg protein) was added to the mixture. The extinction at 334 nm (E2) was again determined after 1 hr of incubation at room temperature, and the lactate content was determined from the difference, E = E2 - E,, according to a standard curve. The standard curve was obtained with lactate concentrations varying between 1 and 20 mM. Utilization and biochemical conversion of14C-labeledglucose by splenic T-cell populations. Accessory cell-depleted T cells (1 X 108/ml) were incubated with conca-
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BY LACTATE
409
navalin A (5 &ml) and 4 &i/ml of D[U-“C]glucose in a total volume of 0.3 ml. Samples of 9 ~1 were removed after certain time intervals; and the labeled components in these samples were determined by ascending thin-layer chromatography (TLC) on cellulose-aluminum plates (Merck 5552) with N-butanol/acetic acid/water/ propionic acid (60: 15:22:3; v/v/v/v). The labeled components were identified and quantitatively determined with a TLC-linear analyzer (Bertholdt LB 2842). RESULTS
L-LactateSubstitutesfor AccessoryCells in Mitogenically Stimulated Cultures ofMurine Splenic T Cells Accessory cell-depleted splenic T-cell preparations were obtained by incubating C3H/Tif spleen cells twice with nylon wool and by subsequent treatment with ammonium chloride solution for removal of erythrocytes. These accessory cell-depleted splenic T-cell preparations produced only minute amounts of TCGF in response to concanavalin A (Pharmacia) (Fig. 1, bottom); but a more than IO-fold increase in TCGF production was observed after addition of 1 X lo5 splenic adherent cells or 2 X 10e2ML-lactate (Fig. 1, bottom). The TCGF production was not reconstituted by an IL- 1 preparation which was highly active in the murine thymocyte proliferation assay(Fig. 1, top). TCGF production by the EL4-6.1 thymoma line ( 12), in contrast, was effectively supported by this IL- 1 preparation (Table 1). This indicates that IL- 1 supports TCGF production in some but not all types of T-lineage cells that are potentially capable of TCGF production. (Control experiments (not shown) established that the IL- 1 preparation had no TCGF activity). Plateau levels of TCGF production were obtained with 4-8 X 10e2M L-lactate, but the co-inducing effect of lactate was demonstrable at a concentration of 1 X 1Op3 A4 (Fig. 2). Plateau levels in parallel cultures with optimal numbers of splenic adherent cells as accessorycells were about eight times higher (i.e., 400 units/ml) than the maximum TCGF production obtained with L-lactate (data not shown). Thus L-lactate substituted only partly for accessorycells in this system. In the same system lactate had no significant effect on the production of interferon. In a typical experiment essentially under the same culture conditions as in Fig. 2, we detected 8 1 units/ml of interferon in cultures without lactate and 46 units/ml in cultures with 3 X lop2 it4 L-lactate. T cells and macrophage-like accessory cells can form intimate contact (5); and T cells are therefore exposed to high concentrations of low-molecular-weight substances which are releasedby these accessorycells. Lactate is releasedby mononuclear phagocytes and certain subsets of macrophages as a consequence of their strong glycolytic activity (20-26). Figure 3 illustrates that 3.6 X lo6 cells in 0.4 ml of Dulbecco’s MEM produce lactate at a linear rate up to a concentration of almost 6 x 1Oe2M L-lactate alter 45 hr. This is compatible with the observation in vivo that in extreme lactacidosis the plasma concentration can exceed 3 X 10e2A4 L-lactate, indicating that lactateproducing cells can releaselactate even against a relatively high extracellular concentration of lactate.
Lactate Functions Also as a Regulatory Signal for Immune Responsesin Vivo and for Unfactionated Lymphocyte Populations in Vitro The production of TCGF is augmented by L-lactate not only in mitogenically stimulated accessory cell-depleted spleen cells, but also in mitogenically stimulated un-
DR6GE ET AL.
I
0
I
I
5xlo-4
I
1x10-3
I
I
2~10-3 5xX)-3 L-bcme (mobalL)
I
1x90-2
I
2xlo-2
I
I
5x10-2 1x10-l
FIG. 2. Augmentation of TCGF production in accessory cell-depleted splenic T-cell populations by graded doses of L-lactate. Accessory cell-depleted splenic T cells from C3H/Tif mice (5 X 106/ml) were incubated with concanavalin A (5 &ml) and with the indicated final concentrations of L-lactate. The culture supematants were harvested after 48 hr and were assayedfor TCGF. Parallel cultures with optimal numbers of splenic adherent cells as accessorycells (plateau level) generated 400 units/ml of TCGF (data not shown).
fractionated murine spleen cells (Fig. 4). L-Lactate was found to mediate a strong dose-dependent augmentation of the TCGF production if added at the start of the culture but mediated only minute effectsif added 20 hr later. The activation of cytotoxic T cells in mixed-leukocyte cultures was found to be augmented by L-lactate if lymph node cells were used as responder and as stimulator cells (Table 2). The cytotoxic response in IG2-containing cultures of lymph node cells or in cultures of spleen cells with or without IL-2 was always a priori relatively high and only moderately augmented by L-lactate. These results suggestthat lactate augments the cytotoxic responsesprimarily by co-inducing the production of interleukin-2 and not by a direct effect on the cytotoxic T-lymphocyte precursor cells. Spleen cells may contain a priori more accessory cells than lymph node cells and may, therefore, be lessdependent on the external application of L-lactate. Finally, L-lactate was also found to augment the in vivo immunization (priming) of DBA/2 mice for a subsequent secondary cytotoxic response in vitro against synge-
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I
0
10
OF T-CELL FUNCTION
I
I
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I
20 30 40 time after initiation of culture (hrs)
I
t
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60
FIG. 3. Releaseof lactate by peritoneal macrophages. C3H/Tif mice received 1 ml of phosphate-buffered saline ip and their peritoneal exudate cells were harvested 1 day later and were incubated at a density of 7.5 x lo6 cells per 0.4 ml in Dulbecco’s MEM for 2 hr. The nonadherent cells were then removed and counted and the remaining adherent macrophages (3.6 X lo6 per well) were again incubated in 0.4 ml of Dulbecco’s MEM. Supematant samples of 0.05 ml were removed at the indicated time points and assayed for lactate as described under Materials and Methods. The remaining culture was reconstituted each time with 0.05 ml of fresh Dulbecco’s MEM.
neic L 5 178 Y ESb-D tumor cells (Fig. 5, top panel). This indicates that L-lactate also determines in vivo the magnitude of TCGF-dependent immune responses.The primary cytotoxic response against the unrelated C57BL/6 stimulator cells in the same cultures (Fig. 5, bottom panel), in contrast, was a priori relatively strong and only slightly augmented after the in vivo treatment with lactate. The injection of L-lactate without ESb-D tumor cells (i.e., without in vivo priming) was not sufficient to mediate a detectable cytotoxic response of the corresponding spleen cells against ESb-D target cells in a subsequent in vitro culture with ESb-D and C57BL/6 stimulator cells (not shown). Biochemical Efects of L-Lactate on Mitogenically Stimulated T Cells In agreement with earlier studies (27-33) we found that our mitogenically stimulated T-cell populations express rapid, strong glycolytic activity and release lactate
412
DRijGE ET AL.
0
I
I
1.6 x 1O-4
6 x 10-4
I
4 x 10-a
I
2 x 10-2
moles/L
Fzc. 4. Effect of L-lactate on the production of TCGF by unfractionated spleen cells. C3H/Tif spleen cells (5 X 106/ml) were incubated with concanavalin A (5 &ml) and the indicated final concentrations of L-lactate (Sigma) for 48 hr in 4-ml cultures. The resulting supematants were then assayedfor TCGF activity. Some of the cultures received L-lactate only 20 hr after incubation. 0, L-lactate at the start; W,L-lactate after 20 hr.
into the supematant (Fig. 6, top panel). This indicates that the T-cell population contains more pyruvate and lactate than it can use by its respiratory metabolism. The possibility that L-lactate or pyruvate may serve as an additional source of energy by the respiratory metabolism is therefore unlikely. This conclusion was supported by additional experiments using cultures with 1 X lo-* A4 r4C-labeledL-lactate or labeled pyruvate. These experiments revealed that the labeled L-lactate was not detectably
TABLE 2 The Activation of Cytotoxic T Cells in Cultures of Allogeneic Lymph Node Preparations Is Augmented by L-Lactate % Wr release Cultured materials
No lactate
4 X 10m3Mlactate
2 X lo-* Mlactate
Lymph node cells Lymph node cells + IL-2 Spleen cells Spleen cells + IL-2
3.3 45.3 46.1 65.8
10.4 47.2 56.6 65.9
27.1 61.7 58.4 67.5
Note. C3H lymph node cells (1 X 10’) or spleen cells (3 X 105)were incubated with irradiated C57BL/6 lymph node cells (7 X 104)or spleen cells (1 X 105),respectively, and with or without 0.02 ml of an IG2containing EL-4 cell supematant in 0.2-ml microcultures for 5 days. Cytotoxic activity was then tested in a 4-hr “Cr-release assaywith 2 X lo4 C57BL/6 target cells.
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A:T ratio n 15.1 0 3:1
n-------.
60.
40 II
20
!! L P
0
2 ‘%
60
6 40
20
0
0.‘lO’l a:1 /li/*\/\ . 0-O 0\ . x 0 0 1 I 0
II
I 4 x 10-3
1 2 x 10-Z
I 1 x 10-I
I 5 x 10-l
moles/L FIG. 5. Effect of L-lactate on the in vivo immunization (priming) for a secondary in vitro cytotoxic response against L 5 178 Y E&-D tumor cells. DBA/2 mice were immunized with 1 X lo6 mitomycin-Ctreated ESb-D tumor cells ip. The mice received also 3 X 0.2 ml of BSS with the indicated concentrations of L-lactate ip at the time of immunization and 2 and 4 hr later. Eight days later, the mice were sacrificed and 2 X 10’ spleen cells were incubated for another 5 days in 5 ml of culture medium with 1 X lo6 mitomycin-C-treated ESb-D cells and 5 X lo6 irradiated C7BL/6 spleen cells. The cytotoxic activity was finally assayedagainst ESb-D target cells (top panel) and C57BL/6 targets (bottom panel) at the indicated attacker:target cell ratios.
utilized and pyruvate was mainly converted into L-lactate (data not shown). Taken together, our experiments thus confirm earlier observations that the glycolytic metabolism is the major source of energy for the lymphocytes after mitogenic stimulation (27-33). The external application of L-lactate and pyruvate was found to inhibit the utilization of glucose (Fig. 6). DISCUSSION Macrophage-like cells are known to play an important role as accessorycells and stimulator cells during lymphocyte activation (l-4); but the molecular basis of this accessory function is not entirely clear. The macrophage product interleukin-1 was previously thought to be the most important mediator especially for the induction of TCGF production by helper T cells (6-9). The experiments in this report show, however, that the production of TCGF by mitogenically activated accessorycell-depleted splenic T lymphocytes or by unfmctionated spleen cells is augmented by L-lactate. L-Lactate was capable of partially replacing the accessorycells in cultures of accessory cell-depleted splenic T-cell populations, whereas interleukin- 1 either alone or in combination with L-lactate had practically no effect on the TCGF production by these splenic T cells.
414
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0
2
7 18 time after start of culture (hrs)
26
20-
0'
I 0
"
I
1.2
I
2.5 moles/L
I
I
10
I
20
I
40
I
80
:X 103)
FIG. 6. Inhibition of glucose utilization by L-lactate and pyruvate. Accessory cell-depleted splenic T-cells (3 X 10’ cells/O.3 ml) were incubated with concanavalin A (5 &ml) and 4 ICi of [‘4C]glucose (A). Some of the cultures contained also 2 X lo-* A4 lactate (0) or pyruvate (Cl) (top) or graded concentrations of lactate (0) and pyruvate (0) (bottom). Supematant samples of 9 ~1were removed at the indicated time points (top) or after 26 hr (bottom). The radioactive materials were subsequently analyzed by ascending thin-layer chromatography. The figure illustrates both the remaining concentration of the labeled glucose (solid lines) and the appearance of lactate (broken lines) in the supematant as a percentage of the total radioactivity applied.
The latter point is clearly at variance with previous reports which suggestedthat IL- 1 induces TCGF production by mitogenically activated accessory cell-depleted peripheral T-cell populations (6-9). One of these reports (7) showed, however, that IL- 1 induced a much more profound augmentation of TCGF production and DNA synthesis in thymocyte populations (i.e., 30-fold) than in adherent cell-depleted splenit T-cell preparations (Sfold); and this suggestedthe possibility that IL-l may be primarily a maturation factor for relatively immature T-lineage cells. This interpretation is compatible with our observations that IL-l was very active in the thymocyte proliferation assay(Fig. 1) and was also capable of supporting the TCGF production in EL4-6.1 thymoma cells (Table 1). The issue whether IL- 1 is absolutely required for T-cell activation has recently been critically reviewed by Oppenheim and colleagues, and both positive and negative arguments have been given (9). Irrespective of the apparently variable contribution of IL- 1 to the accessoryfunction, our experiments strongly indicate that lactate is another particularly important
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mediator by which accessorycells augment helper-T-cell functions. Macrophages can generate concentrations of lactate up to 6 X 10e2M in their extracellular space;and lactate concentrations between 1 X 10e3and 8 X 10M2M were found to mediate a substantial dose-dependent augmentation of the TCGF production in accessorycelldepleted T-cell populations. Moreover, macrophage-like accessory cells come into intimate contact with lymphocytes as judged from microscopic inspection (see Fig. 1 in Ref. (5)) and are therefore expected to expose the lymphocytes to relatively high concentrations of L-lactate. (The experiment in Fig. 3 indicates that each macrophage in the culture produces 1.2 X lo-l3 mol/hr and within 45 hr fills a volume of 10m7ml with a concentration of 6 X 1Od2ML-lactate. One can, therefore, expect that an area of 5 pm around the macrophage (i.e., 1 X 10T8ml) is filled with a concentration of 6 X 1O-2M L-lactate within 4.5 hr under conditions of limited diffusion as they may exist in the tissue.) The conclusion that lactate is an important regulatory signal by which macrophage-like accessory cells control the magnitude of helper-T-cell function is sup ported by a series of in vivo and in vitro experiments. These experiments collectively showed that the external application of lactate regulates also the magnitude of T-cell responsesin vivo and also in cultures with unfi-actionated lymphoid cell populations. The effects of lactate on cytotoxic T-cell responses in vitro are compatible with the interpretation that lactate is required only for the induction of TCGF production but not for the activation of the CTL precursor cells. Cytotoxic responsesin cultures with lymph node responder and lymph node stimulator cells were strongly augmented by the external application of either lactate or IL-2, whereas cultures with splenic responder and stimulator cells were only marginally augmented by additional lactate or IL-2. IL-2-containing cultures of either cell type were only marginally augmented by lactate (Table 2). Also lactate had practically no effect on the interferon production by mitogenically stimulated accessorycell-depleted T-cell populations. The mechanism by which lactate augments IL2 production is not known. The most obvious possibility that lactate or pyruvate may serve as an additional source of energy was rendered extremely unlikely. Our experiments showed in agreement with earlier reports (27-3 1) that Con A-stimulated accessorycell-depleted splenic T cells rapidly utilize glucose by glycolytic metabolism and already release substantial amounts of L-lactate during the first 8 hr after stimulation. The cell population thus contains by itself already more lactate and pyruvate than it can utilize by respiratory energy metabolism. Externally administered 14C-labeledlactate was indeed not utilized to any detectable extent and labeled pyruvate was mainly converted into lactate (data not shown). Our experiments showed, on the other hand, that the augmentation of TCGF production by application of external L-lactate or pyruvate is correlated with a strong inhibition of the glycolytic activity (Fig. 6). ACKNOWLEDGMENTS The expert technical assistanceof Mrs. Heike Schmidt and Mrs. Sabine Nick and the assistanceof Mrs. Ingrid Fryson in the preparation of this manuscript are gratefUlly acknowledged.
REFERENCES 1. Schwab, R., Crow, M. K., Russo, C., and Weksler, M. E. J. Immunol.135,1714,1985. 2. Rosenstreich, D. L., Farrar, J. J., and Dougherty, S. J. Immunof.116, 13 1, 1976.
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ET AL.
3. Devries, J. E., Caviles, A. P., Bont, W. S., and Mendelsohn, J., J. Immunol. 122, 1099, 1979. 4. Thiele, D. L., and Lipsky, P. E., J. Immunol. 129, 1033, 1982. 5. Rosenthal, A. S., Immunol. Rev. 40,136,1978. 6. Larsson, E.-L., Iscove, N. N., and Coutinho, A., Nature (London) 283,664, 1980. 7. Smith, K. A., Lachman, L. B., Oppenheim, J. J., and Favata, M. F., J. Exp. Med. 151,1551,1980. 8. Palacios, R., J. Immunol. 128,337, 1982. 9. Oppenheim, J. J., Kovacs, E. J., Matsushima, K., and Durum, S. K., Immunol. Today 7,45, 1986. 10. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3,645, 1973. 11. Steinmann, R. M., Gutchinov, G., Witmer, M. D., and Nussenzweig, M. C., J. Exp. Med. 157,6 13, 1983.
12. MacDonald, H. R., Lees, R. K., and Bron, C., J. Immunol. 135,3944,1985. 13. Falk, W., Mannel, D. N., and Droge, W., J. Immunol. 130,22 14, 1983. 14. Farrar, J. J., Mizel, S. B., Fuller-Farrar, J., Farrar, W. L., and Hilfiker, M. L., J. Zmmunol. 125,793, 1980.
15. Mosmann, T. R., Bond, M. W., Coffman, R. L., Ohara, J., and Paul, W. E., Proc. Natl. Acad. Sci. USA83,5654, 1986. 16. Femandez-Botran, R., Krammer, P. H., Diamantstein, T., Uhr, J. W., and Vitetta, E. S., J. Exp. Med. 164,580, 1986.
17. In “The Interferon System” (W. E. Stewart II, Ed.), p. 15. Springer-Verlag, New York, 1979. 18. Galli, P., and Driige, W., Eur. J. Immunol. 10,87, 1980. 19. DrBge, W., Eck, H.-P., Kriegbaum, H., and Mihm, S., J. Zmmunol. 137, 1383, 1986. 20. Roos, D., and Balm, A. J. M., In “The Reticuloendothelial System,” Vol. 2, “Biochemistry and metabolism” (A. J. Sbarra and R. R. Strauss,Eds.), p. 189. Plenum, New York/London, 1980. 21. Axline, S. G., Semin. Hematol. 7, 142, 1970. 22. Oren, R., Famham, A. E., Saito, K., Milofsky, E., and Kamovsky, M. L., J. Cell. Biol. 17,487, 1963. 23. Simon, L. M., Robin, E. D., Phillips, J. R., Acevedo, J., Axline, S. G., and Theodore, J., J. Clin. Invest. 59,443, 1977. 24. Para, M., Sagone,A., Balcerzak, S., and LoBuglio, A., Clin. Rex 20,742A, 1972.
25. Roos, D., Reiss, M., Balm, A. J. M., Palache, A. M. Cambier, P. H., and Van der Stijl-Neijenhuis, J. S. In “macrophages and Lymphocytes: Nature, Functions, and Interaction” (M. R. Escobar and H. Friedman, Eds.), pp. 29-36. Plenum, New York, 1979. 26. Hard, G. C., Brit. J. Exp. Puthol. 51,97, 1970. 27. Hedeskov C. J., and Esmann, V., Blood28,163,1966. 28. Roos, D., and Loos, J. A. Biochim. Biophys. Acta 222,565, 1970. 29. Roos, D., De Boer, J. E. G., Huismans, L., and Boom, A. J., Exp. Cell Res. 75, 185, 1972. 30. Roos, D., and Loos, J. A., Exp. Cell Rex 77, 121, 1973. 31. Roos, D., and Loos, J. A., Exp. Cell Res. 77,127, 1973. 32. Wang, T., Foker, J. E., and Tsai, M. Y., Biochem. Biophys. Res. Commun. 95, 13, 1980. 33. Wang, T., Marquardt, C., and Foker, J., Nature (London) 261,702, 1976.
IMMUNOLOGY
108,405-416
(1987)
Regulation of T-Cell Functions by L-Lactate WULF DR~GE, STEFFENROTH, ANNETTEALTMANN, AND SABINEMIHM Institut fir Immunologic und Genetik, DeutschesKrebsforschungszentruum, Im Neuenheimer Feld 280, D-6900 Heidelberg, West Germany Received January 22,1987; acceptedMarch 17, I987 Lactate is a product ofglycolytically active macrophages. After stimulation with concanavalin A accessory cell-depleted splenic T-cell populations were found to produce only minute amounts of T-cell growth factor (TCGF); but substantial amounts of TCGF were produced if the cultures were supplemented either with splenic adherent cells or with lactate but not with interleukin-1 (IL 1). IL- 1 was capable, however, of supporting TCGF production by the thymoma subline EL4-6.1. TCGF production in cultures of accessorycell-depleted splenic T-cell populations was demonstrable with lo-’ ML-lactate, and optimal responses(plateau level) were obtained with 4-6 X lo-* ML-lactate. Cultures of macrophages were found to accumulate up to 5 X lOA2M lactate. Our experiments indicate, therefore, that lactate serves as a regulatory signal by which macrophage-like accessorycells enhance helper-T-cell functions. Lactate is apparently not the only mediator of accessorycell function since plateau levels of TCGF production were markedly lower with lactate than with splenic accessorycells; but L-lactate was found also to determine the magnitude of T-cell-mediated immune responsesin vivo and in cultures of unfractionated lymphocyte populations. The production of interferon in accessory celldepleted and concanavalin A-treated T-cell cultures, however, was not significantly affected by lactate. Concanavalin A-stimulated splenic T-cell populations were found to consume glucose rapidly and to release lactate into the supematant. This indicates that the cells contain more lactate and pyruvate than they can utilize by their respiratory metabolism. The administration ofexternal lactate or pyruvate was found to inhibit the utilization ofglucose by the mitogenically stimulated T cells. o 1987 Academic FWSS, 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)). The molecular nature of the accessorycell function is not entirely clear. Experiments from several laboratories (6-9) suggestedthat interleukin- 1 (IL-l)’ is one of the mediators which are released by accessory cells and which support the activation of helper T cells. The experiments in this report demonstrate, however, that T-cell growth factor (TCGF) production in murine accessory cell-depleted splenic T-cell populations is not reconstituted by interleukin-1, but is reconstituted at least to some extent by the macrophage product L-lactate. The experiments indicate that the delivery of lactate is an important component of accessory ’ Abbreviations used: CTL, cytotoxic T lymphocytes; IL 1, interleukin-1 ; IL-2, interleukin-2; TCGF, T-cell growth factor; TPA, tetradecanoylphorbol acetate. 405 0008-8749187$3.00 Copyright Q 1987 by Academic F’Rs, Inc. All rights of reproduction in any form reserved.
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cell function. Lactate was found to determine the magnitude of immune responses in vivo and in cultures of unfractionated lymphoid cell populations in vitro. MATERIALS AND METHODS Animals. Mice were obtained from the breeding stock of the German Cancer Research Center or purchased from Bomholtgard (Ry, Denmark). Mice 8- 14 weeks old were used in the experiments. Materials. Lactate was obtained from Serva (Heidelberg, FRG), mitomycin C from Sigma, and concanavalin A from Pharmacia. 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 (10). 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. The preparation of splenic adherent cells (AC). The term “splenic adherent cells” was used in this report for a fraction of spleen cells prepared by a procedure that has been reported to enrich mainly for dendritic cells and some immature macrophages ( 11). Spleen cells from three mice (about 3 X lo* cells) were incubated in 10 ml of culture medium in plastic petri dishes with a IO-cm diameter (Greiner, Niirtingen, West Germany, type TC94/ 16) at 37°C. After 2 hr, the supernatant was discarded, the nonadherent cells were washed off carefully by extensive rinsing with warm culture medium or balanced salt solution (BSS), and the adherent cells wer again incubated with 10 ml of fresh culture medium for another 18 hr. The cells that detached spontaneously during this time period (usually 1 to 2 X lo6 cells) were then collected in the supematant and used as the “splenic adherent spleen cell population” in these studies. The IL-l l-dependent IL-2-producing EL-4 thymoma subline. EL4-6.1 has been described elsewhere ( 12) and was kindly provided by Dr. R. McDonald. Interleukin 1. Human IL-l was kindly provided by Dr. W. Falk. It was prepared by incubating human monocytes from huffy coat with pansorbin (Calbiochem) for 24 hr. Assay for TCGF activity. The amount of TCGF in supematants was measured by their ability to support the growth of the T-cell clone W-2 (originally M-2, cloned by Michael Stock, Hannover, West Germany) (13). The culture supematants were titrated by serial two- or threefold dilutions in flat-bottom tissue culture plates (Flow Laboratories) in a volume of 0.1 ml of culture medium without fetal calf serum (FCS). One hundred microliters of W-2 cell suspension (105/ml) in culture medium was added per well, and the plates were incubated at 37°C in moist air with 5% CO* for 20 hr. [3H]Thymidine (1 PCi) in 25 ~1 of RPM1 1640 was added per well, and the plates were incubated for another 4 to 6 hr. The cells were then harvested with a Dunn cell harvester, and the filters were counted in a liquid scintillation counter. Each point was assayedin duplicate. Data are given as TCGF units which are equivalents of the IL-2 activity units of an IL-2 standard, based on the method described by Farrar et al. (14). Units were calculated with an HP 9815 computer using linear regression analysis of the linear part of the titration curve. Control experiments (not shown) have established that lactate (up to a concentration of 2 X lo-’ M) has no
REGULATION
OF T-CELL FUNCTION BY LACTATE
0
o--o
0 /‘1-I no IL-1
407
loo0 reciprocal
titers
I
I
100
10
of IL-l
FIG. 1. TCGF production by accessory cell-depleted splenic T cells is augmented by lactate or splenic adherent cells but not by IL 1. Upper panel: The activity of the IL 1 preparation under test was demonstrated in the murine thymocyte proliferation assay with 1 X IO6C3H-HeJ thymocytes and 0.2 pg PHAM (Sigma) in 0.2-ml microcultures. Bottom panel: Nylon-wool-nonadherent and NH&l-treated C3H/Tif spleen cells (5 X 106)were incubated with 5 pg concanavalin A in 1 ml of culture medium for 58 hr. Some of the cultures also received 2 X lo-’ M L-lactate or 1 X 10’ splenic adherent cells and graded doses of IL-l at the start of the culture period. The supematants were subsequently assayed for TCGF activity. Corresponding concentrations of lactate (up to 2 X lo-* M) had no effect on the TCGF assay, i.e., they had no effect on the DNA synthesis of W-2 cells in cultures with or without TCGF (data not shown). 0, graded concentrations of IL1 only; 0; L-lactate + graded concentrations of IL-l; A, AC + graded concentrations of IL- 1.
effect on the TCGF assay.The assayhas been shown by W. Falk (personal communication) to detect not only interleukin-2 (TCGF-1) activity but also interleukin-4 (TCGF-2) which has been discovered recently ( 15,16) and shown to be identical with B-cell-stimulating factor- 1. Interferon was assayed as described (17). Briefly, 2 X lo4 L929 cells were cultured in 0.1 ml for 24 hr, then mixed with 0.05 ml of threefold serial dilutions of the sample and incubated for 24 hr. The supernatant was then removed and the cells were mixed with 0.1 ml of a vesicular stomatitis virus preparation and incubated for another hour. The supernatant was again removed, 0.2 ml of fresh culture medium was added, and the cultures were incubated for another 48 hr. The supernatants were removed again and the cells were stained with crystal violet. One interferon (IF) unit was defined as the minimal amount of interferon capable of conferring protection to 50% of the cells. All titers are expressedin laboratory units. Activation of cytotoxic T lymphocytes in mixed-lymphocyte cultures (microcultures). Spleen cells (3 X 105) or lymph node cells (1 X 105) were incubated with 1 X lo5 irradiated (1500 rad) allogeneic C57BL/6 spleen cells or 7 X IO4 irradiated
408
DRijGE ET AL. TABLE 1 IL- 1 Supports TCGF Production in Cultures of the Thymoma Line EL4-6.1 IL-2 (units/ml) IL 1 dilution
0.3 ng TPA/ml
1 ng TPA/ml
1:5 1:25 1:125 1:625 1:3125 No. IL- 1
4 15 11 4 0.1 0.0
19 68 53 15 4 0.1
Note. EL4-6.1 cells (2 X 10S/ml)were incubated with the indicated concentration of tetradecanoylphorbolacetate (TPA) and graded concentrations of IL- 1. The supematants were collected after 3 days and assayedfor TCGF.
lymph node cells, respectively, in 0.2 ml of culture medium for 5 days. The culture medium (RPM1 1640, GIBCO) was supplemented with r&ttamine (GIBCO, final concentration 5 mM), streptomycin/penicillin (GIBCO, 100 U/ml), 0.5% Hepes (GIBCO), 10% fetal calf serum (GIBCO), and 3 X 10e5M 2-mercaptoethanol. The cytotoxic activity was determined 5 days later against 2 X lo4 concanavalin A-activated C57BL/6 spleen cell blasts per well as target cells in a 4-hr ‘lCr-release assayas described previously ( 18). The activation of secondary cytotoxic T-cell responses in vitro (macrocultures). Spleen cells (2 X 10’) from immunized mice were incubated as responder cells with 1 X lo6 mitomycin C-treated (Sigma) (80 pg/ml, 45 min) tumor cells and 5 X lo6 irradiated ( 1500 rad) allogeneic stimulator cells in a total volume of 5 ml of culture medium for 5 days at 37°C in 5% C02. The cultures were tested after 5 days for cytotoxic activity against tumor cells and concanavalin A-activated allogeneic spleen cell blasts in a 4-hr “Cr-release assayas described previously ( 18). The tumor line ESb-D. The tumor line was derived from the methylcholanthreneinduced T-cell lymphoma L 5 187Y (DBA/2 origin). The history and properties of this tumor line have been described recently in detail ( 19). Enzymic determination of L-lactate. Cell culture supernatants were incubated with perchloric acid (0.5 M) for 15 min at 4°C. Precipitated protein was removed by highspeed centrifugation and the resulting supematants were subjected to the following procedure: 1.4 ml of glycine buffer (37.5 g glycine (Merck) plus 84 ml of 24% hydrazine hydrate (Merck) dissolved in 1 liter and adjusted to pH 9.0 with NaOH) was mixed with 0.1 ml of @-nicotinamide adenine dinucleotide (Sigma, 30 mg/ml = 40 mM) and 0.02 ml of supematant sample. The extinction of this mixture at 334 nm (E,) was determined in a Gilford 250 spectrophotometer. Finally, 0.0 1 ml of L-lactate dehydrogenase (Serva, from rabbit muscle, 10 mg/ml; 600 U/mg protein) was added to the mixture. The extinction at 334 nm (E2) was again determined after 1 hr of incubation at room temperature, and the lactate content was determined from the difference, E = E2 - E,, according to a standard curve. The standard curve was obtained with lactate concentrations varying between 1 and 20 mM. Utilization and biochemical conversion of14C-labeledglucose by splenic T-cell populations. Accessory cell-depleted T cells (1 X 108/ml) were incubated with conca-
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409
navalin A (5 &ml) and 4 &i/ml of D[U-“C]glucose in a total volume of 0.3 ml. Samples of 9 ~1 were removed after certain time intervals; and the labeled components in these samples were determined by ascending thin-layer chromatography (TLC) on cellulose-aluminum plates (Merck 5552) with N-butanol/acetic acid/water/ propionic acid (60: 15:22:3; v/v/v/v). The labeled components were identified and quantitatively determined with a TLC-linear analyzer (Bertholdt LB 2842). RESULTS
L-LactateSubstitutesfor AccessoryCells in Mitogenically Stimulated Cultures ofMurine Splenic T Cells Accessory cell-depleted splenic T-cell preparations were obtained by incubating C3H/Tif spleen cells twice with nylon wool and by subsequent treatment with ammonium chloride solution for removal of erythrocytes. These accessory cell-depleted splenic T-cell preparations produced only minute amounts of TCGF in response to concanavalin A (Pharmacia) (Fig. 1, bottom); but a more than IO-fold increase in TCGF production was observed after addition of 1 X lo5 splenic adherent cells or 2 X 10e2ML-lactate (Fig. 1, bottom). The TCGF production was not reconstituted by an IL- 1 preparation which was highly active in the murine thymocyte proliferation assay(Fig. 1, top). TCGF production by the EL4-6.1 thymoma line ( 12), in contrast, was effectively supported by this IL- 1 preparation (Table 1). This indicates that IL- 1 supports TCGF production in some but not all types of T-lineage cells that are potentially capable of TCGF production. (Control experiments (not shown) established that the IL- 1 preparation had no TCGF activity). Plateau levels of TCGF production were obtained with 4-8 X 10e2M L-lactate, but the co-inducing effect of lactate was demonstrable at a concentration of 1 X 1Op3 A4 (Fig. 2). Plateau levels in parallel cultures with optimal numbers of splenic adherent cells as accessorycells were about eight times higher (i.e., 400 units/ml) than the maximum TCGF production obtained with L-lactate (data not shown). Thus L-lactate substituted only partly for accessorycells in this system. In the same system lactate had no significant effect on the production of interferon. In a typical experiment essentially under the same culture conditions as in Fig. 2, we detected 8 1 units/ml of interferon in cultures without lactate and 46 units/ml in cultures with 3 X lop2 it4 L-lactate. T cells and macrophage-like accessory cells can form intimate contact (5); and T cells are therefore exposed to high concentrations of low-molecular-weight substances which are releasedby these accessorycells. Lactate is releasedby mononuclear phagocytes and certain subsets of macrophages as a consequence of their strong glycolytic activity (20-26). Figure 3 illustrates that 3.6 X lo6 cells in 0.4 ml of Dulbecco’s MEM produce lactate at a linear rate up to a concentration of almost 6 x 1Oe2M L-lactate alter 45 hr. This is compatible with the observation in vivo that in extreme lactacidosis the plasma concentration can exceed 3 X 10e2A4 L-lactate, indicating that lactateproducing cells can releaselactate even against a relatively high extracellular concentration of lactate.
Lactate Functions Also as a Regulatory Signal for Immune Responsesin Vivo and for Unfactionated Lymphocyte Populations in Vitro The production of TCGF is augmented by L-lactate not only in mitogenically stimulated accessory cell-depleted spleen cells, but also in mitogenically stimulated un-
DR6GE ET AL.
I
0
I
I
5xlo-4
I
1x10-3
I
I
2~10-3 5xX)-3 L-bcme (mobalL)
I
1x90-2
I
2xlo-2
I
I
5x10-2 1x10-l
FIG. 2. Augmentation of TCGF production in accessory cell-depleted splenic T-cell populations by graded doses of L-lactate. Accessory cell-depleted splenic T cells from C3H/Tif mice (5 X 106/ml) were incubated with concanavalin A (5 &ml) and with the indicated final concentrations of L-lactate. The culture supematants were harvested after 48 hr and were assayedfor TCGF. Parallel cultures with optimal numbers of splenic adherent cells as accessorycells (plateau level) generated 400 units/ml of TCGF (data not shown).
fractionated murine spleen cells (Fig. 4). L-Lactate was found to mediate a strong dose-dependent augmentation of the TCGF production if added at the start of the culture but mediated only minute effectsif added 20 hr later. The activation of cytotoxic T cells in mixed-leukocyte cultures was found to be augmented by L-lactate if lymph node cells were used as responder and as stimulator cells (Table 2). The cytotoxic response in IG2-containing cultures of lymph node cells or in cultures of spleen cells with or without IL-2 was always a priori relatively high and only moderately augmented by L-lactate. These results suggestthat lactate augments the cytotoxic responsesprimarily by co-inducing the production of interleukin-2 and not by a direct effect on the cytotoxic T-lymphocyte precursor cells. Spleen cells may contain a priori more accessory cells than lymph node cells and may, therefore, be lessdependent on the external application of L-lactate. Finally, L-lactate was also found to augment the in vivo immunization (priming) of DBA/2 mice for a subsequent secondary cytotoxic response in vitro against synge-
REGULATION
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0
10
OF T-CELL FUNCTION
I
I
411
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I
20 30 40 time after initiation of culture (hrs)
I
t
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60
FIG. 3. Releaseof lactate by peritoneal macrophages. C3H/Tif mice received 1 ml of phosphate-buffered saline ip and their peritoneal exudate cells were harvested 1 day later and were incubated at a density of 7.5 x lo6 cells per 0.4 ml in Dulbecco’s MEM for 2 hr. The nonadherent cells were then removed and counted and the remaining adherent macrophages (3.6 X lo6 per well) were again incubated in 0.4 ml of Dulbecco’s MEM. Supematant samples of 0.05 ml were removed at the indicated time points and assayed for lactate as described under Materials and Methods. The remaining culture was reconstituted each time with 0.05 ml of fresh Dulbecco’s MEM.
neic L 5 178 Y ESb-D tumor cells (Fig. 5, top panel). This indicates that L-lactate also determines in vivo the magnitude of TCGF-dependent immune responses.The primary cytotoxic response against the unrelated C57BL/6 stimulator cells in the same cultures (Fig. 5, bottom panel), in contrast, was a priori relatively strong and only slightly augmented after the in vivo treatment with lactate. The injection of L-lactate without ESb-D tumor cells (i.e., without in vivo priming) was not sufficient to mediate a detectable cytotoxic response of the corresponding spleen cells against ESb-D target cells in a subsequent in vitro culture with ESb-D and C57BL/6 stimulator cells (not shown). Biochemical Efects of L-Lactate on Mitogenically Stimulated T Cells In agreement with earlier studies (27-33) we found that our mitogenically stimulated T-cell populations express rapid, strong glycolytic activity and release lactate
412
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0
I
I
1.6 x 1O-4
6 x 10-4
I
4 x 10-a
I
2 x 10-2
moles/L
Fzc. 4. Effect of L-lactate on the production of TCGF by unfractionated spleen cells. C3H/Tif spleen cells (5 X 106/ml) were incubated with concanavalin A (5 &ml) and the indicated final concentrations of L-lactate (Sigma) for 48 hr in 4-ml cultures. The resulting supematants were then assayedfor TCGF activity. Some of the cultures received L-lactate only 20 hr after incubation. 0, L-lactate at the start; W,L-lactate after 20 hr.
into the supematant (Fig. 6, top panel). This indicates that the T-cell population contains more pyruvate and lactate than it can use by its respiratory metabolism. The possibility that L-lactate or pyruvate may serve as an additional source of energy by the respiratory metabolism is therefore unlikely. This conclusion was supported by additional experiments using cultures with 1 X lo-* A4 r4C-labeledL-lactate or labeled pyruvate. These experiments revealed that the labeled L-lactate was not detectably
TABLE 2 The Activation of Cytotoxic T Cells in Cultures of Allogeneic Lymph Node Preparations Is Augmented by L-Lactate % Wr release Cultured materials
No lactate
4 X 10m3Mlactate
2 X lo-* Mlactate
Lymph node cells Lymph node cells + IL-2 Spleen cells Spleen cells + IL-2
3.3 45.3 46.1 65.8
10.4 47.2 56.6 65.9
27.1 61.7 58.4 67.5
Note. C3H lymph node cells (1 X 10’) or spleen cells (3 X 105)were incubated with irradiated C57BL/6 lymph node cells (7 X 104)or spleen cells (1 X 105),respectively, and with or without 0.02 ml of an IG2containing EL-4 cell supematant in 0.2-ml microcultures for 5 days. Cytotoxic activity was then tested in a 4-hr “Cr-release assaywith 2 X lo4 C57BL/6 target cells.
REGULATION
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OF T-CELL FUNCTION BY LACTATE
A:T ratio n 15.1 0 3:1
n-------.
60.
40 II
20
!! L P
0
2 ‘%
60
6 40
20
0
0.‘lO’l a:1 /li/*\/\ . 0-O 0\ . x 0 0 1 I 0
II
I 4 x 10-3
1 2 x 10-Z
I 1 x 10-I
I 5 x 10-l
moles/L FIG. 5. Effect of L-lactate on the in vivo immunization (priming) for a secondary in vitro cytotoxic response against L 5 178 Y E&-D tumor cells. DBA/2 mice were immunized with 1 X lo6 mitomycin-Ctreated ESb-D tumor cells ip. The mice received also 3 X 0.2 ml of BSS with the indicated concentrations of L-lactate ip at the time of immunization and 2 and 4 hr later. Eight days later, the mice were sacrificed and 2 X 10’ spleen cells were incubated for another 5 days in 5 ml of culture medium with 1 X lo6 mitomycin-C-treated ESb-D cells and 5 X lo6 irradiated C7BL/6 spleen cells. The cytotoxic activity was finally assayedagainst ESb-D target cells (top panel) and C57BL/6 targets (bottom panel) at the indicated attacker:target cell ratios.
utilized and pyruvate was mainly converted into L-lactate (data not shown). Taken together, our experiments thus confirm earlier observations that the glycolytic metabolism is the major source of energy for the lymphocytes after mitogenic stimulation (27-33). The external application of L-lactate and pyruvate was found to inhibit the utilization of glucose (Fig. 6). DISCUSSION Macrophage-like cells are known to play an important role as accessorycells and stimulator cells during lymphocyte activation (l-4); but the molecular basis of this accessory function is not entirely clear. The macrophage product interleukin-1 was previously thought to be the most important mediator especially for the induction of TCGF production by helper T cells (6-9). The experiments in this report show, however, that the production of TCGF by mitogenically activated accessorycell-depleted splenic T lymphocytes or by unfmctionated spleen cells is augmented by L-lactate. L-Lactate was capable of partially replacing the accessorycells in cultures of accessory cell-depleted splenic T-cell populations, whereas interleukin- 1 either alone or in combination with L-lactate had practically no effect on the TCGF production by these splenic T cells.
414
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0
2
7 18 time after start of culture (hrs)
26
20-
0'
I 0
"
I
1.2
I
2.5 moles/L
I
I
10
I
20
I
40
I
80
:X 103)
FIG. 6. Inhibition of glucose utilization by L-lactate and pyruvate. Accessory cell-depleted splenic T-cells (3 X 10’ cells/O.3 ml) were incubated with concanavalin A (5 &ml) and 4 ICi of [‘4C]glucose (A). Some of the cultures contained also 2 X lo-* A4 lactate (0) or pyruvate (Cl) (top) or graded concentrations of lactate (0) and pyruvate (0) (bottom). Supematant samples of 9 ~1were removed at the indicated time points (top) or after 26 hr (bottom). The radioactive materials were subsequently analyzed by ascending thin-layer chromatography. The figure illustrates both the remaining concentration of the labeled glucose (solid lines) and the appearance of lactate (broken lines) in the supematant as a percentage of the total radioactivity applied.
The latter point is clearly at variance with previous reports which suggestedthat IL- 1 induces TCGF production by mitogenically activated accessory cell-depleted peripheral T-cell populations (6-9). One of these reports (7) showed, however, that IL- 1 induced a much more profound augmentation of TCGF production and DNA synthesis in thymocyte populations (i.e., 30-fold) than in adherent cell-depleted splenit T-cell preparations (Sfold); and this suggestedthe possibility that IL-l may be primarily a maturation factor for relatively immature T-lineage cells. This interpretation is compatible with our observations that IL-l was very active in the thymocyte proliferation assay(Fig. 1) and was also capable of supporting the TCGF production in EL4-6.1 thymoma cells (Table 1). The issue whether IL- 1 is absolutely required for T-cell activation has recently been critically reviewed by Oppenheim and colleagues, and both positive and negative arguments have been given (9). Irrespective of the apparently variable contribution of IL- 1 to the accessoryfunction, our experiments strongly indicate that lactate is another particularly important
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mediator by which accessorycells augment helper-T-cell functions. Macrophages can generate concentrations of lactate up to 6 X 10e2M in their extracellular space;and lactate concentrations between 1 X 10e3and 8 X 10M2M were found to mediate a substantial dose-dependent augmentation of the TCGF production in accessorycelldepleted T-cell populations. Moreover, macrophage-like accessory cells come into intimate contact with lymphocytes as judged from microscopic inspection (see Fig. 1 in Ref. (5)) and are therefore expected to expose the lymphocytes to relatively high concentrations of L-lactate. (The experiment in Fig. 3 indicates that each macrophage in the culture produces 1.2 X lo-l3 mol/hr and within 45 hr fills a volume of 10m7ml with a concentration of 6 X 1Od2ML-lactate. One can, therefore, expect that an area of 5 pm around the macrophage (i.e., 1 X 10T8ml) is filled with a concentration of 6 X 1O-2M L-lactate within 4.5 hr under conditions of limited diffusion as they may exist in the tissue.) The conclusion that lactate is an important regulatory signal by which macrophage-like accessory cells control the magnitude of helper-T-cell function is sup ported by a series of in vivo and in vitro experiments. These experiments collectively showed that the external application of lactate regulates also the magnitude of T-cell responsesin vivo and also in cultures with unfi-actionated lymphoid cell populations. The effects of lactate on cytotoxic T-cell responses in vitro are compatible with the interpretation that lactate is required only for the induction of TCGF production but not for the activation of the CTL precursor cells. Cytotoxic responsesin cultures with lymph node responder and lymph node stimulator cells were strongly augmented by the external application of either lactate or IL-2, whereas cultures with splenic responder and stimulator cells were only marginally augmented by additional lactate or IL-2. IL-2-containing cultures of either cell type were only marginally augmented by lactate (Table 2). Also lactate had practically no effect on the interferon production by mitogenically stimulated accessorycell-depleted T-cell populations. The mechanism by which lactate augments IL2 production is not known. The most obvious possibility that lactate or pyruvate may serve as an additional source of energy was rendered extremely unlikely. Our experiments showed in agreement with earlier reports (27-3 1) that Con A-stimulated accessorycell-depleted splenic T cells rapidly utilize glucose by glycolytic metabolism and already release substantial amounts of L-lactate during the first 8 hr after stimulation. The cell population thus contains by itself already more lactate and pyruvate than it can utilize by respiratory energy metabolism. Externally administered 14C-labeledlactate was indeed not utilized to any detectable extent and labeled pyruvate was mainly converted into lactate (data not shown). Our experiments showed, on the other hand, that the augmentation of TCGF production by application of external L-lactate or pyruvate is correlated with a strong inhibition of the glycolytic activity (Fig. 6). ACKNOWLEDGMENTS The expert technical assistanceof Mrs. Heike Schmidt and Mrs. Sabine Nick and the assistanceof Mrs. Ingrid Fryson in the preparation of this manuscript are gratefUlly acknowledged.
REFERENCES 1. Schwab, R., Crow, M. K., Russo, C., and Weksler, M. E. J. Immunol.135,1714,1985. 2. Rosenstreich, D. L., Farrar, J. J., and Dougherty, S. J. Immunof.116, 13 1, 1976.
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ET AL.
3. Devries, J. E., Caviles, A. P., Bont, W. S., and Mendelsohn, J., J. Immunol. 122, 1099, 1979. 4. Thiele, D. L., and Lipsky, P. E., J. Immunol. 129, 1033, 1982. 5. Rosenthal, A. S., Immunol. Rev. 40,136,1978. 6. Larsson, E.-L., Iscove, N. N., and Coutinho, A., Nature (London) 283,664, 1980. 7. Smith, K. A., Lachman, L. B., Oppenheim, J. J., and Favata, M. F., J. Exp. Med. 151,1551,1980. 8. Palacios, R., J. Immunol. 128,337, 1982. 9. Oppenheim, J. J., Kovacs, E. J., Matsushima, K., and Durum, S. K., Immunol. Today 7,45, 1986. 10. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3,645, 1973. 11. Steinmann, R. M., Gutchinov, G., Witmer, M. D., and Nussenzweig, M. C., J. Exp. Med. 157,6 13, 1983.
12. MacDonald, H. R., Lees, R. K., and Bron, C., J. Immunol. 135,3944,1985. 13. Falk, W., Mannel, D. N., and Droge, W., J. Immunol. 130,22 14, 1983. 14. Farrar, J. J., Mizel, S. B., Fuller-Farrar, J., Farrar, W. L., and Hilfiker, M. L., J. Zmmunol. 125,793, 1980.
15. Mosmann, T. R., Bond, M. W., Coffman, R. L., Ohara, J., and Paul, W. E., Proc. Natl. Acad. Sci. USA83,5654, 1986. 16. Femandez-Botran, R., Krammer, P. H., Diamantstein, T., Uhr, J. W., and Vitetta, E. S., J. Exp. Med. 164,580, 1986.
17. In “The Interferon System” (W. E. Stewart II, Ed.), p. 15. Springer-Verlag, New York, 1979. 18. Galli, P., and Driige, W., Eur. J. Immunol. 10,87, 1980. 19. DrBge, W., Eck, H.-P., Kriegbaum, H., and Mihm, S., J. Zmmunol. 137, 1383, 1986. 20. Roos, D., and Balm, A. J. M., In “The Reticuloendothelial System,” Vol. 2, “Biochemistry and metabolism” (A. J. Sbarra and R. R. Strauss,Eds.), p. 189. Plenum, New York/London, 1980. 21. Axline, S. G., Semin. Hematol. 7, 142, 1970. 22. Oren, R., Famham, A. E., Saito, K., Milofsky, E., and Kamovsky, M. L., J. Cell. Biol. 17,487, 1963. 23. Simon, L. M., Robin, E. D., Phillips, J. R., Acevedo, J., Axline, S. G., and Theodore, J., J. Clin. Invest. 59,443, 1977. 24. Para, M., Sagone,A., Balcerzak, S., and LoBuglio, A., Clin. Rex 20,742A, 1972.
25. Roos, D., Reiss, M., Balm, A. J. M., Palache, A. M. Cambier, P. H., and Van der Stijl-Neijenhuis, J. S. In “macrophages and Lymphocytes: Nature, Functions, and Interaction” (M. R. Escobar and H. Friedman, Eds.), pp. 29-36. Plenum, New York, 1979. 26. Hard, G. C., Brit. J. Exp. Puthol. 51,97, 1970. 27. Hedeskov C. J., and Esmann, V., Blood28,163,1966. 28. Roos, D., and Loos, J. A. Biochim. Biophys. Acta 222,565, 1970. 29. Roos, D., De Boer, J. E. G., Huismans, L., and Boom, A. J., Exp. Cell Res. 75, 185, 1972. 30. Roos, D., and Loos, J. A., Exp. Cell Rex 77, 121, 1973. 31. Roos, D., and Loos, J. A., Exp. Cell Res. 77,127, 1973. 32. Wang, T., Foker, J. E., and Tsai, M. Y., Biochem. Biophys. Res. Commun. 95, 13, 1980. 33. Wang, T., Marquardt, C., and Foker, J., Nature (London) 261,702, 1976.