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

Open AccessPublished:December 27, 2001DOI:https://doi.org/10.1074/jbc.M109711200
      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.
      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
      Lithium has a number of effects on various biological processes, including embryonic development, glycogen synthesis, hematopoiesis, and neuronal communication (
      • Phiel C.J.
      • Klein P.S.
      ). 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 (
      • Phiel C.J.
      • Klein P.S.
      ). 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 (
      • Phiel C.J.
      • Klein P.S.
      ). There is an accumulating body of evidence demonstrating that lithium increases activation of the transcription factor activator protein-1 (
      • Yuan P.
      • Chen G.
      • Manji H.K.
      ,
      • Bullock B.P.
      • McNeil G.P.
      • Dobner P.R.
      ,
      • Ozaki N.
      • Chuang D.M.
      ,
      • Chen G.
      • Yuan P.X.
      • Jiang Y.M.
      • Huang L.D.
      • Manji H.K.
      ), 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 (
      • Hoeflich K.P.
      • Luo J.
      • Rubie E.A.
      • Tsao M.S.
      • Jin O.
      • Woodgett J.R.
      ). 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 (
      • Bournat J.C.
      • Brown A.M.
      • Soler A.P.
      ). 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 (
      • Baeuerle P.A.
      • Baichwal V.R.
      ). 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 (
      • Baeuerle P.A.
      • Baichwal V.R.
      ). Although lithium has been shown to potentiate the expression of cytokine and chemokine genes (
      • Beyaert R.
      • Schulze-Osthoff K.
      • Van Roy F.
      • Fiers W.
      ,
      • Beyaert R.
      • De Heyninck K.
      • Valck D.
      • Boeykens F.
      • van Roy F.
      • Fiers W.
      ,
      • Maes M.
      • Song C.
      • Lin A.H.
      • Pioli R.
      • Kenis G.
      • Kubera M.
      • Bosmans E.
      ) as well as inducible nitric-oxide synthase (
      • Feinstein D.L.
      ), 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 (
      • Jobin C.
      • Sartor R.B.
      ). 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.

      DISCUSSION

      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 (
      • Beyaert R.
      • Schulze-Osthoff K.
      • Van Roy F.
      • Fiers W.
      ,
      • Beyaert R.
      • De Heyninck K.
      • Valck D.
      • Boeykens F.
      • van Roy F.
      • Fiers W.
      ,
      • Maes M.
      • Song C.
      • Lin A.H.
      • Pioli R.
      • Kenis G.
      • Kubera M.
      • Bosmans E.
      ,
      • Wu Y.Y.
      • Yang X.H.
      ). 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-κB 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-α 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 (
      • Cario E.
      • Rosenberg I.M.
      • Brandwein S.L.
      • Beck P.L.
      • Reinecker H.C.
      • Podolsky D.K.
      ,
      • Bocker U.
      • Schottelius A.
      • Watson J.M.
      • Holt L.
      • Licato L.L.
      • Brenner D.A.
      • Sartor R.B.
      • Jobin C.
      ,
      • Abreu M.T.
      • Vora P.
      • Faure E.
      • Thomas L.S.
      • Arnold E.T.
      • Arditi M.
      ,
      • Lammers K.M.
      • Jansen J.
      • Bijlsma P.B.
      • Ceska M.
      • Tytgat G.N.
      • Laboisse C.L.
      • van Deventer S.J.
      ). 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 (
      • Aderem A.
      ). 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 (
      • Hallcher L.M.
      • Sherman W.R.
      ,
      • Berridge M.J.
      • Downes C.P.
      • Hanley M.R.
      ) and other phosphomonoesterases (
      • Baraban J.M.
      ) as well as glycogen synthase kinase-3β (
      • Klein P.S.
      • Melton D.A.
      ). Furthermore, other protein kinases, such as protein kinase C (
      • Jope R.S.
      • Williams M.B.
      ) and myristoylated alanine-rich C kinase substrate (
      • Jope R.S.
      • Williams M.B.
      ) as well as G proteins (
      • Avissar S.
      • Schreiber G.
      • Danon A.
      • Belmaker R.H.
      ) 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 (
      • Phiel C.J.
      • Klein P.S.
      ). 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+ (
      • Demaurex N.
      • Grinstein S.
      ,
      • Grinstein S.
      • Wieczorek H.
      ). 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 (
      • Civitelli R.
      • Teitelbaum S.L.
      • Hruska K.A.
      • Lacey D.L.
      ), tumor necrosis factor-α (
      • Vairo G.
      • Royston A.K.
      • Hamilton J.A.
      ), interferon-γ (
      • Prpic V.
      • Yu S.F.
      • Figueiredo F.
      • Hollenbach P.W.
      • Gawdi G.
      • Herman B.
      • Uhing R.J.
      • Adams D.O.
      ), and lipopolysaccharide (
      • Vairo G.
      • Royston A.K.
      • Hamilton J.A.
      ,
      • Orlinska U.
      • Newton R.C.
      ). 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-1α, macrophage inflammatory protein-2 (mouse homolog of IL-8), and IL-12 production by macrophages (
      • Mastronarde J.G.
      • Monick M.M.
      • Gross T.J.
      • Hunninghake G.W.
      ,
      • Németh Z.H.
      • Deitch E.A.
      • Szabó C.
      • Haskó G.
      ,
      • Rolfe M.W.
      • Kunkel S.L.
      • Rowens B.
      • Standiford T.J.
      • Cragoe Jr., E.J.
      • Strieter R.M.
      ). We have recently shown that cytokines activate the IEC inflammatory response via an NHE-dependent mechanism.
      Z. H. Németh, E. A. Deitch, C. Szabó, Z. Fekete, C. J. Hauser, and G. Haskó, unpublished observation.
      Although lithium inhibits NHE function in many systems (
      • Aronson P.S.
      ), 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 (
      • Parker J.C.
      ,
      • Davis B.A.
      • Hogan E.M.
      • Boron W.F.
      ,
      • Kobayashi Y.
      • Pang T.
      • Iwamoto T.
      • Wakabayashi S.
      • Shigekawa M.
      ). 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 (
      • Szabo E.Z.
      • Numata M.
      • Shull G.E.
      • Orlowski J.
      ,
      • Yu F.H.
      • Shull G.E.
      • Orlowski J.
      ,
      • Yun C.H.
      • Tse C.M.
      • Nath S.K.
      • Levine S.A.
      • Brant S.R.
      • Donowitz M.
      ,
      • Numata M.
      • Orlowski J.
      ). Importantly, even in the same cell, lithium can activate or inhibit NHEs, depending on the isoforms expressed (
      • Kobayashi Y.
      • Pang T.
      • Iwamoto T.
      • Wakabayashi S.
      • Shigekawa M.
      ). While monocytes/macrophages express only NHE1 (
      • Demaurex N.
      • Orlowski J.
      • Brisseau G.
      • Woodside M.
      • Grinstein S.
      ), IECs have been shown to express NHE1–4 (
      • Janecki A.J.
      • Montrose M.H.
      • Tse C.M.
      • de Medina F.S.
      • Zweibaum A.
      • Donowitz M.
      ,
      • Maouyo D.
      • Chu S.
      • Montrose M.H.
      ,
      • Turner J.R.
      • Black E.D.
      • Ward J.
      • Tse C.M.
      • Uchwat F.A.
      • Alli H.A.
      • Donowitz M.
      • Madara J.L.
      • Angle J.M.
      ). 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 (
      • Parker J.C.
      ) and Daviset al. (
      • Davis B.A.
      • Hogan E.M.
      • Boron W.F.
      ) 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-κB and MAP kinase activation. Furthermore, this activation of NF-κB and MAP kinases results in the development of an inflammatory response in IECs.

      Acknowledgments

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

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