Lithium Induces NF-κB Activation and Interleukin-8 Production in Human Intestinal Epithelial Cells (2002)

Lithium has been documented to regulate apoptosis and apoptotic gene expression via NF-κB and mitogen-activated protein (MAP) kinase-dependent mechanisms. Since both NF-κB and MAP kinases are also important mediators of inflammatory gene expression, we investigated the effect of lithium on NF-κB- and MAP kinase-mediated inflammatory gene expression. Incubation of human intestinal epithelial cells with lithium induced both enhanced NF-κB DNA binding and NF-κB-dependent transcriptional activity. In addition, lithium stimulated activation of both the p38 and p42/44 MAP kinases. This lithium-induced up-regulation of NF-κB 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-κB and MAP kinase activation. Lithium has been documented to regulate apoptosis and apoptotic gene expression via NF-κB and mitogen-activated protein (MAP) kinase-dependent mechanisms. Since both NF-κB and MAP kinases are also important mediators of inflammatory gene expression, we investigated the effect of lithium on NF-κB- and MAP kinase-mediated inflammatory gene expression. Incubation of human intestinal epithelial cells with lithium induced both enhanced NF-κB DNA binding and NF-κB-dependent transcriptional activity. In addition, lithium stimulated activation of both the p38 and p42/44 MAP kinases. This lithium-induced up-regulation of NF-κB 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-κB and MAP kinase activation. intestinal epithelial cell 5-(N-ethyl-N-isopropyl)-amiloride mitogen-activated protein 5-(N-methyl-N-isobutyl)-amiloride Na+/H+ exchanger 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide interleukin reverse transcription glyceraldehyde-3-phosphate dehydrogenase Lithium has a number of effects on various biological processes, including embryonic development, glycogen synthesis, hematopoiesis, and neuronal communication (1Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Crossref PubMed Scopus (447) Google Scholar). 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 (1Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Crossref PubMed Scopus (447) Google Scholar). 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 (1Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Crossref PubMed Scopus (447) Google Scholar). There is an accumulating body of evidence demonstrating that lithium increases activation of the transcription factor activator protein-1 (2Yuan P. Chen G. Manji H.K. J. Neurochem. 1999; 73: 2299-2309Crossref PubMed Scopus (69) Google Scholar, 3Bullock B.P. McNeil G.P. Dobner P.R. Brain Res. Mol. Brain Res. 1994; 27: 232-242Crossref PubMed Scopus (18) Google Scholar, 4Ozaki N. Chuang D.M. J. Neurochem. 1997; 69: 2336-2344Crossref PubMed Scopus (166) Google Scholar, 5Chen G. Yuan P.X. Jiang Y.M. Huang L.D. Manji H.K. J. Neurochem. 1998; 70: 1768-1771Crossref PubMed Scopus (63) Google Scholar), 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-κB. For example, treatment of mouse embryonic fibroblasts with lithium decreases tumor necrosis factor-α-induced NF-κB transactivation (6Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1200) Google Scholar). On the other hand, consistent with the notion that the effects of lithium are cell type-specific, lithium increases NF-κB activity in the rat pheochromocytoma cell line PC12 (7Bournat J.C. Brown A.M. Soler A.P. J. Neurosci. Res. 2000; 61: 21-32Crossref PubMed Scopus (95) Google Scholar). This increase in NF-κB 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-κB contributes to the protective effect of lithium against apoptosis. While NF-κB is important in regulating apoptotic events, this transcription factor is also a central mediator of inflammatory processes in a wide variety of cell types (8Baeuerle P.A. Baichwal V.R. Adv. Immunol. 1997; 65: 111-137Crossref PubMed Google Scholar). Stimulation of the NF-κB transcription factor system is instrumental in the transcriptional activation of a variety of inflammatory genes including cytokines, chemokines, and the inducible nitric-oxide synthase (8Baeuerle P.A. Baichwal V.R. Adv. Immunol. 1997; 65: 111-137Crossref PubMed Google Scholar). Although lithium has been shown to potentiate the expression of cytokine and chemokine genes (9Beyaert R. Schulze-Osthoff K. Van Roy F. Fiers W. Cytokine. 1991; 3: 284-291Crossref PubMed Scopus (23) Google Scholar, 10Beyaert R. De Heyninck K. Valck D. Boeykens F. van Roy F. Fiers W. J. Immunol. 1993; 151: 291-300PubMed Google Scholar, 11Maes M. Song C. Lin A.H. Pioli R. Kenis G. Kubera M. Bosmans E. Psychopharmacology. 1999; 143: 401-407Crossref PubMed Scopus (67) Google Scholar) as well as inducible nitric-oxide synthase (12Feinstein D.L. J. Neurochem. 1998; 71: 883-886Crossref PubMed Scopus (49) Google Scholar), it is unknown whether these proinflammatory effects are related to NF-κB activation. In the current paper, we investigated the effect of lithium on NF-κB activation and the inflammatory response in human intestinal epithelial cells (IECs).1These cells have recently been demonstrated to exhibit a substantial inflammatory response to various extracellular stimuli including cytokines and bacterial products, in which NF-κB activation plays a central role (13Jobin C. Sartor R.B. Am. J. Physiol. 2000; 278: C451-C462Crossref PubMed Google Scholar). 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-κB and production of the chemokine IL-8. Furthermore, we provide evidence that upstream from or parallel to NF-κB, 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. 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. 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-1β 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. 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. 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 mmdithiothreitol, 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 mmMgCl2, 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 (14Nilsen E.M. Johansen F.-E. Jahnsen F.L. Lundin K.E.A. Scholz T. Brandtzaeg P. Haraldsen G. Gut. 1998; 42: 635-642Crossref PubMed Scopus (142) Google Scholar), 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. 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 (15Haskó G. Szabó C. Németh Z.H. Kvetan V. Pastores S.M. Vizi E.S. J. Immunol. 1996; 157: 4634-4640PubMed Google Scholar). 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 mmMgCl2, 0.2% (v/v) Nonidet P-40, 0.5 mmdithiothreitol, 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 mmHEPES, 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 mmphenylmethylsulfonyl 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-κB consensus oligonucleotide probe used for the EMSA was purchased from Promega. Oligonucleotide probes were labeled with [γ-32P]ATP using kinase (Invitrogen) and in For the EMSA μg of nuclear proteins were with EMSA binding buffer glycerol 20 mm pH 7.9, 1 mm EDTA, and 1 mm as well as and 0.2 bovine serum at for 10 min the addition of the oligonucleotide for an 25 min. The of the binding were tested by with a of the oligonucleotide were resolved using a gel of of and in buffer mm 45 mm 1 for 2.5 h at current (25 were to paper, at °C for min, and to at −70 °C with an For studies, the addition of the were incubated for 30 min with or 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 10 μg of cytosolic protein extracts were on gel San Diego, CA) and to a The membrane was with or MAP kinase and incubated with a (Roche Molecular Biochemicals). were using Western blotting (Invitrogen). For × HT-29 cells were well of a 1 to Cells were with medium containing 25 of (Invitrogen) and 10 DNA including an NF-κB or the San Diego, CA) according to the manufacturer's instructions. This NF-κB of the NF-κB consensus to a from the virus kinase After 5 h of incubation, medium was replaced with McCoy's 5A medium containing 10% fetal bovine serum. Cells were to at °C for 20 h and were to Selectamine medium containing NaCl (140 mm) or a combination of NaCl (60 mm) and LiCl (80 activity was by the System and to μg of an of cell was by the of to Cells in 96-well plates were incubated with for min at Culture medium was by and the cells were in The of of to cells was by of at using a G. Chen E.A. Szabó C. J. 2000; PubMed Scopus Google G. Németh Z.H. Szabó C. J. Immunol. 2000; PubMed Scopus Google Scholar). are as of of the was performed by of followed by as the effect of lithium on the production of IL-8. of NaCl with LiCl stimulated IL-8 production The effect of LiCl was in the mm concentrations of LiCl to further increase the production of IL-8 Lithium was also tested in Caco-2 cells, similar to HT-29 cells, it the of IL-8 The IL-8 concentrations induced by lithium were with by a high of IL-1β we whether the effect of NaCl with LiCl was to a in or an increase in in of mm extracellular NaCl with mm choline to IL-8 with mm LiCl stimulated IL-8 to choline chloride, increase in IL-8 production was observed when NaCl was replaced with the of lithium the in extracellular the of IL-8 production when NaCl is replaced with LiCl. whether the effect of lithium on IL-8 protein was associated with accumulation of IL-8 of NaCl (80 mm) with LiCl stimulated IL-8 mRNA accumulation as by The effect of lithium on IL-8 mRNA accumulation was because lithium to mRNA levels of the gene Because NF-κB is the central of IL-8 gene expression in IECs (13Jobin C. Sartor R.B. Am. J. Physiol. 2000; 278: C451-C462Crossref PubMed Google Scholar), we the effect of lithium on NF-κB activation by both NF-κB DNA binding and NF-κB-dependent transcriptional activity. of HT-29 cells to medium containing mm LiCl and mm NaCl induced an NF-κB DNA binding that was in cells to 140 mm NaCl This as as 15 min after stimulation with 45 min after the and min following stimulation studies indicated that the induced by lithium contained both and while the contained 5 is because both the and the and the the Stimulation of the cells with IL-1β induced a that was in its to the induced by lithium and also contained both and amiloride (300 lithium-induced NF-κB DNA binding in HT-29 After discarding growth medium (140 mm cells were treated with medium containing mm NaCl and mm LiCl for 45 min. Amiloride or its vehicle was added to the cells with medium. are indicated by as determined by cells were treated with medium containing 140 mm IL-1β treatment of HT-29 cells for 45 min induces a DNA binding similar to the induced by lithium. This is of three investigated whether the lithium-induced increase in NF-κB DNA binding with an increase in NF-κB-dependent gene transcription. To this HT-29 cells were with a or the of HT-29 cells to lithium for h induced a substantial increase in activity in the cells with the the we can that lithium NF-κB-dependent transcriptional activity in HT-29 of the p38 and p42/44 MAP kinases is an important in the of cellular to IL-8 production in IECs C. Sartor R.B. J. Immunol. 1999; Google Scholar, J. 2000; PubMed Scopus Google Scholar, Chen J. Immunol. 1997; Google Scholar, E. J. Immunol. 2000; PubMed Scopus Google Scholar). we whether lithium (80 mm NaCl replaced with mm enhanced IL-8 production by a p38 and Western we that lithium induced both p38 and p42/44 activation in HT-29 cells Lithium induced both p38 and p42/44 activation as as 15 min after which the To whether this activation of p38 and p42/44 by lithium to the effect of lithium on IL-8 we investigated whether MAP kinase decreased lithium-induced IL-8 of the cells with the selective p38 inhibitor SB 203580 or the selective p42/44 inhibitor PD 98059 a of the IL-8 response to lithium. Furthermore, of these inhibitors as determined using the assay These that both p38 and p42/44 activation to the effect of lithium on IL-8 production by Since are important membrane proteins that are in mediating the cellular effects of lithium J.C. J. Physiol. PubMed Scopus Google Scholar, E.M. Am. J. Physiol. PubMed Google Scholar, T. T. M. 2000; PubMed Scopus Google Scholar), we tested the that lithium IL-8 production via an of NHE activation with amiloride, or EIPA the effect of lithium on IL-8 production Furthermore, the NHE inhibitors cimetidine, harmaline, and clonidine M. J. J. 2000; PubMed Scopus Google J. J. 1993; PubMed Google Scholar) all the IL-8 response to lithium The effects of NHE inhibitors were because of the NHE inhibitors effect on cell Furthermore, similar to its effect on IL-8 amiloride the lithium-induced increase in IL-8 mRNA that play a central role in the lithium-induced IL-8 response in HT-29 cells similar to its on IL-8 protein and mRNA expression, NHE by amiloride a of the lithium-induced NF-κB DNA binding In this we demonstrate that lithium induces a inflammatory response in human studies have that lithium inflammatory in and (9Beyaert R. Schulze-Osthoff K. Van Roy F. Fiers W. Cytokine. 1991; 3: 284-291Crossref PubMed Scopus (23) Google Scholar, 10Beyaert R. De Heyninck K. Valck D. Boeykens F. van Roy F. Fiers W. J. Immunol. 1993; 151: 291-300PubMed Google Scholar, 11Maes M. Song C. Lin A.H. Pioli R. Kenis G. Kubera M. Bosmans E. Psychopharmacology. 1999; 143: 401-407Crossref PubMed Scopus (67) Google Scholar, 1991; PubMed Scopus Google Scholar). is the to demonstrate that lithium an inflammatory response in the of to this lithium-induced inflammatory response of IECs is the activation min after lithium of the NF-κB In addition, lithium of the of the inflammatory response including the p38 and p42/44 MAP kinase These play a central role in mediating of the inflammatory such as the up-regulation of IL-8 gene expression. is that lithium on all of these various inflammatory and is that lithium an upstream from MAP kinases and transcription For example, it is that lithium stimulates a membrane by its induces a response similar to the observed with lithium. are the and tumor necrosis as well as the which are the membrane for extracellular inflammatory of in IECs E. 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Furthermore, this activation of NF-κB and MAP kinases results in the of an inflammatory response in for the of the