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NDT Advance Access originally published online on December 8, 2005
Nephrology Dialysis Transplantation 2006 21(4):898-910; doi:10.1093/ndt/gfi316
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Experimental Nephrology

Leptospiral membrane proteins stimulate pro-inflammatory chemokines secretion by renal tubule epithelial cells through toll-like receptor 2 and p38 mitogen activated protein kinase

Cheng-Chieh Hung1, Chiz-Tzung Chang1,2, Ya-Chung Tian1, Mai-Szu Wu1, Chun-Chen Yu1, Ming-Jeng Pan3, Alain Vandewalle4 and Chih-Wei Yang1

1 Kidney Institute and 2 Graduate Institute of Clinical Medical Science, Chang Gung Memorial Hospital, 3 Graduate Institute of Veterinary Medicine, National Taiwan University, Taipei, Taiwan and 4 Institut National de la Santé et de la Recherche Médicale (Inserm), Unit 478, Faculty of Medicine Xavier Bichat, BP 416, 75870 Paris Cedex 18, France

Correspondence and offprint requests to: Chih-Wei Yang, Kidney Institute, Taipei, Taiwan Kidney Institute and Department of Nephrology, Chang Gung Memorial Hospital, 199, Tun-Hwa North Road, Taipei 105, Taiwan, Republic of China. Email: cwyang{at}ms1.hinet.net



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Leptospiral membrane proteins extracted from pathogenic Leptospira santarosai serovar Shermani (LMPS) stimulated pro-inflammatory chemokines production in cultured mouse proximal tubule epithelial cells (PTECs) and implicated its role in the pathogenesis of leptospira-induced tubulointerstitial nephritis. PTECs express the functional TLR2 and TLR4, which have been shown to play essential roles in innate immunity. This study investigated the roles of Toll-like receptors (TLRs) and mitogen-activated protein kinases (MAPKs) signalling pathways in the pathogenesis of leptospira-induced tubulointerstitial nephritis.

Methods. The immortalized mouse PKSV-PR late PTECs were used as the model system. The genes expression and secretion of CCL2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and CXCL2/macrophage inflammatory protein-2 (CXCL2/MIP-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). We investigated MAPKs signalling pathways by Western blot and their reciprocal roles by specific inhibitors. A specific TLR2 neutralizing antibody was applied to evaluate the crosstalk between TLR2 and MAPKs.

Results. The LMPS stimulated extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (p38 MAPK), initiated the nuclear transcription factor kappaB (NF-{kappa}B), and enhanced the secretion of CCL2/MCP-1 and CXCL2/MIP-2. The LMPS also unregulated the level of TLR2 mRNA expression in PTECs through time- and dose-dependent effects. The LMPS enhanced the secretion of CCL2/MCP-1 and CXCL8/interleukin-8 (CXCL8/IL-8) in TLR-defective human embryonic kidney (HEK) 293 cells only when transfected with a TLR2 expressing plasmid. The secretions of CCL2/MCP-1 and CXCL2/MIP-2 stimulated by LMPS were significantly reduced by incubating PTECs with SB203580, an inhibitor of p38 MAPK. Furthermore, a neutralizing anti-mouse TLR2 antibody hindered the phosphorylation of p38 and LMPS-stimulated secretion of CCL2/MCP-1 and CXCL2/MIP-2.

Conclusion. These findings demonstrate that activation of p38 MAPK and release of chemokines by LMPS are mediated by TLR2 in renal proximal tubule cells. These results also implicate the crucial role of innate immunity in leptospira-induced tubulointerstitial nephritis.

Keywords: chemokines; leptospiral membrane proteins; mitogen-activated protein kinases; renal proximal tubule cells; receptor 2



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Leptospirosis, a worldwide zoonotic infection with a high incidence rate in tropical regions, has now been identified as an emerging infectious disease [1]. Leptospirosis infection is transmitted via direct contact with infected animals or by ingesting soil or water contaminated with leptospira. Pathogenic leptospira disseminated haematogeneously in an infected host produces a mild infection or a severe infection with multiorgan failure (Weil's syndrome). In the kidney, leptospira colonizes and multiplies in proximal tubule epithelial cells (PTECs) causing tubulointerstitial nephritis and acute renal failure [2].

The initial interaction between pathogens and host cells induce an innate immune response at the infection site. Toll-like receptors (TLRs) have been shown to play a central role in the recognition of bacterial components [3]. To date, 11 TLRs have been identified in mammals and linked to the intracellular signal pathways in the expression of numerous inflammatory cytokines, chemokines, costimulatory proteins and adhesion molecules. Among these inflammatory cytokines, TLR4, in association with CD14, MD2, the myeloid differentiation factor 88 (MyD88), and the Toll receptor-IL-1R domain-containing adapter protein (TIRAP)/MyD-adapter-like (MAL) complex, plays a critical role in the recognition of lipopolysaccharide (LPS), the major constituent of Gram-negative bacterial envelopes [4]. The TLR2 confers responsiveness to other bacterial components such as lipoprotein, petidoglycan (PNG) or lipoteichoic acid. Either TLR1 or TLR6 forms a heterodimer with TLR2 to mediate responsiveness to PNG and zymozan, whereas TLR3, TLR5 and TLR9 mediate signals from RNAs, flagella and bacterial DNA, respectively [4]. The TLR11, expressed in liver, kidney and bladder epithelial cells, plays a crucial role in preventing urinary tract infections [5]. Recognition of bacterial components by TLRs induces cascades of events which activate the nuclear transcription factor kappaB (NF-{kappa}B) and mitogen-activated protein kinases (MAPKs), and the induction of chemokines and cytokines [4]. Renal tubule epithelial cells express TLR1, -2, -3, -4 and -6, indicating that these TLRs may contribute to the activation of immune response during tubulointerstitial injury [6,7]. However, little is currently known regarding the role of TLRs signalling in renal tubule epithelial cells.

The authors have previously shown that leptospiral membrane proteins extracted from pathogenic Leptospira santarosai serovar Shermani (LMPS), the most common pathogenic leptospires encountered in Taiwan, activated NF-{kappa}B and stimulated secretion of chemokines and cytokines by cultured murine PTECs [8,9]. However, the mechanisms of how PTECs recognize leptospira and their downstream signalling pathways remain unclear. Recently, Werts et al. [10] demonstrated that the activation of mouse macrophages by the leptospiral membrane constituents of invasive spirochetes required TLR2. Combined with the fact that TLR2 is constitutively expressed in renal tubule cells, these results engendered an investigation of the reciprocal roles of NF-{kappa}B, MAPKs and TLR2 in the initiation of proinflammatory chemokines secreted by murine PTECs, which are stimulated by leptospiral membrane proteins (LMP).

This work analysed the effects of membrane proteins LMPS on the secretion of the CCL2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and CXCL2/macrophage inflammatory protein-2 (CXCL2/MIP-2) and the expression of TLR2 and activation of NF-{kappa}B and MAPKs in cultured murine PTECs, and in human embryonic kidney (HEK) 293 cells transfected with TLR-expressing plasmids.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents
Fetal calf serum (FCS), Dulbecco's modified Eagle's medium (DMEM) and F-12 nutrient mixture (HAM) were each obtained from the Invitrogen Corporation (Carlsbad, CA, USA). Pam3CSK4 and purified LPS were purchased from Invivogen Inc. (San Diego, USA). Pyrrolidine dithiocarbonate (PDTC), PD98095, SP600125 and SB203580 were obtained from Calbiochem Corporation (La Jolla, CA, USA) and stored in dimethylsulfoxide (DMSO). Anti-phospho-specific and total MAPKs antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA). The anti-mouse TLR2 IgG clone T2.5 neutralizing antibody and isotype mouse IgG conT2 were purchased from Cell Science Inc. (Canton, MA, USA). All other chemicals were purchased from Sigma (St Louis, MO, USA).

Preparation of membrane protein by Triton X-114 extraction
Pathogenic Leptospira santarosai serovar Shermani (LMPS) and non-pathogenic Leptospira biflexa serovar Prato (as a negative control), obtained from the American Type Culture Collection (Manassas, VA, USA), were grown in 10% Ellinghausen McCallough Johnson Harris (EMJH) leptospiral enrichment medium (Detroit, MI, USA). Leptospira were cultured for 5–7 days at 28°C until they reached a cell density of 108/ml as previously described [11]. The LMP from pathogenic LMPS and non-pathogenic L. biflexa serovar Prato (LMPP) were extracted with 1% Triton X-114 using the extraction protocol in Haake et al. [12]. Briefly, culture-attenuated LMPS and L. biflexa serovar Prato organisms were washed in phosphate-buffered saline (PBS) supplemented with 5 mM MgCl2 and then extracted at 4°C in the presence of 1% protein grade Triton X-114, 10 mM Tris (pH 8), 1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, and 10 mM ethylenediaminetetraacetic acid. Insoluble material was removed by centrifugation at 17 000 x g for 10 min. Phase separation was conducted by warming the supernatant to 37°C and subjecting it to centrifugation for 10 min at 2000 x g. The detergent and aqueous phases were separated and precipitated with acetone and then lyophilized. The extracts were further dissolved in sterile H2O, filtered through 0.22 µm membrane filters, and stored at –80°C until use.

Cultured mouse proximal tubule cells
The mouse PKSV-PR late proximal tubule cells used in this study were derived from microdissected PTECs of the kidneys of L-PK/Tag1 transgenic mice. Cells were grown in modified DMEM-HAM F12 1:1 (vol/vol) and 2% heat-inactivated FCS at 37°C as previously described [13]. Experiments were performed on confluent cells between passages 44 and 55. Cells were cultured in a serum-free medium and then directly added LMPS or LMPP into the medium, without changing the medium to prevent activation of any signalling pathways due to medium change. In some experiments, specific inhibitors or neutralizing antibodies were added 1 h before incubating cells with LMPS.

RNA extraction and RT-PCR
Total RNA was extracted from confluent PTECs using the guanidium thiocyanate–phenol–chloroform method (Cinna/Biotecx Laboratories International Inc., Friendwood, TX, USA) and treated with RNase-free DNase I (Boehringer Mannheim, Germany) at 37°C for 30 min. The RNA (1 µg) was reversed-transcribed using the avian myeloblastosis virus reverse transcriptase (RT AMV, Boehringer Mannheim) at 42°C for 60 min. Complementary DNA was amplified for 30–42 cycles in 100 µl total volume containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 10 mM dNTP, 1.5–3.0 mM MgCl2, 1 unit Taq polymerase and 10 pmol of specific PCR primers. The thermal cycling protocol was as follows: 94°C for 1 min, 60°C for 1 min, and 72°C for 3 min. Amplification products were separated on a 4% agarose gel with ethidium bromide and then photographed. Table 1 shows the primer constructs for RT-PCR as previously described in [14,15].


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  		Table 1. Primers used in this study

 
Enzyme linked immunosorbent assay (ELISA)
The amount of mouse/human CCL2/MCP-1, mouse CXCL2/MIP-2 and human CXCL8/interleukine-8 (IL-8) recovered in the supernatants from cultured PTECs or HEK293 cells were measured with commercially available ELISA kits (R&D systems, Minneapolis, USA).

Electrophoretic mobility shift assay (EMSA)
Nuclear proteins were prepared according to the method in Satriano and Schlondorff [16] with slight modifications. Nuclear proteins were assayed for NF-{kappa}B nuclear binding activity by applying NF-{kappa}B consensus oligonucleotides (5'-AGT TGA GGG GAC TTT AGG C-3'; Promega, Madison, WI, USA) using the Digoxigenin EMSA kit (Roche, Sommerville, NJ, USA). A total of 10 µg nuclear proteins were incubated with Digoxigenin-labelled NF-{kappa}B oligonucleotide in a binding mixture to a final volume of 15 µl. After 20 min incubation, protein-DNA complexes were resolved on 8% polyacrylamide gel in 0.5X Tris-borate-EDTA buffer. Gels were then transferred to nylon membranes. After cross-linking, the membrane was washed, blocked and incubated with anti-digoxigenin-alkaline phosphatase. Signal was detected by adding disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricycle [3.3.1.13,7]decan}-4-yl) phenyl phosphate (CSPD) and then exposed to X-ray film. Specific competition control using a 100-fold excess of unlabelled oligonucleotide was added to the binding reaction mixture for 10 min before adding the labelled NF-{kappa}B probe [8].

Western blot analysis
Cells were lysed in lysis buffer containing protease inhibitors for 30 min and centrifuged at 11 000 x g for 15 min. Samples were mixed with 6 x sample buffer, boiled at 95°C for 5 min and loaded onto 10% SDS-polyacrylamine gels. After eletrophoresis, gels were immersed in a transfer buffer, electrotransferred to Immunobilon-P membranes (Millipore Bedford, MA, USA) and blotted with antibodies overnight at 4°C in Tris buffer system (TBS) containing 0.1% Tween 20 and 5% non-fat milk. After washing, membranes were incubated with HRP-conjugated antibodies and signals were detected using chemiluminescence (ECL Amersham, Arlington Heights, IL, USA). To compare equally loaded samples, the initial antibody probe was stripped and re-blotted with the indicated antibodies [17].

HEK293 cells culture and transient transfection
The HEK293 cells (ATCC, Manassas, VA, USA) grown in 6-well plates with DMEM supplemented with 10% FCS were transfected with either pDUNO-hTLR2, pDUO-hTLR4/MD2 or pDUNO empty plasmid (Invivogen Inc., San Diego, USA) alone or in combination using lipofectamine according to LipofectamineTM2000 transfection protocol (Life Technologies Inc., Carlsbad, CA, USA). Transfected cells were cultured for 24 h and then serum-deprived for 12 h prior to incubation with LMPS for an additional 24 h. The mRNAs were then extracted for RT-PCR and supernatants were examined for production of chemokines by ELISA. All experiments were performed a minimum of three times.

Densitometric analysis
The band intensity obtained by RT-PCR or Western blot analyses were quantified by scanning densitometry and normalized to the values of ß-actin, total ERK1/2, total JNK1/2 and total p38, respectively. Densitometric quantification of mRNA expression was normalized to ß-actin. Values were expressed as fold-increase of untreated cell values.

Statistical analyses
Student's t-test was used to determine if there was a significant difference between two groups (P<0.05). When multiple means were compared, significance (P<0.05) was determined by ANOVA followed by Fisher's protected least significant difference (Fisher's PLSD) test. For ANOVA, letter designations are used to indicate significant difference. Means with a common letter designation are not different, and those with a different letter designation are significantly different from all other means with different letter designations. StatView software (SAS Institute, Cart, NC, USA) was used as a statistical tool in this study.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
LMP stimulate the expression of CCL2/MCP-1 and CXCL2/MIP-2
Confluent PTECs were incubated with LMPS or LMPP to identify their effect on CCL2/MCP-1 and CXCL2/MIP-2, the functional murine equivalent of human CXCL8/IL-8, expression and secretion. The LMPS rapidly initiated the mRNA expression of CCL2/MCP-1 and CXCL2/MIP-2 at 4 h as compared with that of untreated cells (Figure 1A, upper panel). The LMPS also induced secretion of CCL2/MCP-1 and CXCL2/MIP-2 higher than that of the control values at 4 h and remained stabilized at their enhanced levels (for CCL2/MCP-1) or slightly decreased (for CXCL2/MIP-2) during the following 24 h (Figure 1A, lower panel). The levels of CCL2/MCP-1 and CXCL2/MIP-2 mRNA expressions and secretion also increased as a function of the LMPS concentrations added for 24 h (Figure 1B, upper and lower panels). In contrast, LMPP, as a negative control, did not stimulate the expression and secretion of CCL2/MCP-1 and CXCL2/MIP-2 from PTECs (Figure 1A and B). The LMPS or LMPP did not influence the viability of LMP-treated cells (92±7% and 93±5%, respectively) as compared with that of untreated cells by MTT assay. Analytical results indicate that LMPS stimulate secretion of CCL2/MCP-1 and CXCL2/MIP-2 without affecting the viability of PTECs.


Figure 1
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  		Fig. 1. LMPS induced CCL2/MCP-1 and CXCL2/MIP-2 mRNA expression and protein secretion in renal tubule epithelial cells. Confluent PTECs grown in serum-free medium for 18 h were then incubated with increasing concentrations of leptospiral membrane proteins (LMP) from pathogenic LMPS and non-pathogenic LMPP (0.1–1 µg/ml) for various periods. Expression of CCL2/MCP-1, CXCL2/MIP-2 and ß-actin mRNAs analysed by RT-PCR in PTECs as a function of time (A, upper panel) or increasing concentrations of LMP (B, upper panel). As a control, ß-actin was utilized as the internal standard. The secretion of CCL2/MCP-1 and CXCL2/MIP-2 recovered in culture supernatants from PTECs stimulated for various periods (A, lower panel) and with varying amounts of LMP (B, lower panel) were measured by ELISA. Values are means±SE of duplicate measurements from three independent experiments. In all cases, values were significantly higher (*P<0.05) than those of untreated cells (Time, 0; LMP, 0).

 
Up-regulated TLR2 mRNA expression by LMP
TLR2 or TLR4, known to be expressed in intact renal tubule epithelial cells [7], were also expressed in cultured murine PTECs. The PTECs constitutively expressed TLR4 and MD2 to levels significantly close to that expressed in the macrophage cell line RAW264.7 (Figure 2A). In comparison with the level of TLR4 mRNA expression, resting PTECs expressed only low levels of TLR2 mRNA. All TLR2 and TLR4 are functional as PTECs stimulated by specific TLR2 (Pam3CSK4) or TLR4 ligands (purified LPS) stimulated a substantial amount of CCL2/MCP-1 secretion (Figure 2B). Adding 0.1 µg/ml LMPS for 4 h induced a significant increase in the levels of TLR2 mRNA, but not of TLR4 mRNA compared with those of untreated PTECs (Figure 2C). Maximal increase in TLR2 mRNA expression was achieved with 0.1–0.2 µg/ml LMPS and the stimulatory effect on TLR2 persisted for at least 24 h (data not shown).


Figure 2
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  		Fig. 2. Effects of LMPS on TLR2 and TLR4 mRNA expression in renal proximal tubule cells. (A) The expression of TLR2 and TLR4 mRNAs was analysed in untreated PTECs and RAW 246.7 cells. (B) The PTECs were challenged without (Con) or with TLR2 (Pam3CSK4) or TLR4 (purified LPS, pLPS) specific ligand. Secretion of CCL2/MCP-1 was measured with an ELISA kit. The PTECs with 0.1 µg/ml LMPS for 4 or 8 h (C, left panel) or with 0.1 or 0.2 µg/ml LMPS for 24 h (C, right panel). ß-actin was used as internal standard. The bars represent the fold-increase in TLR2 (black bars) or TLR4 (open bars) mRNA expression as compared with that of untreated cell values (Time, 0; LMPS, 0). Values are means±SE of duplicate measurements from three independent experiments. *P<0.05 vs Time 0 or LMPS 0 values.

 
Transient expression of TLR2 in HEK293 cells restores cell sensitivity to LMP
The fact that LMPS provoked an up-regulation of TLR2 in PTECs led to an examination of the role of TLR2 in the induction of CCL2/MCP-1 and CXCL8/IL-8 caused by LMPS in cultured human embryonic kidney cells HEK293. These HEK293 cells, which lacked TLRs, were transiently transfected with a human TLR2 (hTLR2) plasmid and an empty vector plasmid for a control. Transiently transfected HEK293 cells were then incubated with LMPS for 24 h and total RNA was then extracted. The amounts of human CCL2/MCP-1 and CXCL8/IL-8 secreted were measured with an ELISA kit. The LMPS significantly enhanced the mRNA levels of CCL2/MCP-1 and CXCL8/IL-8 in TLR2-transfected cells compared with the mRNA levels in empty vector-transfected cells (Figure 3A and B, left panels). Consistent with these findings, LMPS also significantly increased CCL2/MCP-1 and CXCL8/IL-8 secretion (Figure 3A and B, right panels), and failed to stimulate CCL2/MCP-1 and CXCL8/IL-8 secretion in cells transiently transfected with the empty vector-transfected cells (Figure 3A and B). Thus, these experimental results provided strong evidence that the release of inflammatory mediators stimulated by LMPS is predominantly, if not exclusively, mediated by TLR2. Because TLR4 has a crucial role in the recognition of LPS from Gram-negative bacteria, additional experiments were undertaken to determine whether TLR4 is crucial to the recognition of LMPS. Transfected TLR2 and TLR4/MD2 are functional as HEK293 cells stimulated by specific TLR2 (Pam3CSK4) or TLR4 ligands (purified LPS) stimulated a substantial amount of CCL2/MCP-1 and CXCL8/IL-8 secretion (data not shown). Sets of HEK293 cells were transiently transfected with TLR2 and TLR4/MD2 expression plasmids alone or in combination. The LMPS significantly increased the secretion of CCL2/MCP-1 and CXCL8/IL-8 in TLR2-expressing HEK293 cells and not in TLR4/MD-expressing HEK293 cells (Figure 4A and B), strongly suggesting that TLR4 may not be associated with the innate response induced by LMPS.


Figure 3
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  		Fig. 3. Stimulatory effects of LMPS on the induction of CCL2/MCP-1 or CXCL8/IL-8 mRNA expression and protein secretion in HEK293 cells expressing TLR2. The HEK293 cells were transiently transfected (36 h) with an empty plasmid (vector) or with the pDUNO-hTLR2 expression plasmid (hTLR2). Cells were then grown in normal medium for 12 h and in serum-free for an additional 18 h. Transfected cells were then incubated for 24 h with or without 0.2 or 0.4 µg/ml LMPS for 24 h. (A) CCL2/MCP-1 and (B) CXCL8/IL-8 mRNAs expressions were analysed in transfected HEK293 cells incubated without (0) or with 0.2 or 0.4 µg/ml LMPS for 24 h (left panels). The mRNA expression of hTLR2 was also assessed as a transfection control. The secretion of CCL2/MCP-1 (A, right panel) and CXCL8/IL-8 (B, right panel) recovered in culture supernatants from transfected HEK293 cells (vector, black bars; hTLR2, open bars) incubated without (0) or with 0.2 or 0.4 µg/ml LMPS for 24 h were determined with an ELISA kit. Values are means±SE of duplicate measurements from three independent experiments. *P<0.05 vs untreated cell values (LMPS, 0).

 

Figure 4
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  		Fig. 4. Biological response to LMPS is not reconstituted in TLR4-transfectant cells. The HEK293 cells were transiently transfected with empty plasmid (pDUNO), or expression plasmids for TLR2, TLR4/MD2 or TLR2 and TLR4/MD2 (pDUNO-hTLR2 and/or pDUO-hTLR4/MD2). Twenty four hours following transfection, cultured cells recovered in normal cultured medium for 12 h, serum-deprived 16 h, and then stimulated with 0.2 or 0.4 µg/ml LMPs for 24 h. The supernatant was obtained for measurement of (A) CCL2/MCP-1 or (B) CXCL8/IL-8 cytokines release with an ELISA kit. The data are the means±SE from duplicate measurements from three transfections (n = 3). Significant differences (*P<0.05) between the means were determined by ANOVA followed by Fisher's PLSD test and are indicated by different letters (see Materials and methods).

 
Activation of NF-{kappa}B and the phosphorylation of MAP kinases
Further experiments were conducted to clarify the effects of LMPS on NF-{kappa}B activation and phosphorylation of ERK1/2, JNK1/2 and p38 in PTECs. There were significant amounts of nuclear DNA binding of NF-{kappa}B after incubating cells with LMPS for 15 min. The increase in nuclear DNA binding peaked after 60 min and then decreased for longer incubation periods (Figure 5A). The band was specific for NF-{kappa}B nuclear DNA binding as shown by the inhibition of 100-fold excess of cold NF-{kappa}B probe (Figure 5A). The LMPS significantly increased the phosphorylated ERK1/2 after 30 min incubation with LMPS (Figure 5B). Similarly, Western blotting showed that LMPS stimulated JNKs; this finding was determined by the increase in phosphorylated bands detected at 30 min (Figure 5C). Adding LMPS for 30 min also significantly stimulated the phosphorylation of p38 (Figure 5D). In all cases, LMPS which did not affect total ERKs, JNKs and p38 proteins, had maximal stimulatory influence on phosphorylated kinases after 60 min incubation and slowly decreased during the following 60 min.


Figure 5
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  		Fig. 5. LMPS effects on the induction of NF-{kappa}B and phosphorylated MAPKs in renal proximal tubule cells. Confluent PTECs grown in serum-free medium for 18 h were incubated with 0.1 µg/ml LMPS for various periods. The expression of NF-{kappa}B was assessed with EMSA in nuclear extracts. (A) A 100-fold excess of cold competitive NF-kB probe was added to confirm the specificity of NF-{kappa}B nuclear DNA binding (Comp). Total (T) and phosphorylated (p) (B) ERK1/2, (C) JNK1/2 and (D) p38 MAPK were evaluated in cell lysates using specific antibodies directed against total or phosphorylated kinases. Total ERK1/2, JNK1/2 and p38 were determined to verify equal protein loading. The bars signify the densitometric quantification of the immunoblotted bands normalized to that of total ERK1/2, JNK1/2 or p38 and expressed as fold-increases of untreated cell values (bottom of blots). *P<0.05 vs untreated cell values (Time, 0).

 
Stimulatory effect on CCL2/MCP-1 and CXCL2/MIP-2 expression is controlled by p38 MAPKs
The PDTC, although preventing NF-{kappa}B activation by LMPS (Figure 6A), did not impair the stimulatory action of LMPS in the levels of mRNA expression and secretion of CCL2/MCP-1 and CXCL2/MIP-2 by PTECs (Figure 6B and C). Since the LMPS significantly stimulated phosphorylated MAPKs, whether MAPK inhibitors altered the expression of chemokines stimulated by LMPS was evaluated. Preincubating PTECs with 50 µM PD98095, an inhibitor of MAPK p42/p44, fully prevented any stimulatory effect of 0.1 µg/ml LMPS on phosphorylated ERK1/2 (Figure 7A, upper panel). Although PD98095 reduced the LMPS-stimulated CXCL2/MIP-2 mRNA expression and secretion (by 30%, n = 4) when compared with that measured in cells treated with LMPS and the DMSO alone, the ERK/MAPK inhibitor had no inhibitory action on the levels of CCL2/MCP-1 mRNA and protein release stimulated by LMPS (Figure 7A, middle and lower panels). The effect of SP600125, an inhibitor of c-Jun NH2-terminal kinase, was then analysed. SP600125 completely prevented activation of JNKs (Figure 7B, upper panel), and had no effect on the CCL2/MCP-1 or CXCL2/CXCL2/MIP-2 mRNA expression or protein release (Figure 6B, middle and lower panels). Preincubation of PTECs with SB203580, an inhibitor of p38 MAPK, effectively blocked the phosphorylation of p38 (Figure 7C, upper panel), substantially reduced mRNA expression of both CCL2/MCP-1 and CXCL2/MIP-2 and also significantly reduced the secretion of CCL2/MCP-1 by 43% (n = 4) and CXCL2/MIP-2 by 67% (n = 4) stimulated by LMPS (Figure 7C, middle and lower panels).


Figure 6
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  		Fig. 6. Inhibiting NF-{kappa}B nuclear DNA binding does not alter stimulated CCL2/MCP-1 and CXCL2/MIP-2 mRNA expression or protein secretion caused by LMPS. Confluent PTECs grown in serum-free medium for 18 h were then pre-incubated with DMSO (vehicle) or 25 µM PDTC for 1 h prior to adding 0.1 µg/ml LMPS. (A) The expression of NF-{kappa}B was evaluated by EMSA in nuclear extracts 30 min after adding LMPS. (B) Expression of CCL2/MCP-1, CXCL2/MIP-2 and ß-actin mRNAs were analysed by RT-PCR in cells, or secreted CCL2/MCP-1 (black bars) and (C) CXCL2/MIP-2 (open bars) by PTECs incubated without (C) or with 0.1 µg/ml LMPS for 24 h were measured with an ELISA kit. Values are means±SE of duplicate measurements from three independent experiments. There were no significant differences in chemokine production between LMPS, LMPS plus vehicle pre-treated and LMPS plus PDTC pre-treated.

 

Figure 7
Figure 7
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  		Fig. 7. Inhibition of the ERK42/44, JNKs and p38 MAPK pathways individually affected induction of CCL2/MCP-1 and CXCL2/MIP-2 mRNA expression and protein secretion caused by LMPS. Confluent PTECs grown in serum-free medium for 18 h were then incubated with or without DMSO (vehicle) alone, (A) 50 µM PD98095, (B) 40 µM SP600125, or (C) 10 µM SB203580 for 1 h before adding 0.1 µg/ml LMPS. (Upper panels) Cells incubated with or without LMPS for 30 min were lysed and applied to approximate the amount of phosphorylated (p) and total (T) (A) ERK1/2, (B) JNKs and (C) p38 MAPK using specific antibodies. (Middle panels) CCL2/MCP-1, CXCL2/MIP-2 and ß-actin mRNAs expressions were determined by RT-PCR in cells incubated with LMPS (0.1 µg/ml for 24 h) in the presence or absence of DMSO (vehicle) or various kinase inhibitors. (Lower panels) The secretion of CCL2/MCP-1 (open bars) and CXCL2/MIP-2 (black bars) recovered in culture supernatants from PTECs incubated under the conditions previously described were measured with an ELISA kit. Values are means±SE of duplicate measurements from three independent experiments. (*P<0.05 between groups).

 
TLR2 mediates the stimulatory effects on the secretion of CCL2/MCP-1 and CXCL2/MIP-2 and phosphorylation of p38
As LMPS stimulated an up-regulation of TLR2 mRNA in PTECs (Figure 2), the question arises as to whether TLR2 was the specific LMPS receptor. If this was the case, blocking TLR2 should hinder the inductible action of LMPS on the expression and release of chemokines. The PTECs, therefore, were incubated with the anti-TLR2 neutralizing antibody T2.5. This antibody has been shown to limit mouse and human TLR2-mediated cell activation produced by the TLR2-specific stimuli Pam3CSK4 or by B. subtilis [18]. Preincubating PTECs with T2.5 (50 µg/ml) for 1 h prior to adding LMPS substantially prevented the increased secretion of CCL2/MCP-1 and CXCL2/MIP-2 caused by LMPS, whereas the isotype control conT2 antibody, an isotype IgG antibody devoid of activity, had no effect (Figure 8A). Moreover, incubation of PTECs with the T2.5 antibody prevented activation by LMPS of the p38 pathway (Figure 8B). These results strongly indicate that the action of LMPS on the phosphorylation of p38 and induction of chemokines release requires a functional TLR2.


Figure 8
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  		Fig. 8. Inhibition of TLR2 impedes the inducible effects of LMPs on the secretion of CCL2/MCP-1 and CXCL2/MIP-2 and phosphorylated p38 MAPK. Confluent PTECs grown in serum-free medium for 18 h were then incubated with the T2.5 anti-mouse TLR2 neutralizing antibody (50 µg/ml) or the conT2 isotype control devoid of activity for 1 h before adding 0.1 µg/ml LMPS for 24 h. (A) The secretion of CCL2/MCP-1 (black bars) and CXCL2/MIP-2 (open bars) recovered in culture supernatants from PTECs incubated with or without 0.1 µg/ml LMPS in the presence or absence of T2.5 or isotype control conT2 antibodies for 24 h were measured by ELISA. Values are means±SE of duplicate measurements from three independent experiments. (*P<0.05 vs LMPS-treated cell values). Cells were incubated with or without 0.1 µg/ml LMPS for 30 or 60 min in the presence or absence of T2.5 or isotype control conT2 antibodies. (B) The amount of phosphorylated (p) and total (T) p38 MAPK were analysed on cell lysates using specific antibodies. The blots shown signify three experiments.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Leptospiral membranes are primarily composed of lipoproteins, some of which have been characterized, include LipL36, LipL41, and LipL32. Lipoprotein LipL32 is the predominant antigen extracted from Leptospira interrogans and expressed during infection of mammals [12]. The extraction method used in this study enhanced the harvest of leptospiral protein components. Although one cannot exclude a role for contaminant leptospiral LPS in LMPS preparations, it remains that glycolipoproteins are responsible for major leptospiral toxicity [19]. In this line, we had previously shown that the upregulation of chemokines by LMPS in renal tubule cells was significantly decreased by heating or proteinase K digestion. Moreover, polymyxin B, which binds to the lipid A portion of LPS, resulting in an inhibition of LPS activity, did no significantly impair the inducible action of LMPS on chemokine expressions in renal tubule cells [8,9]. Previous animal studies detected leptospiral antigens in renal tubules (mainly proximal tubules), macrophages and endothelial cells from interstitial capillaries [20]. Thus, the close interactions of membrane proteins from pathogenic leptospira with tubule epithelial cells determine the degree of induction and persistence of leptospiral tubulointerstitial nephritis.

This study obtained lines of evidence which demonstrate that TLR2 is essential to the induction of an innate immune response initiated by LMPS in renal PTECs and the resulting enhanced secretion of chemokines. The LMPS increased TLR2 expression in a model of murine PTECs and concomitantly stimulated the expression of chemokines. Blocking TLR2 in murine PTECs with the neutralizing antibody T2.5 hindered the stimulatory action of LMPS on secreted chemokines. This analytical finding strongly indicated that the stimulatory action of pathogenic Leptospira shermani membrane proteins is mediated by TLR2 in renal proximal tubule cells. These findings are coincident with those obtained by Werts et al. [10], who showed that leptospiral LPS and lipoprotein LipL32 from L. interrogans activate human monocytic THP-1 cells through TLR2. Werts et al. also demonstrated that TLR4, a component crucial to recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans [10]. Experimental results from this study also confirm that TLR4 is not required for recognition of LMPS in PTECs which constitutively express TRL4 or in human HEK 293 cells transiently expressing TLR4/MD2. As the expression of human TLR2 and not of TLR4/MD2 was adequate to confer responsiveness to LMPS in HEK293 cells, these findings favour the hypothesis that TLR2 is the principal innate immune receptor for recognition of LMPS in renal epithelial cells. The fact that LMPS rapidly initiate TLR2 gene transcription also indicates that in the process of nephritis, renal epithelial cells become more responsive to leptospiral components by expressing excessive amounts of TLR2.

The flow of leucocytes from peripheral blood to the site of infected tissues is critical in the induction of immune response and is a determining factor in the development of numerous kidney diseases [21]. The major mononuclear cell chemoattractant chemokines have primary roles in recruiting polymorphonuclear cells and lymphocytes. The authors of this study have previously shown that LMPS stimulate certain chemokines in murine thick ascending limbs and proximal tubule cells, indicating that the generation of these chemokines by renal tubule cells is a factor in the onset of acute tubular inflammation and subsequent tubulointerstitial nephritis [8,9]. In human PTECs, CD40 stimulates CXCL8/IL-8 and CCL2/MCP-1 production that play an important role in tubulointerstitial inflammation through activating JNKs and p38 MAPK phosphorylation [22,23]. Analytical results showed that LMPS induce multiple intracellular signalling events: activation of NF-{kappa}B and three distinct MAPKs, ERKs, JNKs and p38 MAPK. The C–C chemokine CCL2/MCP-1-mediated activation of tubular epithelial cells is a consistent with the hypothesis that CCL2/MCP-1 contributes to tubulointerstitial inflammation, which is a principal characteristic of progressive renal disease [24]. Previous studies have demonstrated that LMPS activated NF-{kappa}B pathway, stimulated CCL2/MCP-1 gene expression and chemokine production in renal tubular epithelial cells [8,9]. In this study, we have further defined the important role of MAPK in this activation. The stimulation of CCL2/MCP-1 gene expression is mainly dependent on p38 activation. On the other hand, preincubation of PTECs with PDTC did not hinder the stimulatory action of LMPS on CCL2/MCP-1 indicating that the NF-{kappa}B pathway is not involved in the CCL2/MCP-1 activation. Studies using specific MAPKs inhibitors also show that both p38 and pERK signalling pathways are involved in the induction and secretion of CXCL2/MIP-2 by LMPS (Figure 7). Although inhibition of NF-{kappa}B and JNKs had no effect on up-regulation of CCL2/MCP-1 and CXCL2/MIP-2 by LMPS, the inducible nitric oxide synthase (iNOS) and NO production, primary mediators of inflammatory processes, stimulated by LMPS in PTECs is responsive to NF-{kappa}B and JNKs MAPK inhibition (C.W. Yang, unpublished observation). These results underline the complexity of signal pathways, which are not mutually exclusive, involved in immune response induced by LMPS in renal epithelial cells. Notably, the inhibition of LMPS binding to TLR2 reduced activation of p38 MAPK and subsequent production of CCL2/MCP-1 and CXCL2/MIP-2. That the TLR2-neutralizing antibody prevented the LMPS-stimulated release of CCL2/MCP-1 and CXCL2/MIP-2 generated by renal epithelial cells strongly indicates that the TLR2/p38 MAPK pathway is crucial to the induction of pro-inflammatory cytokines by LMPS. Furthermore, the MEK inhibitor (PD98095) limited the increase in secreted CXCL2/MIP-2 caused by LMPS confirms that CXCL2/MIP-2 secreted by LMPS is in part through ERK pathways.

In conclusion, the experimental results of this study prove that LMPS-stimulated secretion of chemokines by proximal tubular epithelial cells is mediated by TLR2 and requires activation of p38 MAPK. Thus, these findings uphold the notion that TLR2, expressed in mouse renal proximal tubule cells, plays a key role in the induction of the innate response caused by membrane proteins in renal epithelial cells.



   Acknowledgments
 
The authors would like to thank Y.C. Ko, C.T. Huang and H.M. Yu for their technical assistance. The study was supported by a grant from the Taiwan National Health Research Institute (NHRI-EX93-9138SI to C.W. Yang), grants from the Taiwan National Science Council (NSC 93-2314-B-182A-108 to C.C. Hung and NSC 93-2314-B-182A-117 to C.T. Chang) and by a grant from INSERM (France)-NSC (Taiwan) (to A. Vandewalle, M.S. Wu and C.W. Yang).

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 14. 9.05
Accepted in revised form: 14.11.05


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