Inhibition of Cell Proliferation by Bacterial Lipopolysaccharides in TLR4-Positive Epithelial Cells: Independence of Nitric Oxide and Cytokine Release
2005; Elsevier BV; Volume: 124; Issue: 3 Linguagem: Inglês
10.1111/j.0022-202x.2004.23598.x
ISSN1523-1747
AutoresKarin Müller‐Decker, Gwendolin Manegold‐Brauer, Herbert Butz, Detlef Hinz, Dirk Hüttner, Kathleen Richter, Matthias Tremmel, Rico Weißflog, Friedrich Marks,
Tópico(s)Antimicrobial Peptides and Activities
ResumoPhylogenetically conserved toll-like receptors (TLR) recognize “pathogen-associated molecular patterns”. Upon binding of ligands, TLR initiate innate immune response in immune and most likely epithelial cells. The TLR4 isoform is considered as a lipopolysaccharide (LPS) receptor. As shown here, a rat-tongue-derived epithelial cell line RTE2 expressed TLR4 mRNA and functional protein. LPS-treated RTE2 cells responded with the transient expression of inducible nitric oxide synthase (iNOS), an effector protein of TLR4 involved in the innate immune defense of monocytes. iNOS induction occurred along a nuclear factor-κB (NF-κb)-dependent pathway and correlated with the increased production of NO. Moreover, LPS and lipid A were potent inhibitors of proliferation of RTE2 cells, of mouse keratinocytes, and mouse epidermis in vivo. The inhibition depended on lipid A structure, i.e., it was related to the endotoxin activity of LPS and at least in vitro was in part mediated by NF-κB. C57Bl/10 ScCr mice, lacking a functional TLR4, did not respond with growth inhibition, strongly suggesting a TLR4-mediated effect. RTE2 proliferation was also inhibited by transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα), whereas interferon γ (IFNγ) was a weak inhibitor. But the growth-inhibitory effect of LPS on RTE2 cells was not mediated by TNFα, TGFβ, or NO. It is concluded that besides induction of innate immune responses, LPS specifically induces growth arrest in epithelial tongue cells and keratinocytes in vitro and in mouse epidermis in a TLR4-dependent but cytokine- and NO-independent manner. Phylogenetically conserved toll-like receptors (TLR) recognize “pathogen-associated molecular patterns”. Upon binding of ligands, TLR initiate innate immune response in immune and most likely epithelial cells. The TLR4 isoform is considered as a lipopolysaccharide (LPS) receptor. As shown here, a rat-tongue-derived epithelial cell line RTE2 expressed TLR4 mRNA and functional protein. LPS-treated RTE2 cells responded with the transient expression of inducible nitric oxide synthase (iNOS), an effector protein of TLR4 involved in the innate immune defense of monocytes. iNOS induction occurred along a nuclear factor-κB (NF-κb)-dependent pathway and correlated with the increased production of NO. Moreover, LPS and lipid A were potent inhibitors of proliferation of RTE2 cells, of mouse keratinocytes, and mouse epidermis in vivo. The inhibition depended on lipid A structure, i.e., it was related to the endotoxin activity of LPS and at least in vitro was in part mediated by NF-κB. C57Bl/10 ScCr mice, lacking a functional TLR4, did not respond with growth inhibition, strongly suggesting a TLR4-mediated effect. RTE2 proliferation was also inhibited by transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα), whereas interferon γ (IFNγ) was a weak inhibitor. But the growth-inhibitory effect of LPS on RTE2 cells was not mediated by TNFα, TGFβ, or NO. It is concluded that besides induction of innate immune responses, LPS specifically induces growth arrest in epithelial tongue cells and keratinocytes in vitro and in mouse epidermis in a TLR4-dependent but cytokine- and NO-independent manner. ethylene diamin-tetraacetate ethyleneglycol-bis(β-aminoethylether)-tetraacetate fluorescence-activated cell sorting fetal calf serum inhibitory dose interferon γ interleukin-1β N-nitro-L-arginine methylester NG-monomethyl-L-arginine lipopolysaccharide minimal essential medium nuclear factor-κB N-nitro-L-arginine nitric oxide synthase phosphate-buffered saline trishydroxymethyl-aminomethane transforming growth factor β toll-like receptor tumor necrosis factor α Lipopolysaccharides (LPS), the main components of the outer cell wall of Gram-negative bacteria, are ranked among the most biologically active compounds (Raetz and Whitfield, 2002Raetz C.R. Whitfield C. Lipopolysaccharide endotoxins.Annu Rev Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3019) Google Scholar). Upon contact with eukaryotic organisms they evoke dramatic systemic effects that, if worst comes to worst, culminate in endotoxic shock syndrome (Lei et al., 2003Lei M.G. Gao J.J. Morrison D.C. Qureshi N. Pathogenesis of sepsis: Current concepts and emerging therapies.Mo Med. 2003; 100: 524-529PubMed Google Scholar), mainly as a consequence of complement activation and the activation of the innate immune system. The recognition of pathogen-associated molecular patterns (PAMP) such as LPS, lipoteichoic acid of Gram-positive bacteria, or other structural moieties of the outer surface of microorganisms is of major importance for the initiation of the innate immune defense (Janeway and Medzhitov, 2002Janeway C.A. Medzhitov R. Innate immune recognition.Annu Rev Immunol. 2002; 20: 197-216Crossref PubMed Scopus (5716) Google Scholar). Antigen-presenting cells such as macrophages and dendritic cells express PAMP-specific receptors that belong to the Toll-like receptor (TLR) family consisting of at least ten members in mammals (Rock et al., 1998Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. A family of human receptors structurally related to Drosophila Toll.Proc Natl Acad Sci USA. 1998; 95: 588-593Crossref PubMed Scopus (1403) Google Scholar). TLR are mammalian homologues of TOLL-receptors, originally cloned from and characterized in Drosophila. They were shown to regulate dorso-ventral patterning in the larvae (Stein et al., 1991Stein D. Roth S. Vogelsang E. Nusslein-Volhard C. The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal.Cell. 1991; 65: 725-735Abstract Full Text PDF PubMed Scopus (192) Google Scholar) and additionally to initiate anti-microbial defense (Lemaitre et al., 1996Lemaitre B. Nicolas E. Michaut L. Reichhart J.M. Hoffmann J.A. The dorsoventral regulatory casette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults.Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (2831) Google Scholar). All TLR are type I transmembrane receptors consisting of an intracellular domain exhibiting structural homologies to the IL-1 receptor required for downstream signaling and a leucine-rich extracellular domain, involved in recognition of PAMP (Akira and Takeda, 2004Akira S. Takeda K. TOLL-like receptors signalling.Nat Rev Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6246) Google Scholar). LPS is specifically recognized by TLR4 (Poltorak et al., 1998Poltorak A. He X. Smirnova I. et al.Defective LPS-signalling in C3H/HeJ and C57BL/ScCr mice: Mutations in the TLR4 gene.Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6166) Google Scholar; Qureshi et al., 1999Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. Endotoxin-tolerant mice have mutations in the Toll-like receptor 4 (Tlr 4).J Exp Med. 1999; 189: 615-625Crossref PubMed Scopus (1311) Google Scholar, Lien et al., 2000Lien E. Means T.K. Heine H. et al.Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide.J Clin Invest. 2000; 105: 497-505Crossref PubMed Scopus (670) Google Scholar). There is evidence for the existence of endogenous TLR4 ligands including heat shock protein 70 and fibrinogen (Beg, 2002Beg A.A. Endogenous ligands of Toll-like receptors: Implications for regulating inflammatory and immune responses.Trends Immunol. 2002; 23: 509-512Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Cellular responses triggered by their binding to and activation of TLR4 seem to be related to inflammation and need to be further characterized. In the LPS-TLR4-induced signaling cascade, which leads to innate immune responses, the transcriptional activation of nuclear factor-κB (NF-κB)-responsive genes is considered to be a crucial step (Akira and Takeda, 2004Akira S. Takeda K. TOLL-like receptors signalling.Nat Rev Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6246) Google Scholar). LPS effects on macrophages have been found to be mediated by pro-inflammatory cytokines such as IL-1β and tumor necrosis factor α (TNFα), as well as inducible nitric oxide synthase (iNOS), which generates NO from L-arginine (Jaffrey and Snyder, 1995Jaffrey S.R. Snyder S.H. Nitric oxide: A neural messenger.Annu Rev Cell Dev Biol. 1995; 11: 417-440Crossref PubMed Scopus (294) Google Scholar; Werling et al., 2004Werling D. Hope J.C. Howard C.J. Jungi T.W. Differential production of cytokines reactive oxygen and nitrogen by bovine macrophages and dendritic cells stimulated with Toll-like receptor agonists.Immunol. 2004; 111: 41-52Crossref PubMed Scopus (115) Google Scholar). Only recently, it was recognized that both antigen-presenting cells and epithelial cells possess the capacity to contribute to innate immunity via an activation of the TLR-family. In intestinal cells (Cario et al., 2000Cario E. Rosenberg I.M. Brandwein S.L. Beck P.L. Reinecker H.C. Podolsky D.K. Lipopolysaccharid-activates distinct signalling pathways in intestinal epithelial cell lines expressing toll-like receptors.J Immunol. 2000; 164: 966-972Crossref PubMed Scopus (623) Google Scholar) and in human keratinocytes, the expression of TLR4 and TLR2 (Kawai et al., 2002Kawai K. Shimura H. Minagawa M. Ito A. Tomiyama K. Ito M. Expression of functional toll-like receptor 2 on human epidermal keratinocytes.J Dermatol Sci. 2002; 30: 185-194Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar; Song et al., 2002Song P.I. Park Y.M. Abraham T. et al.Human keratinocytes express functional CD14 and toll-like receptor 4.J Invest Dermatol. 2002; 119: 424-432Crossref PubMed Scopus (159) Google Scholar; Pivarcsi et al., 2003Pivarcsi A. Bodai L. Rethi B. et al.Expression and function of Toll-like receptors 2 and 4 in human keratinocytes.Int Immunol. 2003; 15: 721-730Crossref PubMed Scopus (276) Google Scholar) that leads to the production of IL-1 and IL-8 has been reported (Song et al., 2002Song P.I. Park Y.M. Abraham T. et al.Human keratinocytes express functional CD14 and toll-like receptor 4.J Invest Dermatol. 2002; 119: 424-432Crossref PubMed Scopus (159) Google Scholar; Mempel et al., 2003Mempel M. Voelcker V. Kollisch G. et al.Toll-like receptor expression in human keratinocytes: Nuclear factor kappaB controlled gene activation by Staphylococcus aureus is toll-like receptor 2 but not toll-like receptor 4 or platelet activating factor receptor dependent.J Invest Dermatol. 2003; 121: 1389-1396Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Here we report that endotoxic LPS induces iNOS expression and activity in a NF-κB-dependent pathway in a rat-tongue-derived epithelial cell line, RTE2, which is shown to express TLR4 mRNA and protein similar to the tissue of origin. Furthermore, it is shown that LPS species are highly potent non-toxic inhibitors of cell proliferation, both in RTE2 cells and keratinocyte cultures as well as mouse epidermis in vivo. At least in cell culture the anti-proliferative effect of LPS seemed to be mediated directly via the NF-κB pathway, but not indirectly via endogenous production of cytokines such as transforming growth factor β (TGFβ), TNFα, and interferon γ (IFNγ) or via NO. These data may point to TLR4-mediated regulation of epithelial cell homeostasis. By RT-PCR and/or Northern blot analysis, TLR4 mRNA was detected in the rat-tongue-derived epithelial cell line RTE2 (Figure 1a, b) and in various rat tissues including tongue (Figure 1b). In addition, TLR4 protein could be demonstrated in RTE2 cell extracts by means of immunoblotting with a TLR4-specific antibody (Figure 1c) and by immune fluorescence staining of RTE2 cells (Fig S1). To investigate the function of TLR4 in RTE2 cells, the effect of the TLR4 agonist LPS (Escherichia coli 0111:B4) on the expression of iNOS, a well-known effector protein of LPS in macrophages (Aktan, 2004Aktan F. iNOS-mediated nitric oxide production and its regulation.Life Sci. 2004; 75: 639-653Crossref PubMed Scopus (858) Google Scholar), was analyzed. Two types of NOS were distinguished (Hobbs et al., 1999Hobbs A.J. Higgs A. Moncada S. Inhibition of nitric oxide synthase as a potential therapeutic target.Annu Rev Pharmacol Toxicol. 1999; 39: 191-220Crossref PubMed Scopus (539) Google Scholar), namely, a constitutive NOS (cNOS), which is ubiquitously expressed in at least two isoforms (neuronal ncNOS and endothelial ecNOS), and an inducible NOS (iNOS). Immunoblotting using NOS isozyme-specific antibodies revealed that untreated cells expressed iNOS protein in very small amounts that became visible only upon prolonged exposure of the immunoblots (Figure 2a, c). No immune signals for ecNOS and ncNOS could be detected (Figure 2a). When treated with LPS the cells responded with a strong expression of iNOS. This effect was maximal after 4–8 h and depended on the dose with an EC50 of approximately 100 ng LPS per mL (Figure 2b, c). The induction by LPS of iNOS protein correlated with a significant increase in NOS activity as measured in cell-free preparations. In contrast to cNOS, iNOS activity does not depend on Ca2+/calmodulin. In LPS-stimulated cells, an increased Ca2+-independent NOS activity was measured, which was only weakly stimulated by raising the Ca2+ concentration up to 0.6 mM (Figure 2d). In LPS-treated cells, NOS activity measured as citrulline generation or nitrite/nitrate production was suppressed by the NOS inhibitors N-nitro-L-arginine (NNA) and N-nitro-L-arginine methylester (L-NAME) (Marletta, 1994Marletta M.A. Approaches toward selective inhibition of nitric oxide synthase.J Med Chem. 1994; 37: 1899-1907Crossref PubMed Scopus (175) Google Scholar), respectively (Figure 2d, e). TLR4 has been shown to signal via NF-κB in leukocytes, resulting in the nuclear translocation of the transcription factor (Martin and Wesche, 2002Martin M.U. Wesche H. Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family.Biochim Biophys Acta. 2002; 1592: 265-280Crossref PubMed Scopus (322) Google Scholar). As expected, LPS induced the nuclear translocation of NF-κB in RTE2 cells, as shown by immunofluorescence analysis (Figure 3a–c). To examine the role of NF-κB in mediating the LPS-induced iNOS expression, the effect of an NF-κB inhibitory peptide (NF-κB-IP) was checked. This cell-permeable peptide inhibits nuclear translocation of the NF-κB active complex (Lin et al., 1995Lin Y.Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence.J Biol Chem. 1995; 270: 14255-14258Crossref PubMed Scopus (833) Google Scholar). Therefore, RTE2 cells were pre-treated with NF-κB-IP or vehicle solution for 18 h and subsequently challenged with LPS in the presence or absence of NF-κB-IP for further 6 h. Immunoblot analysis of iNOS protein revealed that LPS-stimulated iNOS expression was markedly inhibited by NF-κB-IP, whereas the treatment of control cells with NF-κB-IP alone did not show an effect (Figure 3d). Upon a single treatment of RTE2 with LPS, growth inhibition became apparent, as evident from an increased number of G0/G1 and a decreased number of S phase cells (Figure 4a). The repeated treatment of these cells with endotoxically highly active LPS species from 13 different sources resulted in a pronounced but transient inhibition of proliferation, the course and degree of which were independent of the LPS type (Figure 4b). At days 4–6, the reduction in cell number caused by 500 ng per mL LPS was comparable to the inhibitory effect of 5 ng per mL TGFβ used as a positive control. Afterwards, however, the cells became refractory to repeated endotoxin treatment, reaching the cell number of the control cultures at day 11, i.e., at the time of maximal confluency. Such an effect was not observed upon TGFβ treatment. The inhibitory effects of lipid A, and TGFβ were entirely reversible upon removal of the inhibitors (Fig S2). At doses ≥50 ng per mL LPS inhibition approached a maximum between 50% and 80%, depending on the initial plating density of RTE2 cells and the culture time. In contrast, 5 ng per mL of TGFβ caused an inhibition of up to 90% during the test period (Figure 4c). The inhibitory effect was also monitored by means of pulse-labeling of RTE2 cell DNA with [3H]thymidine (Figure 4d). As a half-maximal inhibitory concentration, 10 ng per mL, approximately 100 pM, was estimated for LPS as compared with 1 ng per mL, i.e., 80 pM, for TGFβ. DNA labeling was also suppressed by LPS (E. coli 0111) or lipid A in primary mouse keratinocytes, in the mouse keratinocyte line HEL 30, and in Swiss-3T3 fibroblasts, with IC50 values of approximately 100 pM. Primary human keratinocytes did not respond to LPS up to a concentration of 10 nM. Both bacterial and synthetic lipid A (C506) inhibited RTE2 cell proliferation (Figure 4c) and DNA labeling (Figure 4d) almost as effectively as LPS. This inhibitory effect critically depended on the lipid A structure. Thus, the biosynthetic lipid A precursor Ia (C406), which is unable to induce several endotoxin effects (Loppnow et al., 1989Loppnow H. Brade H. Dürrbaum I. Dinarello C.A. Kusumoto S. Rietschel E.T. Flad H.D. IL-1 induction-capacity of defined lipopolysaccharide partial structures.J Immunol. 1989; 142: 3229-3238PubMed Google Scholar; Beutler and Rietschel, 2003Beutler B. Rietschel E.T. Innate immune sensing and its roots: The story of endotoxin.Nat Rev Immunol. 2003; 3: 169-176Crossref PubMed Scopus (972) Google Scholar), did not inhibit RTE2 proliferation (Figure 4c). Moreover, LPS and lipid A from Rhodopseudomonas species, which showed a greatly reduced endotoxic efficacy due to an altered lipid A moiety (Mayer et al., 1989Mayer H. Bhat U.R. Masoud H. Radziejewska-Lebrecht J. Widemann C. Krauss J.H. Bacterial lipopolysaccharides.Pure Appl Chem. 1989; 61: 1271-1282Crossref Scopus (58) Google Scholar; Beutler and Rietschel, 2003Beutler B. Rietschel E.T. Innate immune sensing and its roots: The story of endotoxin.Nat Rev Immunol. 2003; 3: 169-176Crossref PubMed Scopus (972) Google Scholar), exhibited only a weak inhibitory effect (Figure 4d). A suppression by Rhodopseudomonas LPS/lipid A or C406 of the anti-proliferative effect of highly endotoxic LPS as found for other LPS effects (Raetz et al., 1991Raetz C.R.H. Ulevitch R.J. Wright S.D. Sibley A.D. Ding A. Nathan C.F. Gram-negative endotoxin: An extraordinary lipid with profound effects on eukaryotic signal transduction.FASEB J. 1991; 5: 2652-2660Crossref PubMed Scopus (457) Google Scholar) was not observed. Whether the growth-inhibitory effect of LPS depended on NF-κB, similar to the induction of iNOS, was addressed. In fact, non-toxic concentrations of NF-κB-IP significantly restored cell growth in RTE2 cells, which were exposed to LPS for 72 h, suggesting that the growth-inhibitory effect of LPS was mediated at least in part by NF-κB (Figure 5). LPS is known to induce cytokines, some of which have been shown to inhibit the growth of keratinocytes in vitro (Shipley et al., 1986Shipley G.D. Pittelkow M.R. Wille J.J. Scott R.E. Moses H.L. Reversible inhibition of normal human prokeratinocyte proliferation by type beta transforming growth factor-growth inhibitor in serum-free medium.Cancer Res. 1986; 46: 2068-2071PubMed Google Scholar; Nickoloff et al., 1988Nickoloff B.J. Riser B.L. Mitra R.S. Dixit V.M. Varani J. Inhibitory effect of gamma interferon on cultured human keratinocyte thrombospondin production, distribution, and biologic activities.J Invest Dermatol. 1988; 91: 213-218Abstract Full Text PDF PubMed Google Scholar; Pillai et al., 1989Pillai S. Bikle D.D. Ecssaler T.E. Aggarwal B.B. 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Hence, dose–response curves were determined for the three most relevant growth-inhibitory cytokines (TGFβ, TNFα, and IFN-γ) in RTE2 cells, resulting in calculated IC50 values of approximately 1 ng per mL (80 pM) for TGFβ, and 10 ng per mL (approx. 600 pM) for TNFα, and >10 μg per mL for IFN-γ. The IC50 for IFN-γ, however, was un-physiologically high (Figure 6a). Using neutralizing antibodies directed against TNFα and TGFβ we were able to rule out their possible mediator function in LPS-induced growth inhibition, since these antibodies were unable to overcome the inhibitory effect of LPS (Figure 6b, c). The treatment of control cultures with neutralizing anti-TNFα antibodies resulted in an increased DNA labeling suggesting a constitutive release of TNFα. but the TNFα levels in the supernatants of untreated and LPS-treated RTE2 cells were below the detection level of a bioassay. IL-1β, another mediator of endotoxin effects, was not included in these experiments, since this cytokine was found to stimulate rather than inhibit keratinocyte proliferation (Maas-Szabowski et al., 1999Maas-Szabowski N. Shimotoyodome A. Fusenig N.E. Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism.J Cell Sci. 1999; 112: 1843-1853Crossref PubMed Google Scholar). Furthermore, the NOS inhibitors L-NAME and NG-monomethyl-L-arginine (L-NMMA) (Marletta, 1994Marletta M.A. Approaches toward selective inhibition of nitric oxide synthase.J Med Chem. 1994; 37: 1899-1907Crossref PubMed Scopus (175) Google Scholar, see also Figure 2e), when tested at concentrations of 0.5–4 mM and 0.1–1 mM, respectively, did not significantly change the cell number in control cultures and were unable to attenuate the anti-proliferative effect of LPS (500 ng per mL) when incubated with RTE2 cells for 120 h (Figure 6d, Fig S3), showing that NO did not influence growth in RTE2 cells. Recently, LPS-induced TLR4 signaling has been reported for keratinocytes (Song et al., 2002Song P.I. Park Y.M. Abraham T. et al.Human keratinocytes express functional CD14 and toll-like receptor 4.J Invest Dermatol. 2002; 119: 424-432Crossref PubMed Scopus (159) Google Scholar). Thus, the mouse epidermis was chosen to study the effect of LPS in vivo. TLR4 mRNA was expressed in this tissue as demonstrated by RT-PCR analysis, and LPS injection was followed by a transient expression of iNOS protein within 3 h (Figure 7). As shown in the table, an intraperitoneal (i.p.) injection of LPS (E. coli) or lipid A (Salmonella) resulted in a concentration-dependent inhibition of DNA labeling in the epidermis of the NMRI mouse strain. This inhibition was observed already at doses of 30–300 μg per kg body weight, whereas symptoms of illness became apparent only at doses >1 mg per kg and lethal effects at even higher doses (≥4 mg per kg). No symptoms of inflammation were observed upon local application of LPS (E. coli 0111; 1 μg) and lipid A (salmonella 0.1 μg) onto the mouse ear (not shown). A similar effect of LPS on inhibition of DNA labeling in epidermis was measured in the LPS-responder mice of the C57Bl/ScSn strain (Table I). Treatment of TLR4-defective, LPS-resistant C57Bl/10 ScCr mice (Qureshi et al., 1999Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. Endotoxin-tolerant mice have mutations in the Toll-like receptor 4 (Tlr 4).J Exp Med. 1999; 189: 615-625Crossref PubMed Scopus (1311) Google Scholar) with LPS even at a dose of up to 1.5 mg per kg, i.p. did not cause a significant suppression of DNA labeling in epidermis in vivo.Table IEffect of an i.p. injection of LPS (Escherichia coli 0111) and lipid A (S. minnesota) on DNA labeling in mouse epidermis in vivoDNA labellingaDNA labelling was performed as described for Figure 4. 100%=58±15 cpm per μg DNA (NMRI),=124±15 (C57Bl/10 ScSn), and=119±22 cpm per μg DNA (C57Bl/10 ScCr). Each value represents the mean of six animals±SD. 4–15 μg DNA per assay. (mean cpm per μg in % of control±SD)LPS-sensitiveTreatmentDose (μg per kg)NMRIC57Bl/10ScSnLPS-resistant C57Bl/10 ScCrSolvent control0100±26100±15100±19LPS3060±5n.d.n.d.30045±1645±23105±2290033±8n.d.n.d.1500n.d.n.d.84±12Lipid A3063±11n.d.n.d.30042±18n.d.n.d.LPS, lipopolysaccharides; i.p., intraperitoneal; n.d., not done.a DNA labelling was performed as described for Figure 4. 100%=58±15 cpm per μg DNA (NMRI),=124±15 (C57Bl/10 ScSn), and=119±22 cpm per μg DNA (C57Bl/10 ScCr). Each value represents the mean of six animals±SD. 4–15 μg DNA per assay. Open table in a new tab LPS, lipopolysaccharides; i.p., intraperitoneal; n.d., not done. In this study, we present data showing that epithelial RTE2 cells from tongue express TLR4 mRNA and functional protein. Stimulating the cells with LPS, a well-documented bacterial ligand of this receptor, resulted in the time- and concentration-dependent induction of iNOS protein. As expected from experiments with LPS-induced iNOS in macrophages (Aktan, 2004Aktan F. iNOS-mediated nitric oxide production and its regulation.Life Sci. 2004; 75: 639-653Crossref PubMed Scopus (858) Google Scholar), this induction occurred in an NF-κB-dependent manner, because iNOS-expression was strongly inhibited by an inhibitor peptide of NF-κB. ecNOS or ncNOS protein were detected neither in control, nor in LPS-stimulated RTE2 cells. In addition, the increased iNOS expression levels correlated with an increased Ca2+-independent NOS activity. Thus, the elevated NO concentrations in LPS-treated cultures were due to iNOS activity. Macrophage-derived NO has been shown to inhibit DNA synthesis in microorganisms due to inactivation of FeS-containing ribonucleotide reductase (Hobbs et al., 1999Hobbs A.J. Higgs A. Moncada S. Inhibition of nitric oxide synthase as a potential therapeutic target.Annu Rev Pharmacol Toxicol. 1999; 39: 191-220Crossref PubMed Scopus (539) Google Scholar). Our data suggest that epithelial tongue cells may contribute to microbial defense, similar to immune and epithelial cells of the epidermis (Pivarcsi et al., 2003Pivarcsi A. Bodai L. Rethi B. et al.Expression and function of Toll-like receptors 2 and 4 in human keratinocytes.Int Immunol. 2003; 15: 721-730Crossref PubMed Scopus (276) Google Scholar). Recently, human keratinocytes in vitro have been shown to possess an NF-κB-dependent killing activity against the human pathogen Candida albicans, which was discussed to be mediated by TLR2 and TLR4 (Pivarcsi et al., 2003Pivarcsi A. Bodai L. Rethi B. et al.Expression and function of Toll-like receptors 2 and 4 in human keratinocytes.Int Immunol. 2003; 15: 721-730Crossref PubMed Scopus (276) Google Scholar). It remains to be clarified whether NO concentrations in LPS-treated RTE2 cells are sufficiently high to kill Gram-negative bacteria upon direct contact in vitro and particularly in vivo. In keratinocytes, the activation of NF-κB has been linked to a cell cycle arrest and a hypoproliferative state of epidermis in vivo (Kaufmann and Fuchs, 2000Kaufmann C.K. Fuchs E. It's got you covered: NF-κB in the epidermis.J Cell Biol. 2000; 149: 999-1004Crossref PubMed Scopus (101) Google Scholar) rather than to a stimulation of proliferation as was described for immune cells (Gerondakis et al., 1998Gerondakis S. Grumont R. Rourke I. Grossmann M. The regulation and roles of Rel/NF-kappa B transcription factors during lymphocyte activation.Curr Opin Immunol. 1998; 3: 353-359Crossref Scopus (179) Google Scholar). Accordingly, LPS indeed impaired cell cycling and growth in RTE2 cells, as shown here. This occurred at least in part along an NF-κB-dependent pathway, since inhibition of NF-κB activation by the NF-κB-IP (Lin et al., 1995Lin Y.Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence.J Biol Chem. 1995; 270: 14255-14258Crossref PubMed Scopus (833) Google Scholar) restored cell growth in LPS-treated cells. Currently, other TLR4-coupled signaling pathways cannot be ruled out as being involved in the observed anti-proliferative effect of LPS. Besides the activation of the IKK (inhibitor of nuclear factor-κB-kinase) complex leading to NF-κB activation, there is evidence that LPS may stimulate other kinase pathways (Akira and Takeda, 2004Akira S. Takeda K. TOLL-like receptors signalling.Nat Rev Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6246) Google Scholar; Huang et al., 2004Huang Q. Yang J. Lin Y. Walker C. Cheng J. Liu Z.G. Su B. Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3.Nat Immunol. 2004; 5: 98-103Crossref PubMed Scopus (220) Google Scholar). LPS turned out to be a potent inhibitor of RTE2 cell proliferation in vitro, exhibiting IC50 values of approximately 10 ng per mL (100 pM), i.e., approximating the efficacy of TGFβ (1 ng per mL corresponding to 80 pM), the most potent inhibitor of keratinocyte proliferation in cell culture. This is a first report on an anti-proliferative effect of LPS. The inhibition of cell proliferation was reversible and not accompanied by apoptosis, the rate of which was determined as the sub-2n-DNA content by FACS analysis (data not shown). The anti-proliferative effect of LPS on RTE2 cells was specifically determined by the lipid A moiety, i.e., correlated with the endotoxin
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