Lactic Acid and Acidification Inhibit TNF
Secretion and Glycolysis of Human
Monocytes
This information is current as
of July 31, 2017.
Katrin Dietl, Kathrin Renner, Katja Dettmer, Birgit
Timischl, Karin Eberhart, Christoph Dorn, Claus
Hellerbrand, Michael Kastenberger, Leoni A.
Kunz-Schughart, Peter J. Oefner, Reinhard Andreesen, Eva
Gottfried and Marina P. Kreutz
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Material
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J Immunol 2010; 184:1200-1209; Prepublished online 21
December 2009;
doi: 10.4049/jimmunol.0902584
http://www.jimmunol.org/content/184/3/1200
The Journal of Immunology
Lactic Acid and Acidification Inhibit TNF Secretion and
Glycolysis of Human Monocytes
Katrin Dietl,*,1 Kathrin Renner,†,1 Katja Dettmer,† Birgit Timischl,† Karin Eberhart,†
Christoph Dorn,‡ Claus Hellerbrand,‡ Michael Kastenberger,* Leoni A. Kunz-Schughart,x
Peter J. Oefner,† Reinhard Andreesen,* Eva Gottfried,*,1 and Marina P. Kreutz*,1
M
onocytes are essential cellular components of the innate
immune system. They originate from the bone marrow
and circulate in the blood for several days before they
extravasate into (inflammatory) tissues or tumor tissues.
Recruitment is thought to be regulated by chemotactic factors
like MCP-1/CCL-2 or M-CSF (1, 2). Besides phagocytosis and Ag
presentation, a major function of monocytes is the secretion of
anti- and proinflammatory cytokines (e.g., IL-1, IL-6, and TNF).
The secretion of cytokines is induced after monocyte activation
with LPS, lipopeptide, flagellin, or tumor cell membranes (3, 4).
*Department of Hematology and Oncology, †Institute of Functional Genomics, and
‡
Department of Internal Medicine I, University of Regensburg, Regensburg; and x
OncoRay–Center for Radiation Research in Oncology, Dresden University of Technology, Dresden, Germany
1
K.D., K.R., E.G., and M.P.K. contributed equally to this work.
Received for publication August 7, 2009. Accepted for publication November 24,
2009.
M.P.K. was supported by the Deutsche Forschungsgemeinschaft (Krm-1418/6-4); P.O.
was supported by BayGene; M.P.K., P.J.O., and C.H. were supported by the Regensburger
Forschungsförderung in der Medizin (ReForM-C); and L.A.K.-S. was supported by the
German Federal Ministry of Education and Research (01ZZ0502). K.E. was funded by the
Austrian Federal Ministry for Education, Science and Culture (GENAU-CH.I.L.D.).
Address correspondence and reprint requests to Dr. Marina Kreutz, Department of
Hematology and Oncology, Regensburg University School of Medicine, Regensburg
93042, Germany. E-mail address: [email protected]
The online version of this paper contains supplemental material.
Abbreviations used in this paper: 7-AAD, 7-aminoactinomycin D; 2-DG, 2-deoxyglucose; GC-MS, gas chromatography-mass spectrometry; HCl, hydrochloric acid; HIF1a, Hypoxia-inducible factor-1a; LA, lactic acid; LC-MS/MS, liquid chromatographytandem mass spectrometry; MCT, monocarboxylate transporter; MCTS, multicellular
tumor spheroid; NaL, sodium L-lactate; PFK-1, 6-phosphofructo-1-kinase; ROS, reactive oxygen species.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902584
Monocyte activation requires energy, is glucose dependent, and
involves stimulation of glycolysis (5). Accordingly, glucose deprivation decreases the expression of TNF, GM-CSF, and the b
form of pro-IL-1 (6, 7). Hypoxia-inducible factor-1a (HIF-1a)
appears to be essential for the regulation of glycolysis in myeloid
cells and controls the inflammatory response by switching the cell
metabolism to glycolysis (8).
The function of tumor-infiltrating monocytes seems to be depressed (9), and intratumoral monocytes show an altered cytokine
profile (e.g., suppression of TNF and an increase in IL-10) (10).
Several tumor-derived factors have been implicated as potential
modulators of monocyte activation including PGE2, gangliosides,
and lactic acid (LA) (11–14).
Lactate accumulates in the tumor microenvironment due to the
accelerated glycolysis of tumor cells, a phenomenon known as
aerobic glycolysis, or Warburg effect (15). In addition, high lactate
levels are found under inflammatory conditions (16). Glycolytic
enzymes such as lactate dehydrogenase and glucose transporters
are upregulated by hypoxia/HIF-1a or oncogenic transformation
(17, 18). As a consequence, tumor cells produce lactate and secrete lactate together with protons, which in turn lowers the pH of
the tumor environment (19). It has been reported that extracellular
lactate in wounds stimulates macrophages to secrete vascular
endothelial growth factor and TGF-b, both known to be immunosuppressive factors (16, 20). In addition, data from Shime et al.
(14) demonstrated that LA upregulates transcription and secretion
of IL-23, a tumor-promoting cytokine, in human monocytes. Samuvel et al. (21) have shown recently that preincubation of myeloid U937 and monocyte-derived macrophages with lactate
increases the expression of inflammatory cytokines (e.g., IL-6 and
IL-8). However, Douvdevani et al. (22) have described that a low
pH and high concentration of lactate in peritoneal dialysis fluids
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High concentrations of lactic acid (LA) are found under various pathophysiological conditions and are accompanied by an
acidification of the environment. To study the impact of LA on TNF secretion, human LPS-stimulated monocytes were cultured
with or without LA or the corresponding pH control. TNF secretion was significantly suppressed by low concentrations of LA (£10
mM), whereas only strong acidification had a similar effect. This result was confirmed in a coculture model of human monocytes
with multicellular tumor spheroids. Blocking synthesis of tumor-derived lactate by oxamic acid, an inhibitor of lactate dehydrogenase, reversed the suppression of TNF secretion in this coculture model. We then investigated possible mechanisms underlying
the suppression. Uptake of [3-13C]lactate by monocytes was shown by hyphenated mass spectrometry. As lactate might interfere
with glycolysis, the glycolytic flux of monocytes was determined. We added [1,2-13C2]glucose to the culture medium and measured
glucose uptake and conversion into [2,3-13C2]lactate. Activation of monocytes increased the glycolytic flux and the secretion of
lactate, whereas oxygen consumption was decreased. Addition of unlabeled LA resulted in a highly significant decrease in [2,3-13
C2]lactate secretion, whereas a mere corresponding decrease in pH exerted a less pronounced effect. Both treatments increased
intracellular [2,3-13C2]lactate levels. Blocking of glycolysis by 2-deoxyglucose strongly inhibited TNF secretion, whereas suppression of oxidative phosphorylation by rotenone had little effect. These results support the hypothesis that TNF secretion by human
monocytes depends on glycolysis and suggest that LA and acidification may be involved in the suppression of TNF secretion in the
tumor environment. The Journal of Immunology, 2010, 184: 1200–1209.
The Journal of Immunology
inhibit macrophage/monocyte TNF and b form of pro-IL-1 release. In addition, we and others have shown that tumor-derived
LA strongly inhibits both the differentiation of monocytes to
dendritic cells (23, 24) and the activation of T cells (25). These
data demonstrate that lactate can have opposing effects on activation in different types of immune cells.
In this study, we examined the effects of tumor-derived LA and
concomitant acidification on human monocytes and investigated the
mechanism underlying the suppression of monocyte activation. We
found that monocytes take up extracellular lactate, which results in
a decreased glycolytic flux and an inhibition of TNF release. Our data
suggest that impairment of glycolysis may contribute to the immunosuppressive effects of LA in wounds and tumors.
Materials and Methods
Chemicals
Isolation and culture of monocytes
Monocytes were isolated by leukapheresis from healthy donors, followed by
density gradient centrifugation over Ficoll/Hypaque and separation by
countercurrent centrifugation (J6M-E centrifuge; Beckmann, Munich,
Germany) as described previously (26). Monocyte purity was $85% as
determined by CD14 expression. Isolated monocytes were cultured for 4–
20 h in RPMI 1640 supplemented with 2% human AB serum (PAN Biotech, Aidenbach, Germany), L-glutamine (2 mmol/L), 50 U/ml penicillin,
and 50 mg/ml streptomycin (all from Life Technologies, Karlsruhe, Germany). To minimize effects from donor variation, all experiments were
performed with monocytes from at least three different healthy donors.
Activation of monocytes
Monocytes were incubated at a density of 0.5 3 106 cells/ml in the presence
of 2, 5, 10, and 20 mM L-LA or sodium L-lactate (NaL). LPS (kindly
provided by Prof. M. Freudenberg, Max Planck Institute of Immunobiology, Freiburg, Germany) was added at a final concentration of 100 ng/ml.
Control samples were cultured with LPS with or without 1% hydrochloric
acid (HCl) added to titrate the pH of the medium to ∼7.1 and ∼6.6, corresponding to the pH of media containing 10 and 20 mM LA, respectively.
2-Deoxyglucose (2-DG), a competitive inhibitor of hexokinase, was added
to monocyte cultures at concentrations of 1 and 10 mM. Rotenone, an inhibitor
of respiratory chain complex I, was added at concentrations of 0.1 and 1 mM.
Determination of cytokines
For the determination of extracellular cytokine concentrations, cell culture
supernatants were harvested after 18–20 h of incubation, filtered, and stored
at 220˚C prior to analysis of cytokines (TNF, IL-6) by means of commercially available ELISAs (R&D Systems, Minneapolis, MN).
Reverse transcription-quantitative real-time PCR
Total RNA was isolated from monocytes using RNeasy Spin Columns from
Qiagen (Hilden, Germany). Reverse transcription was performed with 500
ng RNA in a total volume of 20 ml using an M-MLV Reverse Transcriptase
from Promega (Mannheim, Germany). To quantify TNF expression, the
Mastercyler Ep Realplex (Eppendorf, Hamburg, Germany) was used. For
reverse transcription-quantitative real-time PCR, 1 ml cDNA, 1 ml of TNF
QuantiTect Primer Assay (Qiagen) or 0.5 ml of 18S primers (10 mM), and
5 ml QuantiFast SYBR Green PCR Kit (Qiagen) in a total of 10 ml were
applied. The sequences of the 18S primers were as follows: 18S forward
59-ACCGATTGGATGGTTTAGTGAG-39 and 18S reverse 59-CCTACGGAAACCTTGTTACGAC-39.
Analysis of cell viability by Annexin-V-FITC/7aminoactinomycin D staining
For analysis of cell viability, monocytes were seeded at a concentration of
0.5 3 106 cells/ml in hydrophobic Teflon bags with or without 100 ng/ml
LPS and 20 mM LA. After 18–20 h, cells were harvested, washed with
PBS, counted, and stained with Annexin-V-FITC and 7-aminoactinomycin
D (7-AAD) (both from BD Biosciences, San Jose, CA) according to the
manufacturer’s instructions. Flow cytometric analyses were performed on
a FACSCalibur (BD Biosciences) using BD CellQuestPro for data acquisition and analysis.
Sample preparation for intra- and extracellular metabolite
determination by mass spectrometry
A total of 1 3 107 monocytes were incubated in 6 ml medium in six-well
plates for 18 h with or without LPS (100 ng/ml) in the presence of unlabeled 20 mM NaL or 5–20 mM LA. For pH control, culture medium was
adjusted to pH 6.6 or 7.1 with HCl. For determination of glycolytic flux,
a glucose-free medium supplemented with 2 mg/ml [1,2-13C2]glucose was
used. For determination of lactate uptake, [3-13C]lactate was used. In the
lactate uptake experiments, the pH was adjusted to ∼7.1 with HCl. After
incubation cells were scraped off the plate, counted (CASY, Schärfe
System, Reutlingen, Germany), washed three times with PBS, again
counted, then flash frozen and stored at 280˚C. Cell pellets were homogenized by three freeze and thaw cycles and ultrasonification. To calculate total protein amount in the sample, the volume of the cell pellet was
determined. A subsample for protein determination (Coomassie, Pierce,
Bonn, Germany) was removed and a protease inhibitor mixture was added.
To correct for loss of metabolites during extraction and evaporation,
stable isotope-labeled [U-13C]pyruvate was added to the cell pellet before
extraction of intracellular metabolites with methanol. The initial extraction
was performed with 80% (v/v) aqueous methanol (Merck, Darmstadt,
Germany), the second extraction with pure methanol. Extracts were
combined, dried by evaporation (Hettich Combi-Dancer, Zinsser Analytic,
Frankfurt, Germany), and reconstituted as described below.
For the analysis of excreted metabolites, cells were pelleted by centrifugation, and 2 ml supernatant was removed and stored at 280˚C. Prior to
analysis by the different hyphenated mass spectrometric methods (i.e., gas
chromatography-mass spectrometry [GC-MS] and liquid chromatographytandem mass spectrometry [LC-MS/MS]), proteins in the supernatants
were removed by filtration through 5 kDa cutoff filter tubes (Vivaspin 4,
Sartorius, Goettingen, Germany), and internal standards were added as
described above.
Analysis of glucose, lactate, and amino acid levels in
supernatants by gas chromatography-MS
Analysis of amino acids was performed as previously described (27). An
aliquot of 20 ml cell culture medium was analyzed.
To calculate glucose uptake, glucose concentrations in the cell culture
medium were analyzed by GC-MS. In addition, lactate levels in the
supernatants were determined.
For GC-MS, we used an Agilent model 6890 GC (Agilent, Palo Alto, CA)
equipped with a Mass Selective Detector model 5975 Inert XL and an Auto
Liquid Injector model 7683B. Separation was carried out on an RXI-5MS
column, 30 m 3 0.25 mm inner diameter 3 0.25 mm film thickness
(Restek, Bad Homburg, Germany). Sample injection was performed in
splitless mode at 280˚C using an injection volume of 1 ml. The initial oven
temperature was set at 50˚C, ramped at 8˚C/min to 300˚C, and held for
10 min. Helium was used as carrier gas at a flow-rate of 0.6 ml/min. The
transfer line to the mass spectrometer was kept at 310˚C. The mass
spectrometer was operated in full scan mode from 50–600 m/z with a scan
time of 0.5 s. A 10-ml aliquot of the cell culture medium was spiked with
10 ml of an internal standard solution containing [U-13C]glucose and [U-13
C]lactate (1 mM each). The samples were dried using a vacuum evaporator
and derivatized prior to injection. For derivatization, 50 ml of 10 mg/
ml methoxylamine hydrochloride in pyridine were added and incubated at
60˚C for 60 min, followed by 50 ml of N-methyl-N-(trimethylsilyl)trifluoroacetamide for 60 min at 60˚C. Quantification was performed using
calibration curves corrected with the internal standard. The fragment ion
319 m/z was employed for glucose area calculation, which provides total
glucose in solution, and the fragment ion 219 m/z was used to quantify
unlabeled lactate in the solution.
Hyphenated MS analysis of lactate isotopes for flux analysis
Intra- and extracellular lactate levels were measured by LC-MS/MS. For
determination of relative glucose flux through glycolysis and the pentosephosphate pathway, the conversion of [1,2-13C2]glucose to [2,3-13C2]lactate, derived from glycolysis, and to [3-13C]lactate or [1,3-13C2]lactate,
derived from the pentose-phosphate pathway, was analyzed by ion-pair
LC-MS/MS using sodium-DL-[2,3,3,3-2H4]lactate as internal standard.
The described method (28) was adapted as follows: LC was performed on
an Agilent 1200 SL HPLC system (Agilent) employing a Synergi Hydro-
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Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich
(Deisenhofen, Germany). Methanol (liquid chromatography-mass spectrometry) was from Fisher Scientific (Ulm, Germany). Sodium-DL-[2,3,3,3-2
H4]lactate, [U-13C]pyruvate, [2,3,3,4,4-2H5]glutamic acid, [1,2-13C2]
glucose, and sodium-[3-13C]lactate were from Euriso-top (Saint-Aubin
Cedex, France).
1201
1202
LACTIC ACID INHIBITS TNF SECRETION
RP column 150 mm 3 2.0 mm inner diameter, 4 mm particles, 80 Å pore
size. Eluent A was an aqueous solution of 10 mM tributylamine and 15
mM acetic acid (pH 4.95), whereas eluent B was pure methanol. The flow
rate was set to 350 ml/min; the column was kept at 50˚C. The optimized
gradient conditions were as follows: 0–7 min from 0–90% B and hold for 3
min at 90% B. Prior to each run, the column was equilibrated at 100% A
for 6 min. The HPLC system was coupled to a 4000 Q TRAP triplequadrupole ion-trap mass spectrometer (Applied Biosystems/MDS SCIEX,
Concord, Ontario, Canada) operated in negative ion mode with selected
reaction monitoring. The following selected reaction monitoring transitions were monitored to determine the [13C]label distribution in lactate:
89 . 43, unlabeled lactate; 90 . 43, [1-13C1]lactate; 90 . 44, [2-13C1]
lactate and [3-13C1]lactate; 91 . 44, [1,2-13C2]lactate and [1,3-13C2]lactate; 91 . 45, [2,3-13C2]lactate; and 92 . 45, [1,2,3-13C3]lactate.
Online measurement of oxygen concentration in cell culture
Enzymatic determination of lactate
Results
Lactate levels in cell culture supernatants were measured by means of an
ADVIA1650 (Bayer, Tarrytown, NY) analyzer using reagents from Roche
(Mannheim, Germany). These measurements were performed at the Department of Clinical Chemistry at the University of Regensburg, Regensburg, Germany.
TNF production of monocytes is inhibited by LA
After incubation, intracellular ATP levels were determined in viable cells
immediately after scraping them off the plate and counting by a luciferase
based assay (CellTiter-Glo Luminescent Cell Viability Assay, Promega)
according to the manufacturer’s protocol. Standard dilutions of ATP were
prepared freshly each time to avoid instability.
Determination of reactive oxygen species
Reactive oxygen species (ROS) were determined with a chemiluminescence
assay using a luminometer (Luminometer Sirius, Berthold Detection
Systems, Pforzheim, Germany). A total of 1 3 106 monocytes/100 ml were
incubated in RPMI 1640 without phenol red (PAN Biotech) supplemented
with 2% human AB serum in the presence of 0.1 mg/ml lucigenin. To
stimulate the production of ROS, 0.1 mM PMA was added in the absence
or presence of 20 mM LA. As a control, cells were incubated with medium
titrated with HCl to pH 6.6 (corresponding to the pH of the medium
supplemented with 20 mM LA). The amount of ROS was determined at 3,
30, and 60 min after incubation with the respective substances.
Culture of the melanoma cell line MelIm and generation of
multicellular spheroids and spheroid cocultures
The melanoma cell line MelIm was grown in RPMI 1640 medium (Life
Technologies) supplemented with 10% FCS (BioWhittaker, Walkersville,
MD), L-glutamine (2 mmol/L), 50 U/ml penicillin, and 50 mg/ml streptomycin under standard tissue-culture conditions and regularly tested for
mycoplasma contamination (Mycoplasma detection kit; Biochrom, Berlin,
Germany). Multicellular tumor spheroids (MCTSs) were generated using
the liquid overlay culture technique (29). In brief, 5 3 103 suspended
MelIm melanoma cells from exponentially growing tumor cell monolayers
were cultured on 1% solid agarose in 96-well plates. To investigate the
impact of tumor cell lactate production on monocytes in the coculture,
MCTSs were generated in the presence or absence of 30 or 90 mM oxamic
acid, an inhibitor of lactate dehydrogenase. After 3 d of cultivation, cells
had formed tight aggregates. On day 5, half of the medium was replaced by
a monocyte suspension containing 4 3 104 monocytes per well (final
concentration 2% AB and 5% FCS) and LPS (100 ng/ml). As controls,
monocytes were incubated with 100 ng/ml LPS without tumor cells. After
18–20 h, supernatants were harvested, filtered, and stored at 220˚C.
Monitoring of tumor cell cytostasis
For determination of the cytostatic activity of monocytes, 30,000 MelIm
melanoma cells/well and 100,000 monocytes/well were coincubated in 96well microtiter plates in the presence of 2, 5, and 10 mM LA, respectively,
as well as in medium titrated with HCl to pH ∼7.1 (corresponding to the pH
of the medium supplemented with 10 mM LA) in a final volume of 200
ml/well. Monocytes were stimulated with 100 ng/ml LPS. After 30 min,
cells were pulsed with 0.0185 MBq [methyl-3H]thymidine/well (Amersham Pharmacia, Piscataway, NJ) and harvested after 18 h onto UniFilter
plates using a Wallac harvester (PerkinElmer, Gaithersburg, MD). Incorporated [3H]thymidine was determined by means of a Wallac Betaplate
counter (PerkinElmer). Monocytes alone showed only background levels
of [3H]thymidine incorporation (677 6 355 cpm).
To investigate the influence of LA on TNF production by human
monocytes, we stimulated monocytes with LPS for 18–20 h in the
absence or presence of LA. A significant effect of LA on TNF
secretion was detected at a concentration as low as 5 mM (Student
t test, p , 0.05; Fig. 1A). At the transcriptional level, quantitative
RT-PCR revealed that 10 mM LA significantly (Student t test, p =
0.01) decreased the amount of TNF mRNA after 4 h of incubation
compared with the LPS control (Fig. 1B). In contrast, 20 mM LA
was needed to strongly suppress IL-6 compared with the LPS
control (Fig. 1C; Student t test, p , 0.005).
To demonstrate the effect of LA on monocytes in a more in vivolike model, we used MCTSs generated from the melanoma cell line
MelIm that secretes lactate (24). We blocked lactate production in
tumor cells with oxamic acid, an inhibitor of lactate dehydrogenase. After 5 d, half of the medium was replaced by
a monocyte suspension. In MCTS cocultures, monocytes secreted
∼65% of the TNF as compared with cells cultured without tumor
cells (Fig. 1D). Oxamic acid led to a decrease in the lactate
content of the coculture from ∼3 mM to ∼0.5 mM and reversed
the suppressive effect of the MCTS on TNF secretion (i.e., the
TNF concentration in the coculture supernatant was comparable to
the control culture without tumor cell contact). These results prove
that tumor-derived LA is a significant inhibitor of monocyte TNF
secretion. In contrast, IL-6 secretion was not altered in the MCTS
coculture (data not shown).
To clarify whether LA also alters other monocyte functions, we
analyzed the impact of LA on the ability of monocytes to inhibit
proliferation of MelIm melanoma cells and their capacity to
produce ROS. The cytostatic effect of monocytes after LPS activation was weakly affected by LA (Supplemental Fig. 1), whereas
LA and acidification strongly suppressed ROS production (Supplemental Fig. 2).
LA and acidification have no effect on cell viability
To demonstrate that the observed inhibitory effect of LA on monocytes did not result from cell death, monocytes were cultured for 18–
20 h with or without 20 mM LA in the absence or presence of LPS.
After washing and staining with Annexin-V-FITC and 7-AAD, flow
cytometric analysis was performed. No differences in the number of
viable cells were detected after incubation of monocytes with LA
compared with monocytes cultured without LA. These results
confirm that the reduced levels of TNF and IL-6 in the culture supernatants were not a result of increased cell death (Fig. 2).
The administration of 10 mM and 20 mM LA decreased the pH
of the cell culture medium from 7.5 to ∼7.1 and 6.6, respectively.
To study the effect of acidification on monocyte survival, we decreased the pH of the culture medium by the addition of HCl. Cell
survival of neither stimulated nor unstimulated monocytes was
altered by acidification (Fig. 2).
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Determination of intracellular ATP levels
The SDR SensorDish Reader (PreSens Precision Sensing, Regensburg,
Germany) is a 24-channel oxygen and pH meter. The optical oxygen
(OxoDish) sensor is integrated at the bottom of each well of a 24-well
multidish. The sensors are luminescent dyes embedded in an analytesensitive polymer. The luminescence lifetime of these dyes depends on the
amount of analyte. The sensors are read out noninvasively through the
bottom of the multidish by the SensorDish Reader. The resulting signal is
converted automatically to the respective parameter (dissolved oxygen)
using calibration parameters stored in the software. A total of 5 3 106
monocytes were incubated in 1 ml medium. The SensorDish Reader was
used in the incubator for the whole duration of the 16-h cultivation period,
and measurements were performed in 30 s intervals.
The Journal of Immunology
1203
The effect of LA is partially pH dependent
To investigate whether acidification of the medium, lactate, or
a combination of both is responsible for the observed reduction in
TNF secretion, we studied all conditions in parallel. Acidification
to pH 7.1 had only a minimal effect on the TNF secretion (Fig. 3A),
whereas pH 6.6 decreased TNF levels significantly (Fig. 3B;
Student t test, p = 0.01). The sodium salt of LA, NaL, did not
change the pH of the culture medium, but it decreased the release
of TNF in three out of four experiments. However, this effect was
not statistically significant (Fig. 3A, 3B). When we combined both
treatments and added NaL under low pH conditions, TNF secretion was inhibited. Titration of the culture medium back to pH 7.6
reversed the effect. These findings demonstrate that LA and, to
a lesser extent, NaL inhibit the secretion of the proinflammatory
cytokine TNF by monocytes. At low LA concentrations, both
lactate and mild acidification are necessary for the inhibition of
TNF secretion; at higher LA concentrations, the acidification
seems to be the dominant factor.
Uptake of lactate by monocytes
In an effort to elucidate the molecular mechanisms underlying the
inhibitory effect of LA on monocytes, we investigated the uptake of
lactate by means of LC-MS/MS. Cells were incubated for 18 h with
sodium-[3-13C]lactate in the absence or presence of mere acidification by HCl and LPS. Control cells were cultured without
sodium-[3-13C]lactate. Quantification of intracellular [3-13C]lactate
levels revealed that lactate was taken up by monocytes constitutively
without stimulation (Fig. 4). Mild acidification (pH 7.1) of the
culture medium as well as LPS stimulation alone had only a slight
effect on the uptake. However, the combined administration of LPS
and acidification resulted in a significantly (Student t test, p = 0.034)
increased intracellular level of [3-13C]lactate.
These results demonstrate the uptake of extracellular lactate by
monocytes. Uptake was further increased in the presence of protons. This suggests that lactate was at least partly transported into
monocytes by monocarboxylate transporters (MCTs), which cotransport protons and lactate anions through the plasma membrane
depending on the concentration gradient (30, 31). It is known that
MCTs are expressed in human monocytes, and we confirmed the
expression of MCT-1 and MCT-4 by RT-PCR. The expression was
unaffected by extracellular LA (data not shown).
Monocyte activation alters energy metabolism
It is known that the activation of immune cells such as macrophages
or dendritic cells depends on glycolysis (32, 33). Therefore, we
hypothesized that monocyte activation may also depend on both
glucose uptake and glycolysis. We analyzed glucose metabolism
with or without LPS stimulation. Fig. 5A shows that glucose uptake was not significantly increased after LPS activation; however,
extracellular lactate levels were significantly elevated (Fig. 5A; p =
0.0001). It appeared that endogenous LA could also modulate
TNF secretion. To that end, we determined the secretion of TNF
and lactate after LPS stimulation in a time-course experiment.
TNF secretion was detected early after LPS stimulation with
a maximum at ∼4 h. Lactate showed slower kinetics. After 17 h,
lactate levels had strongly increased, and TNF production had
decreased (Supplemental Fig. 3). These data suggest that endogenous lactate production might indeed be involved in a negative
feedback regulation.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
FIGURE 1. LA inhibits TNF and IL-6 production by human monocytes. Freshly isolated human monocytes were stimulated with 100 ng/ml LPS for 18–
20 h in the absence or presence of 2–10 mM LA. TNF in the supernatants was determined by ELISA. Data represent means 6 SEM (n $ 5). Five and 10
mM LA caused a statistically significant inhibition of the TNF secretion (A; Student t test, pp , 0.05; ppp , 0.005 compared with the LPS control). The
relative amount of TNF mRNA was determined after 4 h. Only 10 mM LA caused a statistically significant inhibition of the TNF mRNA expression after 4
h (B; Student t test, pp = 0.01; control, LPS, LPS + 10 mM LA, n = 4, mean 6 SEM; LPS + 5 mM LA, n = 2). In addition, IL-6 was measured by ELISA in
the monocyte supernatants. A total of 20 mM LA was necessary to significantly inhibit IL-6 secretion compared with the LPS control. Data represent means 6
SEM (n = 7) (C; Student t test, ppp , 0.005). MCTS of the melanoma cell line MelIm were generated in the presence or absence of 30 and 90 mM oxamic acid.
On day 5, half of the medium was replaced by a monocyte suspension, and the coculture was stimulated with 100 ng/ml LPS. Control monocytes were cultured
without tumor cells in the presence of LPS. TNF levels were significantly lower in the coculture compared with the monocyte control culture (D; Student t test,
ppp , 0.005). Addition of oxamic acid increased TNF levels back to the control level. Lactate levels were analyzed with a clinical routine procedure. Data
represent means 6 SEM (n $ 5).
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LACTIC ACID INHIBITS TNF SECRETION
FIGURE 2. LA does not affect the viability of monocytes. Monocytes
were incubated with 100 ng/ml LPS in the presence or absence of 20 mM
LA or medium titrated with HCl to pH 6.6. Cells were stained with AnnexinV-FITC and 7-AAD and analyzed by flow cytometry. One representative
experiment out of three is shown.
High extracellular LA levels inhibit glycolysis in monocytes
A
B
FIGURE 3. Role of acidification in the suppression of TNF secretion by
LA. Freshly isolated human monocytes were stimulated with 100 ng/ml
LPS for 18–20 h in the absence or presence of 10 mM (A) and 20 mM (B)
LA or NaL, respectively. In addition, the pH of the cell culture medium
was lowered to the respective pH (pH 7.1 or 6.6). Furthermore, LA was
titrated back to pH 7.6 by the addition of NaOH (LA pH 7.6). TNF in the
supernatants was determined by ELISA. For 10 mM LA, 10 mM NaL pH
7.1, 20 mM LA pH 6.6, and 20 mM NaL pH 6.6 values are statistically
significant compared with the LPS control (Student t test, pp , 0.05; ppp ,
0.005; pppp , 0.0005). Data are means 6 SEM (n = 4).
FIGURE 4. Uptake of [3-13C]lactate by monocytes. Monocytes were incubated for 18 h with or without 20 mM sodium-[3-13C]lactate Na-[3-13C]L in
the presence or absence of LPS with or without the addition of HCl (pH ∼7.1 and
pH ∼7.5, respectively). The intracellular level of [3-13C]lactate resulting from
uptake of exogenous lactate was determined in the cell lysates by mass spectrometry. Data are means 6 SEM (n = 4). Statistical analysis was performed by
Student t test (pp , 0.05).
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To clarify whether addition of extracellular LA interferes with
monocyte energy metabolism, we investigated the glucose uptake
by activated monocytes. Monocytes were incubated with LPS for
18 h in the absence or presence of LA or NaL. Extracellular LA
reduced the glucose uptake of stimulated monocytes in a dosedependent manner (Fig. 5B), whereas lowering the medium pH to
6.6 was less pronounced.
To assess whether reduced glucose uptake enhanced the uptake
of alternative nutrients, we determined the amount of glutamine in
the culture medium but found no change in the level of glutamine or
its metabolite glutamate after monocyte stimulation or incubation
with LA (Fig. 5C).
Additionally, we analyzed whether the glycolytic flux in monocytes was influenced by LA. To address this issue, we incubated
monocytes with [1,2-13C2]glucose. We determined by LC-MS/MS
the conversion of [1,2-13C2]glucose into [2,3-13C2]lactate, which
originates from glycolysis, and into [1,3-13C2]lactate and [3-13C]
lactate, both of which originate from the pentose-phosphate pathway.
Because the levels of lactate produced by the pentose phosphate
pathway were ∼2–5% of the level originating from glycolysis,
only the [2,3-13C2]lactate is shown in Fig. 6A and 6B. The addition
of extracellular LA dose-dependently resulted in lower extracellular
levels of [2,3-13C2]lactate, whereas the level of intracellular [2,3-13
C2]lactate increased (Fig. 6A, 6B). Furthermore, the addition of 5–
20 mM LA increased the amount of unlabeled intracellular [12C]
lactate, suggesting an influx of LA into monocytes. Lowering the
pH of the culture medium to 6.6 also resulted in a decrease in extracellular levels of [2,3-13C2]lactate and increased intracellular
[2,3-13C2]lactate levels, albeit to a lesser extent compared with
20 mM LA. These results can be explained by the findings of Poole
and Halestrap (30), showing that lactate transport depends on
MCTs. These transporters cotransport protons and lactate anions
through the plasma membrane depending on the concentration
gradient (31). Accordingly, high levels of extracellular LA invert the
lactate gradient between extracellular milieu and cytoplasm and in
turn lead to an influx of lactate.
Theadditionof10mM2-DG,acompetitiveinhibitorofhexokinase,
the enzyme catalyzing the first and rate limiting step of glycolysis, also
led to a decrease in extracellular levels of [2,3-13C2]lactate, whereas
intracellular lactate levels remained unchanged (Fig. 6A, 6B).
Apart from energy production via glycolysis, ATP is produced
through oxidative phosphorylation. Therefore, we analyzed oxygen
consumption of monocytes with or without stimulation. LPS
stimulation resulted in a decreased oxygen consumption, indicating
a shift from respiration to anaerobic glycolysis during monocyte
activation (Fig. 6C). The reduced glycolytic flux in response to the
addition of LA was paralleled by an induction of respiration
during the first 2–3 h. In contrast to LA, acidification of the culture
medium suppressed the respiration of monocytes.
In line with these results, 2-DG decreased the glycolytic flux and in
parallel increased oxygen consumption (Fig. 6D). These data suggest that an inhibition of glycolysis during monocyte activation
leads to an increased oxidative phosphorylation as monocytes try to
rebalance their disturbed energy supply via increased respiration.
To investigate whether the described effect of LA on glucose
metabolism also affects intracellular ATP levels, we incubated
The Journal of Immunology
A
1205
To examine the importance of oxidative phosphorylation for
TNF production, we incubated monocytes with rotenone, an inhibitor of the respiratory chain complex I. Rotenone decreased the
oxygen consumption (Fig. 6D) and lowered the ATP level (Fig.
7C) of human monocytes but had only a marginal effect on TNF
secretion (Fig. 7B).
Our results show that the disturbance of the glycolytic flux but
not an interruption of the respiratory chain results in an impaired
monocyte TNF secretion. This demonstrates that the TNF secretion
of monocytes strongly depends on glycolysis.
B
Discussion
FIGURE 5. Metabolic changes during monocyte activation and inhibition of glucose uptake by LA. Monocytes were incubated with or
without LPS for 18 h. Glucose and lactate levels in the supernatants were
determined by GC-MS. Glucose uptake was calculated relative to the
medium control without monocytes. Data are means 6 SEM (n = 4) (A;
control versus LPS NS for glucose levels; pppp = 0.0001 for lactate,
Student t test). The influence of extracellular LA (5–20 mM), 20 mM NaL,
or pH 6.6 on glucose uptake was analyzed by GC-MS and calculated
relative to the medium control (B; mean 6 SD, n = 3; Student t test, pp ,
0.05, ppp , 0.005). Molar levels of glutamine and its metabolite glutamate
in the supernatants were analyzed by GC-MS (C; mean 6 SD, n = 3; no
significant difference between LPS and LPS + LA was detected).
monocytes with or without LPS and LA and determined ATP with
a bioluminescent assay after 18 h. Interestingly, the ATP level of
nonstimulated and stimulated monocytes did not significantly differ
from each other, but 20 mM LA diminished intracellular ATP levels
(Fig. 7A). Acidification lowered the ATP level to a similar extent
compared with LA. However, this effect most likely depended
more on the decreased respiration rate and to a lesser extent on the
diminished glycolysis, as LA decreased glycolysis more efficiently (in terms of glucose uptake and extracellular lactate). A
summary of the presented data and a comparison between the
effects of LA and acidification is shown in Table I. We conclude
that the effect of LA seems to consist of two parts: the effect of
acidification and the effect of lactate itself.
Inhibition of glycolysis by 2-DG inhibits monocyte TNF
secretion
To further investigate the impact of impaired glycolysis on
monocyte TNF secretion, we incubated monocytes with 2-DG and
LPS. The addition of 2-DG led to a dose-dependent inhibition of
TNF secretion and diminished ATP levels (Fig. 7B, 7C).
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C
Low pH and lactate accumulation are characteristic features of the
tumor microenvironment and inflammatory conditions in wounds.
Little is known about the biological effects of pH and lactate on
infiltrating monocytes, neutrophils, and T cells. In this report, we
investigated the effect of LA and pH variation on human monocytes and analyzed the underlying mechanisms. LA decreased in
a dose-dependent manner the capacity of monocytes to produce
TNF. A low pH (∼6.6) in the absence of LA also inhibited the
secretion of TNF. Accordingly, paracrine lactate secretion in
a tumor spheroid coculture model led to an inhibition of TNF
secretion and this effect was reversed by oxamic acid, an inhibitor of lactate dehydrogenase. However, the inhibition of IL-6
was only found when we added high LA concentrations and was
not detected in the coculture model with tumor spheroids. Intratumoral analyses revealed that LA concentrations can reach
values up to 40 mM (34). However, in our spheroid cultures, LA
levels normally did not exceed 10 mM. As TNF seems to be
more sensitive to the suppression compared with IL-6, LA may
reduce TNF but not IL-6 secretion in vivo under pathophysiological conditions. Similar results for TNF secretion were obtained by Douvdevani et al. (22), who studied the influence of
peritoneal dialysis fluid on human macrophages and monocytes
and suggested that the inhibitory effect of the dialysate is due to
the low pH and high lactate content. Bidani et al. (35) found that
a pH #7 suppressed the TNF release by rabbit alveolar macrophages. In contrast, Bellocq et al. (36) and Jensen et al. (37)
reported that pH 7.0 and LA elevated TNF in rat macrophages.
Shime et al. (14) recently demonstrated that LA enhanced the
transcription and production of IL-23 in a pH-dependent manner.
In addition, a recent paper by Samuvel et al. (21) reported that
preincubation of U937 or macrophages with sodium lactate resulted in an increase in the secretion of inflammatory cytokines
like IL-6 and IL-8. However, culture conditions and cells used
for analysis in both studies differed from our experimental setting. Therefore, one may speculate that the observed discrepancies are due to species variations, different cell types used for
the analysis, or differences in the culture conditions. In our tumor spheroid model, we investigated the effect of LA on freshly
infiltrating blood monocytes during short-time exposure to LA,
whereas the data by Samuvel et al. (21) may reflect a situation
where monocytes/macrophages are exposed to lactate for a prolonged time period and become adapted to the lactate in the
tumor environment.
To exclude the possibility that the inhibition of cytokine secretion by LA is based on the induction of apoptosis, we performed
Annexin/7-AAD staining. In our hands, pH values in the range of
6.5–7.0 and concentrations of LA up to 20 mM had no effect on
cell viability. Accordingly, Jensen et al. (37) demonstrated that
lactic acidosis at 15 mM did not reduce cell viability, whereas
Bidani et al. (35) showed that the viability of macrophages declined only at pH values #6.0.
1206
LACTIC ACID INHIBITS TNF SECRETION
A
B
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FIGURE 6. Inhibition of the glycolytic
flux and modulation of oxygen consumption by LA, 2-DG, and rotenone. The
effect of extracellular LA on the glycolytic
flux was measured by incubating monocytes in glucose-free medium supplemented with [1,2-13C2]glucose in the
presence or absence of 100 ng/ml LPS and
LA, NaL, or 2-DG (10 mM), an inhibitor
of glycolysis. The secretion of [2,3-13C2]
lactate into the culture supernatant
(A) and the level of accumulated intracellular [2,3-13C2]lactate (B) resulting
from [1,2-13C2]glucose metabolism were
determined by LC-MS/MS. Monocytes
were cultured in the presence or absence
of LPS and/or 5–20 mM LA. Extracellular
lactate was significantly diminished for 10
mM LA, 20 mM LA, pH 6.6, NaL, and 2DG compared with the LPS control (A;
mean 6 SD, n = 3; Student t test, pp ,
0.05; ppp , 0.005; pppp , 0.0005).
Intracellular lactate was significantly elevated for 5–20 mM LA and pH 6.6
compared with the control (B; mean 6
SD, n = 3; Student t test, pp , 0.05;
ppp , 0.005; pppp , 0.0005). To analyze the effect of LA and pH 6.6 on
oxygen consumption, monocytes were
incubated in RPMI medium for 16 h.
Oxygen consumption was analyzed in
24-well multidishes (C; OxoDish). One
representative experiment out of four is
shown. In addition, the effect of 10 mM
2-DG and 0.1 mM rotenone, an inhibitor
of the respiratory chain complex I, on
LPS-stimulated monocytes was studied.
As a control, monocytes without LPS
stimulation were used (D). One representative experiment out of three is
shown.
C
D
Next, we investigated the mechanism underlying the inhibitory
effect and analyzed whether monocytes take up lactate. At physiological pH, LA almost entirely dissociates to the lactate anion,
which cannot cross the plasma membrane by free diffusion but
requires a transport system. MCTs cotransport protons and lactate
and other monocarboxylates dependent on the gradient between the
extracellular and intracellular milieu (30, 31). The expression of
MCTs has been demonstrated in several immune cells including
monocytes (38).
Without stimulation, we found a constitutive uptake of [3-13C]
lactate, which was further enhanced by the combined treatment with
LPS and mild acidification of the medium. This indicates the involvement of MCTs in the uptake of lactate by activated monocytes.
A pH-dependent uptake of lactate was also demonstrated by Loike
The Journal of Immunology
A
1207
Table I. Summary of the presented data concerning the suppression of
20 mM LA and acidification (pH 6.6) relative to the LPS control
a
TNF in supernatant
Glucose uptakeb
Extracellular lactatec
Intracellular ATPd
O2 consumptione,f
20 mM LA
pH 6.6
***
**
***
*
0–4 h ↑/4–16 h ↓
*
*
*
*
0–16 h ↓
a
Fig. 3B.
Fig. 5B.
Fig. 6A.
d
Fig. 7A.
e
Fig. 6C.
f
O2 consumption was measured with the SDR SensorDish Reader (PreSens Precision Sensing; ↑, increase; ↓, decrease relative to the LPS control).
pp , 0.05; ppp , 0.005; pppp , 0.0005.
b
c
B
FIGURE 7. Inhibition of glycolysis by 2-DG but not suppression of
respiration by rotenone disturbs monocyte TNF secretion and the possible
impact of intracellular ATP. To determine ATP levels, freshly isolated
human monocytes were stimulated with 100 ng/ml LPS for 18–20 h in the
absence or presence of 5–20 mM LA or 20 mM NaL. In addition, the
medium pH was lowered to the respective pH (pH 7.1 or 6.6). Intracellular
ATP levels were measured in cell lysates. Data represent the mean of five
independent experiments 6 SEM. Data for 20 mM LA, pH 6.6, and 20
mM NaL differ significantly from the LPS control (A; Student t test, pp ,
0.05). Monocytes were incubated with 100 ng/ml LPS in the absence or
presence of 1 and 10 mM 2-DG, an inhibitor of glycolysis, or 0.1 and 1
mM rotenone, an inhibitor of respiratory chain complex I. TNF was
measured after 18–20 h in the supernatant by ELISA; 10 mM 2-DG but not
rotenone caused a significant decrease in TNF levels (B; mean 6 SEM, n =
6; Student t test, pp , 0.05). To analyze a possible correlation between
intracellular ATP levels and TNF, we measured intracellular ATP levels in
cell lysates after 18 h and found that 10 mM 2-DG as well as 0.1 and 1 mM
rotenone significantly decreased ATP levels in monocytes (C; mean 6
SEM, n = 4; Student t test, pp , 0.05; ppp , 0.005).
et al. (39) in murine macrophages, and they also described that the
lactate uptake was greater in elicited than in resident macrophages,
suggesting a role of activation in the regulation of the transporters.
Beside MCTs, other transporters might also be involved in lactate
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C
uptake and export in a proton-independent manner (e.g., via the sodium coupled low-affinity transporter for monocarboxylates) (40).
We hypothesized that the uptake and accumulation of lactate in
monocytes would suppress the glycolytic flux and thereby impair
monocyte TNF secretion. Monocyte, macrophage, and dendritic
cell activation is energy-dependent and involves stimulation of
glycolysis (5, 32, 33). LPS induces 6-phosphofructo-2-kinase,
which in turn synthesizes fructose 2,6-bisphosphate, the most
potent stimulator of 6-phosphofructo-1-kinase (PFK-1) (5). Elevated activity of PFK-1 was also observed in activated rat macrophages together with an increased lactate release (32).
Therefore, PFK-1 expression and regulation seem to be important
for monocyte activation, and monocytes may upregulate glycolysis via regulatory enzymes such as PFK-1.
Next, we analyzed uptake and metabolism of glucose and its
importance for monocyte TNF secretion. After stimulation with
LPS, we found no significant increase in glucose uptake, but an
increased conversion of glucose into lactate. These findings suggest
an accelerated glycolysis to meet the increased energy demand
after LPS stimulation. In line with this hypothesis, it is known that
monocyte activation requires stimulation of glycolysis via HIF-1a
(5, 8). In addition, data by Roiniotis et al. (41) show that not only
activation but also differentiation of monocytes into macrophages
is associated with enhanced glycolysis.
In our hands, glucose uptake was reduced in response to extracellular LA and also resulted in a decreased secretion of [2,3-13
C2]lactate produced from [1,2-13C2]glucose. Acidification in the
range of pH 6.6 had a moderate effect on both parameters. The
reduced glucose uptake by monocytes and the lowered levels of
extracellular [2,3-13C2]lactate indicate that LA and concomitant
acidification inhibit the glycolytic flux.
Besides the lowered glycolytic flux, high extracellular LA
concentrations led to a significant accumulation of intracellular
lactate. The accumulation of intracellular lactate was most likely
caused by an influx of extracellular lactate and a block in the efflux
of [2,3-13C2]lactate produced from [1,2-13C2]glucose during glycolysis. MCTs transport lactate between the extracellular and intracellular milieu, and this transport is crucially dependent on the
presence of protons (30, 31). Accordingly, the addition of extracellular LA and acidification did not only lead to a block in the
export of [2,3-13C2]lactate by MCTs, but also to an influx of
lactate into the cell.
It is known that lactate inhibits PFK-1, a key regulatory enzyme
of glycolysis, and favors the dissociation of active PFK-1 tetramers
into less active dimers (42). In the light of our findings, intracellular lactate accumulation could suppress PFK-1 and thereby
reduce the glycolytic flux in monocytes.
1208
Acknowledgments
We thank Alice Peuker, Gabi Hartmannsgruber, Monika Wehrstein,
Alexandra Müller, Ireen Ritter, Jan Linnemann, and Nadine Nürnberger
for excellent technical support.
Disclosures
The authors have no financial conflicts of interest.
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Based on the hypothesis that glycolysis is essential for immune
cell activation, MCTs also play a crucial role because lactate has to
be exported from the cell to allow an undisturbed glycolytic flux.
Accordingly, blocking of MCT-1 by pharmacological intervention
suppressed the glycolytic flux in T cells and resulted in a decreased
T cell proliferation and activation (43).
Inhibition of hexokinase activity by the addition of 2-DG suppressed glycolysis and TNF release by human monocytes. Incubation of monocytes with rotenone, an inhibitor of the oxidative
phosphorylation, exerted little effect on LPS-stimulated TNF secretion. Similar results were obtained by Jantsch et al. (33), who
blocked dendritic cell activation by the addition of 2-DG and
demonstrated that activation crucially depends on glycolysis even
in the presence of oxygen. Furthermore, data by Cramer et al. (8)
suggest a basic role of glycolysis for the inflammatory response of
myeloid cells as functional inactivation of HIF-1a significantly
reduced lactate production and TNF secretion.
In summary, our results show that TNF secretion of monocytes
depends both on glycolysis and export of its end product lactate.
Tumor-derived LA and concomitant acidification could modulate
and disturb monocyte cytokine production by blocking lactate
export and glycolytic flux. Together with other observations on the
effects of LA on T cells, these results suggest that selective targeting of glycolysis in tumor cells could restore immune cell
activation and immune response against tumor cells.
LACTIC ACID INHIBITS TNF SECRETION
The Journal of Immunology
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