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J. Biol. Chem., Vol. 277, Issue 10, 7713-7719, March 8, 2002

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Lithium Induces NF-B Activation and Interleukin-8 Production in Human Intestinal Epithelial Cells^*

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

Received for publication, October 9, 2001, and in revised form, December 3, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Lithium has been documented to regulate apoptosis and apoptotic gene expression via NF-B and mitogen-activated 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 Na⁺/H⁺ exchangers, because selective blockade of Na⁺/H⁺ 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 lithium 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-(N-ethyl-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 inhibitor 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 µg of RNA was transcribed in a 20-µl reaction containing 10.7 µl of RNA, 2 µl of 10× PCR buffer, 2 µl of 10 mM dNTP mix, 2 µl of 25 mM MgCl₂, 2 µl of 100 mM dithiothreitol, 1 µl of 50 µM oligo(dT)₁₆, and 0.3 µl of murine leukemia virus reverse transcriptase (50 units/µl). 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 µl) was amplified using the Expand High Fidelity PCR System (Roche Molecular Biochemicals). The PCR mix (25 µl) contained 2-3 µl of cDNA, water, 2.5 µl of PCR buffer, 1.5 µl of 25 mM MgCl₂, 1 µl of 10 mM dNTP mix, 0.5 µl of 10 mM oligonucleotide primer (each), and 0.2 µl 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 µM; 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 µg 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 mM dithiothreitol) as well as 120 ng/µl poly(dI)·poly(dC), 0.4 ng/µl 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 µg 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 peroxidase-conjugated 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 µl/ml of LipofectAMINE 2000 reagent (Invitrogen) and 10 µg/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 µg of protein.

Measurement of Mitochondrial Respiration-- Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondria-dependent 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 µl). 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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).

View larger version (18K): [in this window] [in a new window]   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.

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 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.

View larger version (8K): [in this window] [in a new window]   Fig. 2.   The presence of lithium, but not the reduction in extracellular Na+, 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.

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.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Lithium induces up-regulation of IL-8 mRNA levels in HT-29 cells. Amiloride (300 µM) 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.

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 following 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).

View larger version (60K): [in this window] [in a new window]   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.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   A, amiloride pretreatment (300 µM) 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.

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.

View larger version (14K): [in this window] [in a new window]   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/µg of protein. Data are mean ± S.E. of n = 14-16 wells from two separate experiments. **, p < 0.01.

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.

View larger version (24K): [in this window] [in a new window]   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.

To examine whether this activation of p38 and p42/44 caused 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 (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.

View larger version (18K): [in this window] [in a new window]   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.

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 (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 lithium-induced NF-B DNA binding complex (Fig. 5).

View larger version (21K): [in this window] [in a new window]   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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we demonstrate that lithium induces a full-blown 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 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 NHE-mediated 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 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.

	ACKNOWLEDGEMENT

We thank Dr. John Reeves for helpful discussions during the preparation of the manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The 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.

Published, JBC Papers in Press, December 27, 2001, DOI 10.1074/jbc.M109711200

² Z. H. Németh, E. A. Deitch, C. Szabó, Z. Fekete, C. J. Hauser, and G. Haskó, unpublished observation.

	ABBREVIATIONS

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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