THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 10, Issue of March 8, pp. 7713–7719, 2002
Printed in U.S.A.
Lithium Induces NF-␬B Activation and Interleukin-8 Production in
Human Intestinal Epithelial Cells*
Received for publication, October 9, 2001, and in revised form, December 3, 2001
Published, JBC Papers in Press, December 27, 2001, DOI 10.1074/jbc.M109711200
Zoltán H. Németh, Edwin A. Deitch, Csaba Szabó, Zoltán Fekete, Carl J. Hauser,
and György Haskó‡
From the Department of Surgery, UMD-New Jersey Medical School, Newark, New Jersey 07103
Lithium has a number of effects on various biological processes, including embryonic development, glycogen synthesis,
hematopoiesis, and neuronal communication (1). Lithium exerts its cellular effects by targeting a variety of enzymes that
require metal ions for catalysis or enzymes that transport
metal ions between cellular compartments (1). Alteration of the
activity of these enzymes by lithium results in changes in gene
expression and/or secretory activity by cells, both in a cell
type-specific manner. The modulatory effects of lithium on
gene expression often involve actions on the activation of transcription factor systems as well as upstream regulatory factors
such as protein kinases and phosphatases (1). There is an
accumulating body of evidence demonstrating that lithium increases activation of the transcription factor activator protein-1
(2–5), an effect that is preceded by accumulation of the phosphorylated, active form of c-Jun N-terminal kinase. Furthermore, recent studies have demonstrated that lithium can also
interfere with the activation of NF-␬B. For example, treatment
of mouse embryonic fibroblasts with lithium decreases tumor
necrosis factor-␣-induced NF-␬B transactivation (6). On the
other hand, consistent with the notion that the effects of lith* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
‡ To whom correspondence should be addressed: Dept. of Surgery,
UMD-New Jersey Medical School, 185 S. Orange Ave., University
Heights, Newark, NJ 07103. Tel.: 973-972-8694; Fax: 973-972-6803;
E-mail: haskoge@umdnj.edu.
This paper is available on line at http://www.jbc.org
ium are cell type-specific, lithium increases NF-␬B activity in
the rat pheochromocytoma cell line PC12 (7). This increase in
NF-␬B activity in PC12 cells observed with lithium administration is associated with a decreased apoptosis in these cells,
suggesting that lithium-induced activation of the antiapoptotic
molecule NF-␬B contributes to the protective effect of lithium
against apoptosis.
While NF-␬B is important in regulating apoptotic events,
this transcription factor is also a central mediator of inflammatory processes in a wide variety of cell types (8). Stimulation of the NF-␬B transcription factor system is instrumental
in the transcriptional activation of a variety of inflammatory
genes including cytokines, chemokines, and the inducible
nitric-oxide synthase (8). Although lithium has been shown to
potentiate the expression of cytokine and chemokine genes
(9 –11) as well as inducible nitric-oxide synthase (12), it is
unknown whether these proinflammatory effects are related
to NF-␬B activation.
In the current paper, we investigated the effect of lithium
on NF-␬B activation and the inflammatory response in human intestinal epithelial cells (IECs).1 These cells have recently been demonstrated to exhibit a substantial inflammatory response to various extracellular stimuli including
cytokines and bacterial products, in which NF-␬B activation
plays a central role (13). Our results demonstrate that similar to these classical inflammatory stimuli, lithium induces a
strong inflammatory response in IECs as indicated by the
activation of NF-␬B and production of the chemokine IL-8.
Furthermore, we provide evidence that upstream from or
parallel to NF-␬B, p38 and p42/44 mitogen-activated protein
(MAP) kinases as well as the membrane protein Na⫹/H⫹
exchangers (NHEs) play an important role in mediating the
proinflammatory effects of lithium.
MATERIALS AND METHODS
Cell Lines—The human colon cancer cell lines HT-29 and Caco-2
were obtained from American Type Culture Collection (Manassas, VA).
HT-29 cells were grown in modified McCoy’s 5A medium supplemented
with 10% fetal bovine serum (Invitrogen). Caco-2 cells were grown in
Dulbecco’s modified Eagle’s medium with high glucose containing 10%
fetal bovine serum.
Drugs and Reagents—Amiloride HCl was obtained from Research
Biochemicals Inc. (Natick, MA). LiCl, choline chloride, mannitol, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-(Nethyl-N-isopropyl)-amiloride (EIPA), 5-(N-methyl-N-isobutyl)-amiloride (MIA), cimetidine, harmaline, and clonidine were purchased from
Sigma. Human IL-1␤ was obtained from R&D Systems. The p38 inhib1
The abbreviations used are: IEC, intestinal epithelial cell; EIPA,
5-(N-ethyl-N-isopropyl)-amiloride; MAP, mitogen-activated protein;
MIA, 5-(N-methyl-N-isobutyl)-amiloride; NHE, Na⫹/H⫹ exchanger;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IL,
interleukin; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Lithium has been documented to regulate apoptosis
and apoptotic gene expression via NF-␬B and mitogenactivated protein (MAP) kinase-dependent mechanisms. Since both NF-␬B and MAP kinases are also
important mediators of inflammatory gene expression,
we investigated the effect of lithium on NF-␬B- and
MAP kinase-mediated inflammatory gene expression.
Incubation of human intestinal epithelial cells with
lithium induced both enhanced NF-␬B DNA binding
and NF-␬B-dependent transcriptional activity. In addition, lithium stimulated activation of both the p38
and p42/44 MAP kinases. This lithium-induced up-regulation of NF-␬B and MAP kinase activation was associated with an enhancement of interleukin-8 mRNA
accumulation as well as an increase in interleukin-8
protein release. These proinflammatory effects of lithium were, in large part, mediated by activation of
Naⴙ/Hⴙ exchangers, because selective blockade of
Naⴙ/Hⴙ exchangers prevented the lithium-induced intestinal cell inflammatory response. These results
demonstrate that lithium stimulates inflammatory
gene expression via NF-␬B and MAP kinase activation.
7714
Lithium Induces the Gut Epithelial Cell Inflammatory Response
mM dithiothreitol) as well as 120 ng/␮l poly(dI)䡠poly(dC), 0.4 ng/␮l
single-stranded DNA, and 0.2 mg/ml bovine serum albumin at room
temperature for 10 min before the addition of the radiolabeled oligonucleotide for an additional 25 min. The specificities of the binding reactions were tested by incubating samples with a 50-fold molar excess of
the unlabeled oligonucleotide probe. Protein-nucleic acid complexes
were resolved using a nondenaturing polyacrylamide gel consisting of
4% acrylamide (29:1 ratio of acrylamide/bisacrylamide) and run in 0.5⫻
TBE buffer (45 mM Tris-HCl, 45 mM boric aid, 1 mM EDTA) for 2.5 h at
constant current (25 mA). Gels were transferred to Whatman 3M paper,
dried under vacuum at 80 °C for 40 min, and exposed to photographic
film at ⫺70 °C with an intensifying screen. For supershift studies,
before the addition of the radiolabeled probe, samples were incubated
for 30 min with isotype control, p65, or p50 antibody (Ab) (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA).
Western Blot Analysis—After discarding growth medium, HT-29
cells were treated with Selectamine medium containing NaCl (140
mM) or a combination of NaCl (60 mM) and LiCl (80 mM) for varying
lengths of time. 10 ␮g of cytosolic protein extracts were separated on
8 –16% Tris/glycine gel (Novex, San Diego, CA) and transferred to a
nitrocellulose membrane. The membrane was probed with anti-phospho-p38 or anti-p42/44 MAP kinase Ab (Promega, Madison, WI) and
subsequently incubated with a secondary horseradish peroxidaseconjugated donkey anti-rabbit antibody (Roche Molecular Biochemicals). Bands were detected using ECL Western blotting detection
reagent (Invitrogen).
Transient Transfection and Luciferase Activity—For transient transfections, 2– 4 ⫻ 105 HT-29 cells were seeded per well of a 24-well tissue
culture dish 1 day prior to transient transfection. Cells were transfected
with serum-free RPMI 1640 medium containing 25 ␮l/ml of LipofectAMINE 2000 reagent (Invitrogen) and 10 ␮g/ml plasmid DNA including an NF-␬B luciferase promoter construct or the empty vector
(CLONTECH, San Diego, CA) according to the manufacturer’s instructions. This NF-␬B luciferase vector contains four tandem copies of the
NF-␬B consensus sequence fused to a TATA-like promoter region from
the herpes simplex virus thymidine kinase promoter. After 5 h of
incubation, medium was replaced with McCoy’s 5A medium containing
10% fetal bovine serum. Cells were allowed to recover at 37 °C for 20 h
and subsequently were exposed to Selectamine medium containing
NaCl (140 mM) or a combination of NaCl (60 mM) and LiCl (80 mM).
Luciferase activity was measured by the Luciferase Reporter Assay
System (Promega, Madison, WI) and normalized relative to ␮g of
protein.
Measurement of Mitochondrial Respiration—Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondriadependent reduction of MTT to formazan. Cells in 96-well plates were
incubated with MTT (0.5 mg/ml) for 60 min at 37 °C. Culture medium
was removed by aspiration, and the cells were solubilized in Me2SO
(100 ␮l). The extent of reduction of MTT to formazan within cells was
quantitated by measurement of optical density at 550 nm using a
Spectramax 250 microplate reader (16, 17).
Statistical Evaluation—Values throughout are expressed as
mean ⫾ S.E. of n observations. Statistical analysis of the data was
performed by one-way analysis of variance followed by Dunnett’s test,
as appropriate.
RESULTS
Lithium Induces IL-8 Protein Secretion by HT-29 and Caco-2
Cells—We first examined the effect of lithium on the production of IL-8. Replacement of NaCl with equimolar LiCl
stimulated IL-8 production (Fig. 1). The effect of LiCl was
concentration-dependent in the 0 – 60 mM range. However,
higher concentrations of LiCl failed to further increase the
production of IL-8 (Fig. 1). Lithium was also tested in Caco-2
cells, where, similar to HT-29 cells, it increased the release of
IL-8 (data not shown). The IL-8 concentrations induced by
lithium were comparable with those elicited by a high dose of
IL-1␤ (20 ng/ml; data not shown).
Next, we determined, whether the stimulatory effect of NaCl
replacement with LiCl was due to a decrease in external Na⫹
concentration or an increase in Li⫹ concentration. As shown, in
Fig. 2, replacement of 80 mM extracellular NaCl with 80 mM
choline chloride failed to up-regulate IL-8 production, whereas
replacement with 80 mM LiCl stimulated IL-8 production. Similar to choline chloride, no increase in IL-8 production was
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itor SB 203580 and p42/44 inhibitor PD 98059 were purchased from
Calbiochem. SB 203580, PD 98059, amiloride, cimetidine, harmaline,
and clonidine were dissolved in 0.5% Me2SO.
IL-8 Measurement—After discarding the growth medium, confluent
layers of HT-29 or Caco-2 cells in 96-well plates were treated with
Selectamine medium (Invitrogen) containing 140 mM NaCl or with the
same medium with all or part of the NaCl replaced isosmotically with
LiCl, choline chloride, or mannitol. The cells were incubated with Selectamine medium for 18 h. To test the effect of NHE or MAP kinase
inhibitors, these inhibitors or their vehicle were added to the cells
immediately following treatment with Selectamine medium, followed
by an incubation period of 4 –18 h. Because harmaline was toxic to the
cells when the incubation lasted for 18 h, the effect of this agent on
LiCl-induced IL-8 production was tested 4 h after stimulation. Furthermore, the effect of both MIA and EIPA was tested 4 h after stimulation
with LiCl. Human IL-8 levels were determined from the cell supernatants using commercially available enzyme-linked immunosorbent assay kits (BD Pharmingen, San Diego, CA) and according to the manufacturer’s instructions.
RNA Isolation and RT-PCR—After discarding growth medium,
HT-29 cells were treated with Selectamine medium containing NaCl
(140 mM) or a combination of NaCl (60 mM) and LiCl (80 mM) for 4 h. At
the end of the incubation, total RNA was isolated from cells using
TRIzol Reagent (Invitrogen). Reverse transcription of the RNA was
performed using murine leukemia virus reverse transcriptase from
PerkinElmer Life Sciences. 5 ␮g of RNA was transcribed in a 20-␮l
reaction containing 10.7 ␮l of RNA, 2 ␮l of 10⫻ PCR buffer, 2 ␮l of 10
mM dNTP mix, 2 ␮l of 25 mM MgCl2, 2 ␮l of 100 mM dithiothreitol, 1 ␮l
of 50 ␮M oligo(dT)16, and 0.3 ␮l of murine leukemia virus reverse
transcriptase (50 units/␮l). The reaction mix was incubated at 42 °C for
15 min for reverse transcription. Thereafter, the reverse transcriptase
was inactivated at 99 °C for 5 min. RT-generated DNA (1–5 ␮l) was
amplified using the Expand High Fidelity PCR System (Roche Molecular Biochemicals). The PCR mix (25 ␮l) contained 2–3 ␮l of cDNA,
water, 2.5 ␮l of PCR buffer, 1.5 ␮l of 25 mM MgCl2, 1 ␮l of 10 mM dNTP
mix, 0.5 ␮l of 10 mM oligonucleotide primer (each), and 0.2 ␮l of enzyme.
cDNA was amplified using the following primers and conditions: IL-8
(14), 5⬘-ATGACTTCCAAGCTGGCCGTGGCT-3⬘ (sense) and 5⬘-TCTCAGCCCTCTTCAAAAACTTCTC-3⬘ (antisense); GAPDH, 5⬘-CGGAGTCAACGGATTTGGTCGTAT-3⬘ (sense) and 5⬘-AGCCTTCTCCATGGTGGTGAAGAC-3⬘ (antisense); an initial denaturation at 94 °C for 5 min,
27 and 23 cycles of 94 °C for 30 s for IL-8 and GAPDH, respectively;
58 °C for 45 s and 72 °C for 45 s; and a final dwell at 72 °C for 7 min. The
expected PCR products were 289 bp (for IL-8) and 306 bp (for GAPDH).
PCR products were resolved on a 1.5% agarose gel and stained with
ethidium bromide.
NF-␬B Electromobility Shift Assay (EMSA) and Supershift Assay—
After discarding growth medium, HT-29 cells in six-well plates were
treated with Selectamine medium containing NaCl (140 mM) or a combination of NaCl (60 mM) and LiCl (80 mM) for varying lengths of time,
and nuclear protein extracts were prepared as described previously
(15). To determine the effect of NHE blockade, cells were treated with
amiloride (300 ␮M; Sigma) or its vehicle (0.5% Me2SO) concomitantly
with the addition of Selectamine medium. All nuclear extraction procedures were performed on ice with ice-cold reagents. Cells were
washed twice with PBS and harvested by scraping into 1 ml of PBS and
pelleted at 2,000 ⫻ g for 15 min. The pellet was resuspended in three
packed cell volumes of cytosolic lysis buffer (20% (v/v) glycerol, 10 mM
HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 0.2% (v/v)
Nonidet P-40, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl
fluoride) and incubated for 15 min on ice with occasional vortexing and
then homogenized using a pellet pestle. After centrifugation at 3,000 ⫻
g for 15 min, supernatants (cytosolic extracts) were saved for Western
blotting studies, while the pellet was further processed to obtain nuclear extracts. Two cell pellet volumes of nuclear extraction buffer (20%
(v/v) glycerol, 20 mM HEPES, pH 7.9, 420 mM NaCl, 0.5 mM EDTA, 1.5
mM MgCl2, 50 mM glycerol phosphate, 0.5 mM dithiothreitol, and 0.2 mM
phenylmethylsulfonyl fluoride) was added to the nuclear pellet and
incubated on ice for 20 min with occasional vortexing. Nuclear proteins
were isolated by centrifugation at 14,000 ⫻ g for 30 min. Protein
concentrations were determined using the Bio-Rad protein assay. Nuclear extracts were aliquoted and stored at ⫺70 °C until used for EMSA.
The NF-␬B consensus oligonucleotide probe used for the EMSA was
purchased from Promega. Oligonucleotide probes were labeled with
[␥-32P]ATP using T4 polynucleotide kinase (Invitrogen) and purified in
MicroSpin G-50 columns (Amersham Biosciences). For the EMSA analysis, 12 ␮g of nuclear proteins were preincubated with EMSA binding
buffer (10% glycerol (v/v), 20 mM Tris-HCl, pH 7.9, 1 mM EDTA, and 1
Lithium Induces the Gut Epithelial Cell Inflammatory Response
7715
FIG. 3. Lithium induces up-regulation of IL-8 mRNA levels in
HT-29 cells. Amiloride (300 ␮M) treatment inhibits lithium-induced
IL-8 mRNA accumulation. Lanes 1 and 2, control (140 mM NaCl); lanes
3 and 4, lithium (80 mM of extracellular NaCl replaced with 80 mM
LiCl); lanes 5 and 6, amiloride plus lithium. GAPDH levels were not
affected by both lithium and amiloride treatment. IL-8 and GAPDH
mRNA levels were quantitated using semiquantitative RT-PCR. This
figure is representative of three separate experiments.
FIG. 1. Lithium stimulates IL-8 production by HT-29 cells. Cells
were incubated with medium, in which NaCl was replaced with equimolar amounts of LiCl. Supernatants for IL-8 measurement were taken
18 h after replacement of growth medium with NaCl- or LiCl-containing
medium. Data are mean ⫾ S.E. of n ⫽ 6 –12 wells from two different
experiments. *, p ⬍ 0.05; **, p ⬍ 0.01.
FIG. 2. The presence of lithium, but not the reduction in extracellular Naⴙ, causes the augmentation of IL-8 production
when NaCl is replaced with LiCl. Replacement of 80 mM of extracellular NaCl with 80 mM choline chloride fails to up-regulate IL-8
production, whereas replacement with 80 mM LiCl stimulates IL-8
production. Supernatants for IL-8 measurement were taken 18 h after
treatment with NaCl, LiCl, or choline chloride-containing medium.
Data are mean ⫾ S.E. of n ⫽ 6 –12 wells from two different experiments.
**, p ⬍ 0.01.
observed when NaCl was replaced with isosmolar mannitol
(data not shown). Thus, the presence of lithium but not the
reduction in extracellular Na⫹ causes the augmentation of IL-8
production when NaCl is replaced with LiCl.
Lithium Stimulates IL-8 mRNA Accumulation in HT-29
Cells—We next examined whether the effect of lithium on IL-8
protein synthesis was associated with increased accumulation
of IL-8 mRNA. Replacement of NaCl (80 mM) with equimolar
LiCl stimulated IL-8 mRNA accumulation as measured by
RT-PCR (Fig. 3). The stimulatory effect of lithium on IL-8
mRNA accumulation was selective, because lithium failed to
affect mRNA levels of the housekeeping gene GAPDH.
Lithium Induces NF-␬B Activation in HT-29 Cells—Because
NF-␬B is the central regulator of IL-8 gene expression in IECs
(13), we examined the effect of lithium on NF-␬B activation by
measuring both NF-␬B DNA binding and NF-␬B-dependent
transcriptional activity. Exposure of HT-29 cells to medium containing 80 mM LiCl and 60 mM NaCl induced an NF-␬B DNA
binding complex that was not seen in cells exposed to 140 mM
NaCl (Fig. 4). This complex could be detected as early as 15 min
after stimulation with lithium, became maximal 45 min after the
stimulus, and had almost completely disappeared 90 min follow-
ing stimulation (Fig. 4). Supershift studies indicated that the
band induced by lithium contained both p65 and p50, while the
lower, constitutive band contained p50 but not p65 (Fig. 5A).
That is because both the p50 and p65 Abs shifted the upper
complex, and only the p50 Ab shifted, albeit only partially, the
lower complex. Stimulation of the cells with IL-1␤ induced a
complex that was identical in its position to the one induced by
lithium and also contained both p50 and p65 (Fig. 5B).
We next investigated whether the lithium-induced increase
in NF-␬B DNA binding corresponded with an increase in NF␬B-dependent gene transcription. To this end, HT-29 cells were
transiently transfected with a NF-␬B-luciferase reporter construct or the empty vector. Exposure of HT-29 cells to lithium
for 16 h induced a substantial increase in luciferase activity in
the cells transfected with the NF-␬B-luciferase reporter construct but not the empty vector (Fig. 6). Therefore, we can
conclude that lithium activates NF-␬B-dependent transcriptional activity in HT-29 cells.
Both p38 and p42/44 Activation Contribute to Lithium-induced IL-8 Production in HT-29 Cells—Activation of the p38
and p42/44 MAP kinases is an important step in the cascade of
cellular events leading to IL-8 production in IECs (18 –21).
Therefore, we examined whether lithium (80 mM NaCl replaced
with 80 mM LiCl) enhanced IL-8 production by a mechanism
involving p38 and p42/44. Using Western blotting, we found
that lithium induced both p38 and p42/44 activation in HT-29
cells (Fig. 7). Lithium induced both p38 and p42/44 activation
as early as 15 min after stimulation, which remained increased
during the 90-min observation period.
To examine whether this activation of p38 and p42/44 caused
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FIG. 4. Lithium induces NF-␬B DNA binding in HT-29 cells.
After discarding growth medium (140 mM NaCl), cells were treated with
medium containing 60 mM NaCl and 80 mM LiCl for the indicated time
periods. NF-␬B DNA binding was assessed using EMSA. This figure is
representative of two separate experiments.
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Lithium Induces the Gut Epithelial Cell Inflammatory Response
FIG. 5. A, amiloride pretreatment (300
␮M) inhibits lithium-induced NF-␬B DNA
binding in HT-29 cells. After discarding
growth medium (140 mM NaCl), cells
were treated with medium containing 60
mM NaCl and 80 mM LiCl for 45 min.
Amiloride or its vehicle was added to the
cells together with LiCl-containing medium. NF-␬B-specific complexes are indicated by arrows as determined by Ab supershifting. Control cells were treated
with medium containing 140 mM NaCl. B,
IL-1␤ (20 ng/ml) treatment of HT-29 cells
for 45 min induces a DNA binding complex similar to the one induced by lithium. This figure is representative of three
separate experiments.
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FIG. 6. Lithium stimulation increases NF-␬B-dependent transcriptional activity. HT-29 cells were transiently transfected with a
NF-␬B-luciferase promoter construct, following which the cells were
incubated for 16 h with either medium containing 140 mM NaCl or
medium containing 80 mM LiCl and 60 mM NaCl. NF-␬B-dependent
transcriptional activity was determined using the luciferase assay
(pNF-␬B-Luc). This figure also shows that lithium does not influence
luciferase activity in cells transfected with an enhancerless construct
(pTAL-Luc). Specific activity is expressed as units/␮g of protein. Data
are mean ⫾ S.E. of n ⫽ 14 –16 wells from two separate experiments. **,
p ⬍ 0.01.
FIG. 7. Lithium induces both p38 and p42/44 activation in
HT-29 cells. After discarding growth medium (140 mM NaCl), cells
were treated with medium containing 60 mM NaCl and 80 mM LiCl for
the indicated time periods. p38 and p42/44 activation was determined
using Western blotting. This figure is representative of two separate
experiments.
by lithium contributed to the stimulatory effect of lithium on
IL-8 production, we next investigated whether MAP kinase
inhibition decreased lithium-induced IL-8 production. Treatment of the cells with either the selective p38 inhibitor SB
203580 (Fig. 8A) or the selective p42/44 inhibitor PD 98059
(Fig. 8B) produced a concentration-dependent blunting of the
IL-8 response to lithium. Furthermore, neither of these inhibitors caused any toxicity, as determined using the MTT assay
FIG. 8. Treatment of HT-29 cells with the selective p38 inhibitor
SB 203580 (A) or the selective p42/44 inhibitor PD 98059 (B) suppresses lithium-induced IL-8 production. Cells were incubated with
medium containing 140 mM NaCl or medium containing 80 mM LiCl and
60 mM NaCl in the presence or absence of MAP kinase inhibitors. Supernatants for IL-8 measurement were taken 18 h after replacement of
growth medium with NaCl or LiCl-containing medium. Data represent
the mean ⫾ S.E. of n ⫽ 12 wells from two separate experiments. *, p ⬍
0.05; **, p ⬍ 0.01. Dark bar, no lithium; light bars, lithium.
(data not shown). These data indicate that both p38 and p42/44
activation contribute to the stimulatory effect of lithium on
IL-8 production by IECs.
Lithium-induced IL-8 Production by HT-29 Cells Is Dependent on NHE Activation—Since NHEs are important membrane
proteins that are involved in mediating the cellular effects of
Lithium Induces the Gut Epithelial Cell Inflammatory Response
7717
lithium (22–24), we tested the possibility that lithium increased IL-8 production via an NHE-dependent mechanism.
Inhibition of NHE activation with amiloride, MIA, or EIPA
almost completely abrogated the stimulatory effect of lithium
on IL-8 production (Fig. 9, A–C). Furthermore, the nonselective
NHE inhibitors cimetidine, harmaline, and clonidine (25, 26)
all blunted the IL-8 response to lithium (Fig. 9, D and E). The
effects of NHE inhibitors were specific, because none of the NHE
inhibitors had any effect on cell viability (data not shown). Furthermore, similar to its effect on IL-8 production, amiloride reduced the lithium-induced increase in IL-8 mRNA levels, confirming that NHEs play a central role in the lithium-induced IL-8
response in HT-29 cells (Fig. 3). Finally, similar to its action on
IL-8 protein synthesis and mRNA expression, NHE inhibition by
amiloride caused a nearly complete disappearance of the lithiuminduced NF-␬B DNA binding complex (Fig. 5).
DISCUSSION
In this study, we demonstrate that lithium induces a fullblown inflammatory response in human IECs. Numerous studies have reported that lithium enhances inflammatory events
in immunostimulated macrophages and lymphocytes (9 –11,
27). However, our study is the first one to demonstrate that
lithium activates an inflammatory response even in the absence of conventional immunostimulatory/inflammatory stimuli. Central to this lithium-induced inflammatory response of
IECs is the early activation (15 min after lithium treatment) of
the NF-␬B system. In addition, lithium activates several of the
early intracellular cascades characteristic of the IEC inflammatory response including the p38 and p42/44 MAP kinase
systems. These early pathways play a central role in mediating
later events of the IEC inflammatory response, such as the
up-regulation of IL-8 gene expression.
It is unlikely that lithium acts on all of these various inflammatory events and enzyme cascades independently. A more
plausible scenario is that lithium targets an early pathway
upstream from MAP kinases and transcription factors. For
example, it is conceivable that lithium stimulates a membrane
receptor whose ligation by its ligands normally induces a response similar to the one observed with lithium. Possible candidates are the IL-1 and tumor necrosis factor-␣ as well as the
Toll receptors, which are the major membrane structures responsible for relaying extracellular inflammatory signals toward intracellular effector sites of inflammation in IECs (21,
28 –30). If lithium acted via one of these receptors, then we
would expect lithium to cause an inflammatory response in
monocytes/macrophages in the absence of extracellular inflammatory stimuli such as bacterial products or cytokines, because
monocytes/macrophages express all of the cytokine and Toll
receptors that are present on IECs (31). However, because, as
described above, lithium alone fails to induce inflammatory
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FIG. 9. Treatment of HT-29 cells with the selective NHE inhibitors amiloride (A), MIA (B), or EIPA (C) suppresses lithium-induced
IL-8 production. Treatment of HT-29 cells with the non-amiloride NHE inhibitors cimetidine (D), clonidine (D), or harmaline (E) reproduces the
suppressive effect of selective NHE inhibitors on the production of IL-8 by lithium-induced HT-29 cells. When harmaline, MIA, or EIPA was used,
supernatants for IL-8 measurement were taken 4 h after lithium treatment. In the case of amiloride, cimetidine, and clonidine, IL-8 levels were
measured from supernatants obtained 18 h after challenge with lithium. Data are mean ⫾ S.E. of n ⫽ 6 –12 wells from two different experiments.
*, p ⬍ 0.05; **, p ⬍ 0.01. Dark bar, no lithium (medium containing 140 mM NaCl); light bars, lithium (medium containing 80 mM LiCl and 60 mM
NaCl).
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Lithium Induces the Gut Epithelial Cell Inflammatory Response
2
Z. H. Németh, E. A. Deitch, C. Szabó, Z. Fekete, C. J. Hauser, and
G. Haskó, unpublished observation.
have evolved as a positive feedback signal during IEC
activation.
In summary, our experiments using IECs demonstrate that
the intracellular targets of lithium’s action involve both NF-␬B
and MAP kinase activation. Furthermore, this activation of
NF-␬B and MAP kinases results in the development of an
inflammatory response in IECs.
Acknowledgment—We thank Dr. John Reeves for helpful discussions
during the preparation of the manuscript.
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activity in monocytes/macrophages, it appears improbable that
the proinflammatory effects of lithium in IECs are mediated by
inflammatory membrane receptors.
The best characterized targets of lithium are inositol monophosphatase (32, 33) and other phosphomonoesterases (34) as
well as glycogen synthase kinase-3␤ (35). Furthermore, other
protein kinases, such as protein kinase C (36) and myristoylated alanine-rich C kinase substrate (36) as well as G proteins
(37) have also been documented to be the targets of lithium’s
action. Lithium regulates these pathways with an EC50 value
of 0.1–2 mM (1). The fact that lithium stimulated IEC IL-8
production with an EC50 value of ⬃30 mM suggests that the
above pathways are unlikely to be involved in the proinflammatory effects of lithium in IECs.
Na⫹/H⫹ exchangers (antiporters, NHEs) are a family of
ubiquitous plasma membrane transport proteins that catalyze the exchange of extracellular Na⫹ for intracellular H⫹
(38, 39). Recent evidence indicates that NHEs also regulate
inflammatory processes. NHEs are rapidly activated in response to a variety of inflammatory signals, such as IL-1 (40),
tumor necrosis factor-␣ (41), interferon-␥ (42), and lipopolysaccharide (41, 43). On the other hand, inhibition of NHEs
suppresses inflammatory responses, including IL-8 production by monocytes and respiratory epithelial cells as well as
macrophage inflammatory protein-1␣, macrophage inflammatory protein-2 (mouse homolog of IL-8), and IL-12 production by macrophages (44 – 46). We have recently shown that
cytokines activate the IEC inflammatory response via an
NHE-dependent mechanism.2 Although lithium inhibits
NHE function in many systems (47), recent studies have
shown that lithium, in the concentration range in which it
activates IEC IL-8 production, can also activate NHEs in a
cell type-specific fashion (22–24). This fact, coupled with the
observation that NHE inhibition almost completely prevented the proinflammatory effects of lithium, demonstrates
a key role for these proteins in mediating the IEC inflammatory response to lithium. Recent molecular cloning studies
have confirmed that NHEs constitute a gene family from
which seven mammalian isoforms (NHE1, NHE2, NHE3,
NHE4, NHE5, NHE6, and NHE7) have been cloned and
sequenced (25, 26, 48, 49). Importantly, even in the same cell,
lithium can activate or inhibit NHEs, depending on the isoforms expressed (24). While monocytes/macrophages express
only NHE1 (50), IECs have been shown to express NHE1– 4
(51–53). Thus, it is possible that the differential regulation of
inflammation in monocytes/macrophages and IECs by lithium is due to the differential expression of NHEs in these cell
types.
Since plasma lithium concentrations that induce NHE activation as well as IEC IL-8 production are fatal in humans, it is
improbable that the lithium-induced activation of IEC inflammatory activity has any physiological significance. However, as
both Parker (22) and Davis et al. (23) suggested, lithium may
mimic a physiological stimulus, most probably extracellular
hyperosmolarity, that is capable of activating NHEs. Given our
recent observation that extracellular hyperosmolarity stimulates the IEC inflammatory response in a similar NHE-dependent manner as lithium,2 it can be proposed that extracellular
hyperosmolarity is the physiological stimulus for the NHEmediated inflammatory response in IECs. Furthermore, because the cytokine-induced IEC inflammatory response also
has an NHE-dependent component,2 we speculate that the
mechanism of NHE promotion of inflammatory processes may
Lithium Induces the Gut Epithelial Cell Inflammatory Response
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