Toll-like receptor 2 mediates early inflammation by leptospiral outer membrane proteins in proximal tubule cells
2006; Elsevier BV; Volume: 69; Issue: 5 Linguagem: Inglês
10.1038/sj.ki.5000119
ISSN1523-1755
AutoresChih‐Wei Yang, Cheng Chieh Hung, Ming‐Kung Wu, Ya‐Chung Tian, C.-T. Chang, Michael Pan, Alain Vandewalle,
Tópico(s)Immune Response and Inflammation
ResumoTubulointerstitial nephritis is a cardinal renal manifestation in leptospirosis and LipL32, the major lipoprotein component of leptospiral outer membrane proteins (OMPs), induces a robust inflammatory response in cultured renal proximal tubule cells through a nuclear factor-κB-related pathway. Here, we investigated whether Toll-like receptor (TLR), known to play a pivotal role in innate immunity, could mediate the inflammatory response induced by leptospiral OMPs in renal proximal tubule cells. TLR expression was analyzed by flow cytometry and indirect immunofluorescence in cultured mouse proximal tubule (pyruvate kinase simian virus 40-proximal straight (PKSV-PR)) cells. Reverse transcription-competitive polymerase chain reaction and enzyme-linked immunosorbent assay were undertaken to analyze the inducible effects of inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1 also termed CCL2) by pathogenic and non-pathogenic leptospiral OMPs and recombinant lipoproteins in either PKSV-PR cells or TLR-transfected human embryonic kidney (HEK) 293 cells. Anti-TLR antibodies were used for blocking experiments. Leptospira santarosai serovar Shermani OMPs and LipL32 induced a significant increase in TLR2 but not TLR4 expression in PKSV-PR cells. The increase in iNOS and CCL2/MCP-1 mRNA expressions could be prevented by an anti-TLR2 antibody, but not by an anti-TLR4 antibody. Furthermore, leptospiral OMPs stimulated both CCL2/MCP-1 mRNA and secreted protein in transfected HEK 293 cells with a TLR2-expressing plasmid, but had no effect in cells with a TLR4-expressing plasmid. In conclusion, these findings indicate that the stimulation of iNOS and CCL2/MCP-1 caused by pathogenic leptospiral OMPs, in particular LipL32, in proximal tubule cells requires TLR2 for the early inflammatory response. Tubulointerstitial nephritis is a cardinal renal manifestation in leptospirosis and LipL32, the major lipoprotein component of leptospiral outer membrane proteins (OMPs), induces a robust inflammatory response in cultured renal proximal tubule cells through a nuclear factor-κB-related pathway. Here, we investigated whether Toll-like receptor (TLR), known to play a pivotal role in innate immunity, could mediate the inflammatory response induced by leptospiral OMPs in renal proximal tubule cells. TLR expression was analyzed by flow cytometry and indirect immunofluorescence in cultured mouse proximal tubule (pyruvate kinase simian virus 40-proximal straight (PKSV-PR)) cells. Reverse transcription-competitive polymerase chain reaction and enzyme-linked immunosorbent assay were undertaken to analyze the inducible effects of inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1 also termed CCL2) by pathogenic and non-pathogenic leptospiral OMPs and recombinant lipoproteins in either PKSV-PR cells or TLR-transfected human embryonic kidney (HEK) 293 cells. Anti-TLR antibodies were used for blocking experiments. Leptospira santarosai serovar Shermani OMPs and LipL32 induced a significant increase in TLR2 but not TLR4 expression in PKSV-PR cells. The increase in iNOS and CCL2/MCP-1 mRNA expressions could be prevented by an anti-TLR2 antibody, but not by an anti-TLR4 antibody. Furthermore, leptospiral OMPs stimulated both CCL2/MCP-1 mRNA and secreted protein in transfected HEK 293 cells with a TLR2-expressing plasmid, but had no effect in cells with a TLR4-expressing plasmid. In conclusion, these findings indicate that the stimulation of iNOS and CCL2/MCP-1 caused by pathogenic leptospiral OMPs, in particular LipL32, in proximal tubule cells requires TLR2 for the early inflammatory response. Leptospirosis is a re-emerging and widespread zoonosis in many tropical regions.1.Pereira M.M. Matsuo M.G. Bauab A.R. et al.A clonal subpopulation of Leptospira interrogans sensu stricto is the major cause of leptospirosis outbreaks in Brazil.J Clin Microbiol. 2000; 38: 450-452PubMed Google Scholar, 2.Farr R.W. Leptospirosis.Clin Infect Dis. 1995; 21: 1-6Crossref PubMed Scopus (329) Google Scholar In the infected host, pathogenic leptospires can disseminate hematogeneously and invade kidneys, which represents one of the most frequently affected organs, and through the effects of leptospiral endotoxins and immunologic responses favor the development of tubulointerstitial nephritis.2.Farr R.W. Leptospirosis.Clin Infect Dis. 1995; 21: 1-6Crossref PubMed Scopus (329) Google Scholar, 3.Sitprija V. Pipatanagul V. Mertowidjojo K. et al.Pathogenesis of renal disease in leptospirosis: clinical and experimental studies.Kidney Int. 1980; 17: 827-836Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 4.Yang C.W. Wu M.S. Pan M.J. Leptospirosis renal disease.Nephrol Dial Transplant. 2001; 16: 73-77Crossref PubMed Scopus (128) Google Scholar Leptospiral outer membrane proteins (OMPs) and lipopolysaccharide (LPS) are the major antigens that confer immunity to leptospires and are thought to be involved in host–pathogen interactions.5.Vinh T. Adler B. Faine S. Ultrastructure and chemical composition of lipopolysaccharide extracted from Leptospira interrogans serovar copenhageni.J Gen Microbiol. 1986; 132: 103-109PubMed Google Scholar We have previously shown that pathogenic leptospiral OMPs activate the expression of proinflammatory genes related to tubulointerstitial nephritis in mouse renal medullary thick ascending limb cells.6.Yang C.W. Wu M.S. Pan M.J. et al.Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells.J Am Soc Nephrol. 2000; 11: 2017-2026Crossref PubMed Google Scholar We have also shown that LipL32, the major leptospiral outer membrane lipoprotein, can induce a robust inflammatory response in cultured mouse renal proximal tubule cells.7.Yang C.W. Wu M.S. Pan M.J. et al.The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.J Am Soc Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (94) Google Scholar The identification of virulent OMP components have represented an important step in the understanding of the pathogenesis of leptospirosis tubulointerstitial nephritis.4.Yang C.W. Wu M.S. Pan M.J. Leptospirosis renal disease.Nephrol Dial Transplant. 2001; 16: 73-77Crossref PubMed Scopus (128) Google Scholar, 8.Barnett J.K. Barnett D. Bolin C.A. et al.Expression and distribution of leptospiral outer membrane components during renal infection of hamsters.Infect Immun. 1999; 67: 853-861Crossref PubMed Google Scholar Microbial invasion of the host triggers a series of events designed to control and eventually eliminate the infection. A number of pattern recognition receptors, which recognize defined conserved microbial structures or products of microbial metabolism, have been shown to play a key role in innate immunity. Among them, the family of Toll-like receptors (TLRs) participate in the innate defense against a variety of bacterial infections by initiating acute inflammatory responses.9.Medzhitov R. Preston-Hurlburt P. Janeway Jr, C.A. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature. 1997; 388: 394-397Crossref PubMed Scopus (4208) Google Scholar, 10.Takeda K. Kaisho T. Akira S. Toll-like receptors.Annu Rev Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4551) Google Scholar Recent studies have also shown that renal tubule cells express TLRs,11.Tsuboi N. Yoshikai Y. Matsuo S. et al.Roles of toll-like receptors in C–C chemokine production by renal tubular epithelial cells.J Immunol. 2002; 169: 2026-2033Crossref PubMed Scopus (214) Google Scholar mainly TLR2 and TLR4, that are upregulated following the reperfusion of ischemic kidneys.12.Wolfs T.G. Buurman W.A. van Schadewijk A. et al.In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation.J Immunol. 2002; 168: 1286-1293Crossref PubMed Scopus (379) Google Scholar TLR4 recognizes LPS of Gram-negative bacteria,13.Chow J.C. Young D.W. Golenbock D.T. et al.Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction.J Biol Chem. 1999; 274: 10689-10692Crossref PubMed Scopus (1540) Google Scholar whereas TLR2 provides responsiveness to bacterial lipoproteins and peptidoglycan of Gram-positive bacteria.14.Takeuchi O. Hoshino K. Kawai T. et al.Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components.Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2695) Google Scholar, 15.Yoshimura A. Lien E. Ingalls R.R. et al.Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2.J Immunol. 1999; 163: 1-5PubMed Google Scholar, 16.Lien E. Sellati T.J. Yoshimura A. et al.Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.J Biol Chem. 1999; 274: 33419-33425Crossref PubMed Scopus (773) Google Scholar Recognition of bacterial components by their respective TLRs will allow the activation of the nuclear factor-κB pathway and the stimulation of chemokines and cytokines production.17.Re F. Strominger J.L. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells.J Biol Chem. 2001; 276: 37692-37699Crossref PubMed Scopus (546) Google Scholar Although leptospirosis is an important cause of acute renal failure, the mechanism of renal dysfunction caused by this microorganism is not fully understood. We have shown that leptospiral OMPs and the main antigenic component, LipL32 lipoprotein, certainly play a role in the pathogenesis of leptospirosis renal disease by mediating the activation of the nuclear factor-κB-associated pathways and the release of cytokines.6.Yang C.W. Wu M.S. Pan M.J. et al.Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells.J Am Soc Nephrol. 2000; 11: 2017-2026Crossref PubMed Google Scholar, 7.Yang C.W. Wu M.S. Pan M.J. et al.The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.J Am Soc Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (94) Google Scholar Werts et al.18.Werts C. Tapping R.I. Mathison J.C. et al.Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism.Nat Immunol. 2001; 2: 346-352Crossref PubMed Scopus (550) Google Scholar have provided the first demonstration that TLR2 is required for the activation of macrophages by leptospiral outer membrane constituents. Because this TLR is highly expressed in renal tubule cells, the question arises as to whether TLR2 participates in the inflammatory response triggered by pathogenic leptospires in renal proximal epithelial tubule cells. To answer this question, experiments were carried out to analyze the expression of TLR2 stimulated by leptospiral OMPs and its participation in the induction of the inflammatory response caused by pathogenic leptospiral OMPs in cultured mouse proximal tubule PKSV-PR cells19.Cartier N. Lacave R. Vallet V. et al.Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene.J Cell Sci. 1993; 104: 695-704Crossref PubMed Google Scholar and in human embryonic kidney (HEK) 293 cells transiently expressing TLR2. The results of this study indicate that OMPs from pathogenic leptospires and purified LipL32 increased the expression of TLR2 and stimulated the release of the monocyte chemoattractant protein-1 (CCL2/MCP-1) in renal proximal tubule cells. These results strongly suggest thus that TLR2 initiate the inflammatory response caused by pathogenic leptospires in renal tubule epithelial cells. We first analyzed the effects of leptospiral OMPs on TLR2 and TLR4 expression in cultured mouse PKSV-PR cells by using flow cytometry. Adding 0.2 μg/ml OMPs extracted from Leptospira santarosai serovar Shermani for 48 h increased TLR2 expression by 28% as compared to untreated cells (P<0.05). In contrast, the stimulation by leptospiral OMPs did not induce any increase in the expression of TLR4 (Figure 1a). Consistent with these results, indirect immunofluorescence studies revealed that leptospiral OMPs induced a marked redistribution of the cytoplasmic TLR2 at the cell peripheries of proximal tubule cells, without affecting the weak cytoplasmic TLR4 staining (Figure 1b). The levels of mRNA expression for TLR2, TLR4, and β-actin were then analyzed by using reverse transcriptase (RT)-competitive polymerase chain reaction (PCR). Incubation of PKSV-PR cells with 0.2, 0.3, 0.5, or 1 μg/ml leptospiral OMPs for 48 h induced significant 1.8-, 1.8-, 1.9-, and 1.8-fold increase, respectively, in the level of TLR2/β-actin mRNA expression as compared to that of untreated cells. Conversely, no changes in TLR4/β-actin mRNA levels were observed when incubating cells with increasing concentrations of leptospiral OMPs (Figure 2a). The levels of TLR2 and β-actin mRNA expression were further analyzed with the two recombinant LipL32 and LipL41 leptospiral lipoproteins. Incubating cells with increasing concentrations (0.6–12 ng/ml) of LipL32 for 48 h induced significant 1.5- to 1.9-fold increases in the level of TLR2/β-actin mRNA expression as compared to that of untreated cells. Conversely, no changes in TLR2/β-actin mRNA levels were observed when incubating cells with increasing concentrations of recombinant LipL41 (Figure 2b). To ensure that the observed increase in TLR2 expression was directly related to pathogenic leptospiral OMPs, additional experiments were carried out using OMPs extract from another pathogenic serovar Leptospira interrogans serovar Bratislava, and compared to that from the non-pathogenic serovar Leptospira biflexa serovar Patoc. PKSV-PR cells were incubated with these different OMPs extracts (0.2 μg/ml) for 48 h. The results from RT competitive-PCR revealed that both OMPs extracted from L. santarosai serovar Shermani and L. interrogans serovar Bratislava induced significant (P<0.05) 1.8- and 1.6-fold increases, respectively, in the level of TLR2/β-actin mRNA, whereas OMPs from L. biflexa serovar Patoc did not affect the levels (Figure 3a). Overall these results suggested that leptospiral OMPs activate the innate immune receptor TLR2 in renal proximal tubule cells. To answer whether LipL32, a major component of pathogenic leptospires, could per se stimulate the expression of TLR2 in renal proximal tubule cells, cultured PKSV-PR cells were therefore incubated with the recombinant LipL32 alone or with antisera raised against LipL32 (anti-LipL32). The recombinant LipL32 (2.4 ng/ml for 48 h) stimulated by 2.7-fold the level of TLR2 mRNA expression in confluent cultures of PKSV-PR cells. Co-incubation of the cells with recombinant LipL32 and the anti-LipL32 antibody partially prevented the increase in TLR2 expression: the anti-LipL32 antibody significantly reduced (P<0.05) by 50% the expression of TLR2 mRNA (Figure 3b). As control, replacement of the anti-LipL32 antibody by preimmune serum had almost no effect on the induction of TLR2 mRNA expression induced by LipL32. Altogether, these results indicate that LipL32 can directly affect TLR2 gene expression in renal proximal tubule cells. Adding L. santarosai serovar Shermani OMPs (0.2 μg/ml for 48 h) to cultured PKSV-PR cells significantly increased the expression of inducible nitric oxide synthase (iNOS) (× 6.3-fold, P<0.01) and CCL2/MCP-1 (× 2.8-fold, P<0.05) mRNAs as compared to those measured in untreated cells. PKSV-PR cells were further incubated with L. santarosai serovar Shermani OMPs alone or with antisera raised against TLR2 and/or TLR4 (gifts from Tularik Inc., San Francisco, CA, USA) to evaluate the respective roles of TLR2 and TLR4. Preincubation of the cells with the anti-TLR2 antibody significantly reduced (P<0.05) by 46 and 69% the increase in iNOS and CCL2/MCP-1 mRNAs, respectively, caused by leptospiral OMPs. On the other hand, the anti-TLR4 antibody did not significantly reduce the levels of iNOS and CCL2/MCP-1 stimulated by leptospiral OMPs. Furthermore, preincubating cells with the anti-TLR4 antibody plus the anti-TLR2 antibody did not further reduce the increased levels of iNOS and CCL2/MCP-1 mRNAs caused by leptospiral OMPs. These findings indicate thus that TLR2, but not TLR4, is specifically involved in the induction of iNOS and CCL2/MCP-1 caused by leptospiral OMPs (Figure 4a). Cultured renal proximal tubule cells were then further incubated with the recombinant LipL32 (2.4 ng/ml) and the anti-TLR2 antiserum. Recombinant LipL32 significantly increased the mRNA levels of iNOS by 2.0-fold (P<0.05) and CCL2/MCP-1 by 1.7-fold (P<0.05), respectively. In contrast, preincubation of the cells with the anti-TLR2 antiserum prevented (P<0.05) the increase in iNOS and CCL2/MCP-1 caused by LipL32 by 52 and 35%, respectively. Again, the replacement of the anti-TLR2 antibody by the preimmune serum had almost no inhibitory action on LipL32-stimulated iNOS or CCL2/MCP-1 expressions (Figure 4b). These results strongly suggest that TLR2 mediates the induction of tubulointerstitial nephritis-related mRNAs caused by leptospiral OMPs or LipL32. Confluent PKSV-PR cells grown in serum-free medium for 24 h were then incubated with leptospiral OMPs (0.2 μg/ml) for various periods. Expression of TLR2, CCL2/MCP-1, and β-actin mRNAs was analyzed by competitive RT-PCR as a function of time. The TLR2 mRNA was activated within 2 h of stimulation followed by the increase of CCL2/MCP-1 mRNA and a progressive increase of CCL2/MCP-1 secreted protein measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatant (Figure 5). To further assess the role of TLR2 in the induction of CCL2/MCP-1 caused by leptospiral OMPs, HEK 293 cells normally lacking TLR2 and TLR4 were transiently transfected with TLR-expressing plasmids. We first checked that HEK 293 cells transfected with an empty vector do not express TLR2 or TLR4. In contrast, strong TLR2 or TLR4 mRNA expressions were detected in TLR2- or TLR4-transfected cells, respectively. Interestingly, leptospiral OMPs only increased the expression of CCL2/MCP-1 mRNA levels in TLR2-transfected cells but not in TLR4-transfected cells or in empty vector-transfected cells (Figure 6a and b). Additional experiments were then carried out on HEK 293 cells transiently transfected with TLR2- or TLR4-expressing plasmids or with both plasmids and incubated with two concentrations (0.2 and 0.4 μg/ml) of leptospiral OMPs. In both cases, OMPs significantly increased (P<0.05) the CCL2/MCP-1 mRNA expression by 3.1- to 3.4-fold, respectively (Figure 7a). Consistent with these results, 0.2 and 0.4 μg/ml OMPs also significantly increased by 1.9- and 2.1-fold, respectively, the secretion of the CCL2/MCP-1 protein recovered in medium supernatants from HEK 293 cells transfected with the TLR2-expressing plasmid (Figure 7b). Conversely, HEK 293 cells transfected with the TLR4-expressing plasmid remained unresponsive to leptospiral OMPs. Additionally, double transfection of TLR2 and TLR4-expressing plasmids did not result in further increase in CCL2/MCP-1 expression (mRNA and protein) as compared to that achieved with the TLR2-expressing plasmid alone (Figure 7a and b). Thus, these experiments provide additional evidence that the innate immunity TLR2 pathway mediates the induction of CCL2/MCP-1 caused by pathogenic leptospiral OMPs in proximal tubule epithelial cells. The results of this study provide lines of evidence that TLR2 mediates the early inflammatory response caused by pathogenic leptospires in renal proximal tubule cells. At least 11 TLRs have been identified so far in immune cells and in a variety of epithelial cells.20.Cario E. Brown D. 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Buurman W.A. van Schadewijk A. et al.In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation.J Immunol. 2002; 168: 1286-1293Crossref PubMed Scopus (379) Google Scholar, 24.Anders H.J. Banas B. Schlondorff D. Signaling danger: Toll-like receptors and their potential roles in kidney disease.J Am Soc Nephrol. 2004; 15: 854-867Crossref PubMed Scopus (310) Google Scholar TLR2 and TLR-4, the main receptors of Gram-positive and -negative bacteria,25.Medzhitov R. Toll-like receptors and innate immunity.Nat Rev Immunol. 2001; 1: 135-145Crossref PubMed Scopus (3030) Google Scholar, 26.Yang R.B. Mark M.R. Gray A. et al.Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling.Nature. 1998; 395: 284-288Crossref PubMed Scopus (1074) Google Scholar are constitutively expressed in both proximal and distal tubular renal epithelial cells in vivo.27.Kirschning C.J. Schumann R.R. TLR2: cellular sensor for microbial and endogenous molecular patterns.Curr Top Microbiol Immunol. 2002; 270: 121-144Crossref PubMed Scopus (168) Google Scholar Renal inflammation caused by warm ischemia–reperfusion was shown to cause a marked increase in TLR2 and -4 mRNA synthesis dependent on the action of tumor necrosis factor-α and interferon-γ.12.Wolfs T.G. Buurman W.A. van Schadewijk A. et al.In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation.J Immunol. 2002; 168: 1286-1293Crossref PubMed Scopus (379) Google Scholar In the kidney, TLR4 is required for the resistance to Gram-negative bacteria, as assessed by the persistence of Gram-negative bacteria in experimental models of pyelonephritis induced in C3H/HeJ mice harboring a non-functional TLR4, when compared to the rapid renal clearance of bacteria in wild-type counterparts.28.Hagberg L. Hull R. Hull S. et al.Difference in susceptibility to Gram-negative urinary tract infection between C3H/HeJ and C3H/HeN mice.Infect Immun. 1984; 46: 839-844PubMed Google Scholar TLR2 has a broader specificity, as it recognizes microbial components from bacteria, mycobacteria, mycoplasma, and also spirochetes. Werts et al.18.Werts C. Tapping R.I. Mathison J.C. et al.Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism.Nat Immunol. 2001; 2: 346-352Crossref PubMed Scopus (550) Google Scholar have demonstrated that the activation of macrophages by leptospiral LPS requires CD14 and TLR2. The components of the leptospiral outer membrane certainly determine the virulence of pathogenic leptospires. Many of the most abundant proteins in the spirochetal outer membrane are lipoproteins that participate in the pathogenesis of tubulointerstitial nephritis.8.Barnett J.K. Barnett D. Bolin C.A. et al.Expression and distribution of leptospiral outer membrane components during renal infection of hamsters.Infect Immun. 1999; 67: 853-861Crossref PubMed Google Scholar, 29.Haake D.A. Chao G. Zuerner R.L. et al.The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection.Infect Immun. 2000; 68: 2276-2285Crossref PubMed Scopus (372) Google Scholar Previous studies have shown that the leptospiral outer membrane has a relatively complex protein profile.30.Nunes-Edwards P.L. Thiermann A.B. Bassford P.J.J. Stamm L.V. Identification and characterization of the protein antigens of Leptospira interrogans serovar hardjo.Infect Immun. 1985; 48: 492-497PubMed Google Scholar, 31.Zuerner R.L. Knudtson W. Bolin C.A. Trueba G. Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona.Microb Pathog. 1991; 10: 311-322Crossref PubMed Scopus (67) Google Scholar, 32.Brown J.A. LeFebvre R.B. Pan M.J. Protein and antigen profiles of prevalent serovars of Leptospira interrogans.Infect Immun. 1991; 59: 1772-1777PubMed Google Scholar Outer membrane antigenic proteins along with leptospiral LPS are expressed in infected kidneys,8.Barnett J.K. Barnett D. Bolin C.A. et al.Expression and distribution of leptospiral outer membrane components during renal infection of hamsters.Infect Immun. 1999; 67: 853-861Crossref PubMed Google Scholar suggesting their roles in the induction and persistence of leptospirosis tubulointerstitial nephritis. The OMP extraction method used in this study enhanced the harvest of lipoprotein components. Although one cannot exclude a role for contaminant leptospiral LPS in OMPs preparations, it remains that glycolipoprotein are responsible for major leptospiral toxicity.33.Vinh T. Adler B. Faine S. Glycolipoprotein cytotoxin from Leptospira interrogans serovar copenhageni.J Gen Microbiol. 1986; 132: 111-123PubMed Google Scholar In this line, we had previously shown that heat and proteinase K digestion of leptospiral OMP extracts significantly reduced their inducible action on iNOS and CCL2/MCP-1 expressions in renal tubule cells.6.Yang C.W. Wu M.S. Pan M.J. et al.Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells.J Am Soc Nephrol. 2000; 11: 2017-2026Crossref PubMed Google Scholar Moreover, polymyxin B, which inhibits the LPS activity, did no significantly impair the inducible action of leptospiral OMPs (data not shown). To further rule out any participation of contaminant LPS from Escherichia coli preparations in experiments using LipL32 and LipL41, the recombinant lipoproteins were treated with endotoxin-removing gel. LipL32, but not LipL41, stimulated in a dose-dependent manner the expression of TLR2 mRNA in proximal tubule cells. These results are in agreement with a previous study, which showed that LipL41 did not stimulate iNOS or CCL2/MCP-1 expression in proximal tubule cells.7.Yang C.W. Wu M.S. Pan M.J. et al.The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.J Am Soc Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (94) Google Scholar Overall, these results strongly suggest that LipL32 represents the main leptospiral outer membrane lipoprotein responsible for the induction of the proinflammatory response in renal tubule cells. Here, we also show that the neutralizing anti-TLR2 antibody used, but not the anti-TLR4 antibody, inhibits the inducible action of leptospiral OMPs on the iNOS and CCL2/MCP-1 downstream products. The results from TLR transfection studies also provide additional evidence that TLR2 is the innate receptor for leptospiral OMPs in renal proximal tubule cells. In HEK 293 cells that do not express TLRs, the transient expression of TLR2 allows leptospiral OMPs to increase the expression levels of CCL2/MCP-1. In contrast, no stimulation of CCL2/MCP-1 could be detected in TLR4-transfected HEK 293 cells. Thus, these results strongly suggest that TLR2 mediates the induction of the inflammatory response of renal epithelial cells caused by pathogenic leptospires. This is the first demonstration that pathogenic leptospires, which specifically invade and reside in the kidney, may trigger an innate immune response through TLR2-dependent pathway in proximal tubule cells. Chemotactic chemokines responsible for the recruitment of leukocytes to the site of inflammation are upregulated in a variety of glomerular and interstitial renal injuries.34.Tang W.W. Qi M. Warren J.S. Van G.Y. Chemokine expression in experimental tubulointerstitial nephritis.J Immunol. 1997; 159: 870-876PubMed Google Scholar, 35.Lloyd C.M. Dorf M.E. Proudfoot A. et al.Role of MCP-1 and RANTES in inflammation and progression to fibrosis during murine crescentic nephritis.J Leukocyte Biol. 1997; 62: 676-680Crossref PubMed Scopus (92) Google Scholar We have previously shown that leptospiral OMPs and LipL32 stimulate the expression of CCL2/MCP-1 and RANTES (regulated upon activation normal T-cell expressed and secreted) in association with increased generation of iNOS and tumor necrosis factor-α.6.Yang C.W. Wu M.S. Pan M.J. et al.Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells.J Am Soc Nephrol. 2000; 11: 2017-2026Crossref PubMed Google Scholar, 7.Yang C.W. Wu M.S. Pan M.J. et al.The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.J Am Soc Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (94) Google Scholar These findings have revealed that leptospiral OMPs are able to provoke an inflammatory response in injured renal tubule cells and thereby permit the recruitment of infiltrating cells into the renal interstitium.6.Yang C.W. Wu M.S. Pan M.J. et al.Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells.J Am Soc Nephrol. 2000; 11: 2017-2026Crossref PubMed Google Scholar, 7.Yang C.W. Wu M.S. Pan M.J. et al.The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.J Am Soc Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (94) Google Scholar Recent studies have also shown that the expression of TLR2 in human HEK 293 cells mediates the activation of nuclear factor-κB and enables stress-activated mitogen-activated protein kinase p38 phosphorylation in response to LPS, membrane lipoproteins or Gram-positive bacterial products.36.Vasselon T. Hanlon W.A. Wright S.D. Detmers P.A. Toll-like receptor 2 (TLR2) mediates activation of stress-activated MAP kinase p38.J Leukocyte Biol. 2002; 71: 503-510PubMed Google Scholar In accordance with these findings, we found that leptospiral OMPs stimulate the phosphorylated mitogen-activated protein kinase p38 in proximal tubule cells (manuscript in preparation). Together with the present findings, these results therefore support the hypothesis that pathogenic leptospiral OMPs conceivably initiate tubulointerstitial nephritis through the components contained in the outer membrane that binds to TLR2 in proximal tubule cells. The present results also show that TLR4 may not be required in the recognition of leptospiral OMPs. These results are in accordance with previous studies which showed that the LPS isolated from L. interrogans as well as LipL32 stimulate the production of IL-8 and tumor necrosis factor-α in human macrophages via a TLR2-mediated pathway, and that TLR2-deficient mice are insensitive to leptospiral LPS.18.Werts C. Tapping R.I. Mathison J.C. et al.Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism.Nat Immunol. 2001; 2: 346-352Crossref PubMed Scopus (550) Google Scholar Koizumi et al.37.Koizumi N. Watanabe H. Identification of a novel antigen of pathogenic Leptospira spp. that reacted with convalescent mice sera.J Med Microbiol. 2003; 52: 585-589Crossref PubMed Scopus (28) Google Scholar have also shown that TLR4-deficient C3H/HeJ mice remain sensitive to leptospiral infection, suggesting that TLR4 is not a major mediator in leptospirosis infection. Consistent with these studies, the present work provides additional evidence that TLR2 represents the main pattern recognition receptor recognizing pathogenic leptospiral OMPs. In conclusion, the present findings provide a basis for understanding the molecular mechanisms controlling the innate immune response caused by leptospirosis in renal tubule cells, which will serve for future studies devoted to the understanding of the pathogenesis of microorganism-induced tubulointerstitial nephritis. Two frequently encountered pathogenic leptospires serovar L. santarosai serovar Shermani (ATCC number 43286™) and L. interrogans serovar Bratislava (ATCC number 23578™), and a non-pathogenic L. biflexa serovar Patoc (ATCC number 23582™), were obtained from ATCC (Rockville, MD, USA) and grown in 10% Ellinghausen McCullough Johnson Harris leptospiral enrichment medium (Difco, Detroit, MI, USA). The OMPs were extracted with 1% Triton X-114 and more than 90% of OMPs were extracted in the Triton X-114 detergent phase.6.Yang C.W. Wu M.S. Pan M.J. et al.Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells.J Am Soc Nephrol. 2000; 11: 2017-2026Crossref PubMed Google Scholar Standard recombinant DNA procedures were undertaken to clone LipL32 and LipL41 as described.7.Yang C.W. Wu M.S. Pan M.J. et al.The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.J Am Soc Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (94) Google Scholar LipL32, but not LipL41, can induce a robust inflammatory response in cultured mouse proximal tubule cells.7.Yang C.W. Wu M.S. Pan M.J. et al.The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.J Am Soc Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (94) Google Scholar Thus, LipL41 was used to compare the effect of LipL32 in TLR activation. The recombinant proteins were passed through an endotoxin-removing gel, Detoxigel (Pierce Biotechnology Inc., Rockford, IL, USA), to remove possible contamination by LPS, and controlled by the Limulus Amebocyte Lysate assay. The PKSV-PR cells employed herein were derived from isolated late proximal tubules (PR) dissected from the kidney of an L-PK/Tag1 transgenic mouse as specified earlier.19.Cartier N. Lacave R. Vallet V. et al.Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene.J Cell Sci. 1993; 104: 695-704Crossref PubMed Google Scholar Experiments were undertaken on exponentially growing or confluent cells between the 40 and 55th passages. Cells were shifted to a serum-free medium 24 h before adding OMP extract or recombinant lipoproteins to the cell culture medium for 48 h. Total RNA was extracted to undergo RT-PCR, and supernatant was collected to measure the amounts of protein. All measurements were made at least in triplicate. PKSV-PR cells (1 × 106/ml) were incubated or not with leptospiral OMPs for 48 h. After washing, 2 × 105 cells were resuspended in 200 μl phosphate-buffered saline containing 1% bovine serum albumin and 0.01% sodium azide. The cells were preincubated with 1 μg/ml of Fc block (BD PharMingen, San Diego, CA, USA) before incubating them with a monoclonal rat anti-mouse TLR2 antibody or a monoclonal rat anti-mouse TLR4 antibody (eBioscience, San Diego, CA, USA). Similar experiments were performed using a rat immunoglobulin G2a isotype control antibody. Cells were washed and incubated with a fluorescein isothiocyanate-labeled anti-rat immunoglobulin G antibody and analyzed using a FACS Calibur System (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) flow cytometer. The data were analyzed using Win MDI 2.8 software. Confluent cultures of PKSV-PR cells incubated or not with leptospiral OMP were first incubated with rabbit anti-mouse TLR2 or rat monoclonal anti-mouse TLR4 antibodies (eBioscience, San Diego, CA, USA) followed by fluorescein isothiocyanate species-specific secondary antibodies and were examined using an inverted microscope equipped with epifluorescence optics. RNA was extracted from confluent proximal tubule cells using RNA-zol (Cinna/Biotecx Laboratories International Inc., Friendwood, TX, USA) and was reverse transcribed as described.6.Yang C.W. Wu M.S. Pan M.J. et al.Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells.J Am Soc Nephrol. 2000; 11: 2017-2026Crossref PubMed Google Scholar The sets of iNOS, CCL2/MCP-1, and β-actin primers used for RT-PCR were the same as described previously.38.Yang C.W. Striker G.E. Chen W.Y. et al.Differential expression of glomerular extracellular matrix and growth factor mRNA in rapid and slowly progressive glomerulosclerosis: studies in mice transgenic for native or mutated growth hormone.Lab Invest. 1997; 76: 467-476PubMed Google Scholar, 39.Yang C.W. Yu C.C. Ko Y.C. Huang C.C. Aminoguanidine reduces glomerular inducible nitric oxide synthase (iNOS) and transforming growth factor-beta 1 (TGF-beta1) mRNA expression and diminishes glomerulosclerosis in NZB/WF1 mice.Clin Exp Immunol. 1998; 113: 258-264Crossref PubMed Scopus (37) Google Scholar The mouse TLR2 primer pair was 5′-GTG CCA CCA TTT CCA CGG GC-3′(sense) and 5′-CAA AAC ACT TCC TGC TGG CC-3′ (antisense), yielding a 501 bp PCR product. The mouse TLR4 primer pair was 5′-AGT GGG TCA AGG AAC AGA AGC A-3′(sense) and 5′-CTT TAC CAG CTC ATT TCT CAC C-3′ (antisense), yielding a 311 bp PCR product. PCR primers were also used to detect human cDNA in HEK 293 cells. The human TLR2 primer pair was 5′-GCC AAA GCT TTG ATT GAT TGG-3′(sense) and 5′-TTG AAG TTC TCC AGC TCC TG-3′ (antisense), yielding a 347 bp PCR product. The human TLR4 primer pair was 5′-TGC GGG TTC TAC ATC AAA-3′(sense) and 5′-CCA TCC GAA ATT ATA AGA AAA GTC-3′ (antisense), yielding a 413 bp PCR product. The human CCL2/MCP-1 primer pair was 5′-CCT GCT GTT ATA ACT TCA CC-3′(sense) and 5′-ACA TCC CAG GGG TAG AAC TG-3′ (antisense), yielding a 450 bp PCR product. The human β-actin primer pair was 5′-CCC CAG GCA CCA GGG CGT GAT-3′(sense) and 5′-GGT CAT CTT CTC GCG GTT GGC CTT GGG GTT-3′ (antisense), yielding a 263 bp PCR product. The PCR products were analyzed initially by amplifying at the exponential phase. Competitive PCR assays were performed to evaluate the mRNA levels and the mean values of each measured mRNA/β-actin ratio were further expressed as a percentage change of the control values in each experiment.40.Yang C.W. Vlassara H. Peten E.P. et al.Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease.Proc Natl Acad Sci USA. 1994; 91: 9436-9440Crossref PubMed Scopus (279) Google Scholar, 41.Yang C.W. Hattori M. Vlassara H. et al.Overexpression of transforming growth factor-beta 1 mRNA is associated with up-regulation of glomerular tenascin and laminin gene expression in nonobese diabetic mice.J Am Soc Nephrol. 1995; 5: 1610-1617PubMed Google Scholar Competitive PCR was performed to measure mouse TLR2, CCL2/MCP-1, iNOS, β-actin, and for human CCL2/MCP-1, β-actin, using deletion cDNA mutant. HEK 293 cells were incubated without or with L. santarosai serovar Shermani OMPs and the CCL2/MCP-1 protein recovered in culture medium was measured by ELISA using the commercially available kit Quantikine (R&D systems, Minneapolis, MN, USA). Antiserum to LipL32 was prepared by immunizing New Zealand White rabbits with purified His6-LipL32 fusion proteins. These proteins were expressed by E. coli BL21 (DE3) pLysS transformed with the pRSET plasmid that contained the LipL32 gene as described previously.7.Yang C.W. Wu M.S. Pan M.J. et al.The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.J Am Soc Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (94) Google Scholar The pUNO-hTLR2, pDUO-MD2/TLR4, and pUNO empty plasmids were obtained from InvivoGen Inc. (San Diego, CA, USA). The HEK 293 cells (ATCC, Manassas, VA, USA) were plated into six-well tissue culture plates and maintained in Dulbecco's minimum essential medium supplemented with 10% FCS. Cells were transfected using the Lipofectamine™ 2000 transfection protocol (Life Technologies Inc., Carlsbad, CA, USA) with 4.0 μg pUNO, pUNO-hTLR2 or/and pDUO-MD2/TLR4, and 6.0 μl Lipofectamine. Transfected cells were selected in medium containing 10 μg/ml blasticidin S (InvivoGen, San Diego, CA, USA) and maintained in 1–3 μg/ml blasticidin S-supplemented medium. Transfected cells were cultured in normal medium for 36–48 h, and then in serum-free medium for additional 18 h before adding leptospiral OMPs for 24 h. Total mRNA was then extracted for RT-PCR and supernatants were tested for CCL2/MCP-1 by ELISA. All of the transfection experiments were repeated at least twice. All measurements were made at least in triplicate and the results were expressed as means±s.e.m. Differences among groups were analyzed by the unpaired Student's t-test or analysis of variance when appropriate. A P<0.05 was considered significant. This study was supported by grants from the National Health Research Institute and the National Science Council of the Republic of China, Taiwan, and by a grant for INSERM-NSC, France (to A Vandewalle, M-S Wu, and C-W Yang). We thank Yi-Ching Ko, Chung-Tseng Huang, Hsiau-Mai Yu, and Kuo-Hsiang Kuo for their excellent technical support.
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