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MCP-1 Induces Inflammatory Activation of Human Tubular Epithelial Cells: Involvement of the Transcription Factors, Nuclear Factor-B and Activating Protein-1

Christiane Viedt^*, Ralph Dechend, Jianwei Fei^*, Gertrud M. Hänsch, Jörg Kreuzer^* and Stephan R. Orth

*Department of Internal Medicine, Division of Cardiology, Ruperto Carola University, Heidelberg, Germany; Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Germany; Institute of Immunology, Ruperto Carola University, Heidelberg, Germany; Division of Nephrology and Hypertension, University Hospital of Berne (Inselspital), Berne, Switzerland.

Correspondence to Dr. Stephan R. Orth, Division of Nephrology and Hypertension, University Hospital of Berne (Inselspital), CH-3010 Berne, Switzerland. Phone: 0041-31-6322130; Fax: 0041-31-6329734; E-mail: stephan.orth{at}insel.ch

	Abstract

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ABSTRACT. Monocyte chemoattractant protein–1 (MCP-1) is a potent chemokine synthesized by several cell types, e.g., inflammatory cells, such as monocytes, and resident renal cells, such as human tubular epithelial cells (TECs). Besides induction of monocyte recruitment, MCP-1 has been suggested to induce non-leukocytes to produce cytokines and adhesion molecules. Inflammation of the tubulointerstitium is a hallmark of many renal diseases and contributes to progression of renal failure; the purpose therefore of this study was to investigate the influence of MCP-1 on markers of inflammatory activation in human TECs. MCP-1 stimulated interleukin-6 (IL-6) secretion and intercellular adhesion molecule-1 (ICAM-1) synthesis in a time- and dose-dependent manner. In parallel, MCP-1 increased IL-6 and ICAM-1 mRNA expression in human TECs. Pretreatment with pertussis toxin, GF109203X, BAPTA-AM, and pyrrolidine dithiocarbamate inhibited MCP-1–dependent IL-6 and ICAM-1 synthesis, suggesting the involvement of Gi-proteins, protein kinase C, intracellular Ca²⁺, and nuclear factor–B (NF-B) in MCP-1 signaling. Using electrophoretic gel mobility shift assay, we observed that MCP-1 stimulated binding activity of NF-B. Binding activity of the activator protein-1 (AP-1), which has been implicated to regulate induction of the IL-6 gene together with NF-B, was also stimulated by MCP-1. In the present experiments, NF-B and AP-1 were involved in the MCP-1–mediated induction of IL-6, as demonstrated by cis element double-stranded (decoy) oligonucleotides (ODN). In contrast to IL-6 release, MCP-1–induced ICAM-1 expression was predominantly dependent on NF-B activation. These results document for the first time that MCP-1 induces an inflammatory response in human TECs. This may be an important new mechanism in the pathogenesis of tubulointerstitial inflammation.

	Introduction

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Independently of the underlying renal disease, tubulointerstitial renal damage correlates best with the loss of renal function and the risk of progression to end-stage renal failure (1). Resident renal cells, such as tubular epithelial cells (TECs), might have an important role in the tubulointerstitial inflammatory process observed in many renal diseases (2). Human TECs in vitro produce large amounts of cytokines (interleukin-6 [IL-6] and tumor necrosis factor– [TNF-]) (3–5), chemokines (IL-8, monocyte chemoattractant protein-1 [MCP-1]) (6,7), and adhesion molecules (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) (8). Elevated levels of cytokines and chemokines have been documented in both resident renal cells and infiltrating inflammatory cells in various forms of glomerulonephritis and tubulointerstitial nephritis and have been suggested to contribute to progressive renal failure in vivo (9). Thus, TECs may be involved in renal inflammatory processes.

The CC chemokine MCP-1 is secreted by mononuclear cells and various non-leukocytic cells, including endothelial cells, vascular smooth muscle cells (VSMC), and resident renal cells, such as mesangial cells and TECs (6,7,10,11). Recent studies revealed that MCP-1 plays an important role in the pathogenesis of progressive glomerular and tubulointerstitial lesions in different animal models of renal damage and human renal diseases (9).

To date, besides induction of monocyte recruitment in glomerular and tubulointerstitial inflammation, CC chemokines have not been shown to activate resident renal cells. The latter may indeed occur, because MCP-1 is able to activate non-leukocytes, i.e., VSMC (12). Therefore, the current study was designed to examine whether MCP1 has a proinflammatory effect on human TECs and, if so, to define the mechanisms involved.

The present data demonstrate that MCP-1 induces an inflammatory response in human TECs via activation of the transcription factors, NF-B and AP-1, by inducing expression of the proinflammatory cytokine, IL-6, and the adhesion molecule, ICAM-1.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Human recombinant MCP-1, IL-8, and RANTES, as well as monoclonal anti–MCP-1, anti-CC chemokine receptor 1 (CCR1), anti-CCR2, anti-CCR3, anti-CCR5, and anti–IL-6 were purchased from R&D Systems (Minneapolis, MN). Pertussis toxin (PTX), pyrrolidine dithiocarbamate (PDTC), and BAPTA-AM were from Calbiochem (La Jolla, CA). GF109203X was from Biomol (Hamburg, Germany). Phenylmethylsulfonyl fluoride (PMSF), NP-40, and insulin were from Sigma (Deisenhofen, Germany). 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc) and E-64 were purchased from Roche (Mannheim, Germany). Anti-cytokeratin and anti–ICAM-1 (CD54:FITC) were from Serotec (Biozol, Eching, Germany). IgG1 (IgG1:FITC) was from Becton Dickinson (Heidelberg, Germany). Polyclonal antibodies to c-Jun, c-Fos, I-B, and NF-B proteins (i.e., p65, p50) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). NF-B and AP-1 oligonucleotides were obtained from MWG (Ebersberg, Germany). Cell culture media and supplements were from Life Technologies SRL (Karlsruhe, Germany).

Human Tubular Epithelial Cell (TEC) Culture
Human TECs were isolated as described previously in detail (13). Cells were grown in DMEM with heat-inactivated fetal calf serum (FCS) (10%), 1% L-glutamine, penicillin (100 U/ml)/streptomycin (100 µg/mL), 1% vitamin mix, and 0.1% insulin (= standard growth medium). TECs were identified by: (1) characteristic polygonal, typical cobblestone morphology; (2) positive staining with antibodies to aminopeptidase M and angiotensinase A (14) and to CD10 (15); (3) immunofluorescence staining for cytokeratin. The cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂. Cells were grown as subconfluent monolayers and used in passages 4 to 5. Before the experiments, the cells were washed with phosphate-buffered saline (PBS) and grown in serum-free medium (2% Ultroser as serum substitute in DMEM) for 24 h to render TECs quiescent.

To determine the direction of IL-6 secretion, i.e., whether it is secreted mainly to the apical or the basolateral compartment, TECs were seeded into cell culture inserts (Falcon, Heidelberg, Germany) with 0.4-µM pores (1.6 x 10⁶ pores/cm²) and kept for 24 h in standard growth medium to form confluent monolayers. Thereafter, the media of both compartments were changed to serum-free medium containing MCP-1 (10 ng/ml) in either the apical or the basolateral bathing. Because MCP-1 is a small peptide (8 kD), it may leak across the cell culture monolayer. Thirty minutes after stimulation, the medium of both compartments was therefore replaced by fresh serum-free medium to minimize potential leakage. Supernatants of the apical and the basolateral compartment were collected separately after 48 h, and cytokine content was determined by enzyme-linked immunosorbent assay (ELISA) (vide infra). Values are corrected for differences in medium volume of the respective compartments.

Human Monocyte Culture
Monocytes were isolated from human blood samples using Ficoll-Paque followed by adhesion separation in RPMI-1640 with 10% FCS over night. The adherent monocytes were incubated in RPMI-1640 (without FCS) in the presence or absence of MCP-1 for 24 h.

Determination of IL-6 Release
Cells were seeded in standard growth medium at a concentration of 1 x 10⁵ cells/well in 24-well plates for 24 h. Thereafter, cells were kept in serum-free medium for further 24 h. After stimulation, IL-6 was determined in cell culture supernatants by ELISA (Quantikine Immunoassay, R&D Systems) according to the manufacturer‘s instructions.

Determination of MCP-1 Release
TECs were grown in 24-well plates to confluency and kept in serum-free medium for 24 h. Thereafter, cells were kept in serum-free medium for further 24, 48, or 72 h, respectively. MCP-1 was determined in cell culture supernatants by ELISA (Quantikine Immunoassay) according to the manufacturer‘s instructions.

Flow Cytometry Analyses
For flow cytometry, TECs were cultured in six-well plates and stimulated with MCP-1 in either the presence or absence of inhibitors. After stimulation, TECs were washed with PBS at 4°C and then detached with trypsin/EDTA. Trypsinization was stopped by the addition of PBS containing 1% NaN₃ and 10% FCS. Thereafter, cells were centrifuged and incubated for 10 min at 4°C in PBS containing 1% NaN₃ and 1% goat serum. Cells were washed again with PBS/1% NaN₃ and incubated with FITC-conjugated mouse anti-ICAM-1 (CD54), anti-CCR2 or IgG1 (isotype control), respectively (30 min at 4°C). Then, cells were washed with PBS/1% NaN₃ and fixed in 1% paraformaldehyde. Flow cytometry analyses were performed with FACScan (10,000 cells per sample) (Becton-Dickinson, Mountain View, CA). After correction for nonspecific binding (isotype control), specific mean fluorescence intensity was measured.

RNA-Isolation and Reverse Transcriptase–PCR
Total RNA from TECs was extracted by Chomczynski‘s method, using TRIZOL (Life Technologies SRL). After isolation, the integrity of RNA samples was checked by gel electrophoresis in 1% agarose gel stained with ethidium bromide. Specific cDNA was reverse transcribed from 1 µg RNA in 20 µl of reaction mixture containing 2.5 mmol/L dATP, dTTP, dCTP, and dGTP, 100 pmol/L random hexamers, and 20 U of MMLV (Roche, Mannheim, Germany). Incubation was carried out at 37°C for 60 min. An aliquot of cDNA was disolved in 40 µl of the reaction mixture containing 10 x PCR buffer (final Mg²⁺ concentration, 1.5 mmol/L), 2.5 mmol/L dATP, dTTP, dCTP, and dGTP, 10 pmol/L upstream and downstream primers (IL-6: 5'-ATGAACTCCTTCTCCACAAGCGC-3' and 5'-GAAGAGCCCTCAGGCTGGACTG-3'; ICAM-1: R&D Systems [see manufacturer‘s information]; GAPDH: 5'-CCACCCATGGCAAATTCCATGGCA-3' and 5'-TGCTAAGCAGTTGGTGGTGCAGGAG-3') and 1 U Taq polymerase (Roche). The amplification profile consisted of an initial denaturation at 94°C for 5 min followed by denaturation at 94°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 60 s. For semiquantitative analyses, the linearity of amplification of IL-6, ICAM-1, and GAPDH cDNA depending on cycle number was established in pilot experiments. It was 28 cycles for IL-6, 30 for ICAM-1, and 30 for GAPDH. Amplification products were electrophoretically separated in agarose gel (containing ethidium bromide) and quantified by use of a densitometer (Bio-Rad Laboratories, München, Germany). GAPDH was used as an internal standard. The IL-6 signal was normalized by comparison with the GAPDH signal from the same sample, and values expressed as x-fold change compared with unstimulated controls (1.0).

CCR2 RT-PCR
Total RNA from TECs and monocytes was extracted, and specific cDNA was reverse transcribed from 1 µg of RNA as described by Denger et al. (16). Primers were synthesized according to published sequences (17). PCR products were analyzed by gel electrophoresis, and PCR products were cloned and sequenced by MWG (Ebersberg, Germany).

Biotinylated MCP-1 Binding Studies
Binding of MCP-1 to human TECs was measured using Fluorokine kits (R&D Systems) according to the manufacturer‘s instructions. Briefly, cells were grown to subconfluence and then depleted of serum for 24 h. Cells were harvested with trypsin/EDTA, washed thrice, and resuspended in PBS. Biotinylated MCP-1 or negative control protein was incubated at 4°C with 1 x 10⁵ cells, and binding was detected by using avidin-FITC. Cells were washed and analyzed by flow cytometry on a FACScan. The specificity of MCP-1 binding was tested either by preincubating the biotinylated MCP-1 with anti-human MCP-1 antibody for 15 min at 37°C or by the addition of 100-fold excess of nonbiotinylated MCP-1.

NF-B and AP-1 Electrophoretic Mobility Shift Assay (EMSA) Preparation of Nuclear Extracts
For EMSA, nuclear protein extracts were prepared according to the method of Schreiber et al. (18), with minor modifications. After MCP-1 stimulation, 2 x 10^-6 cells were washed twice with PBS (4°C) and scraped into 400 µl of hypotonic buffer (10 mmol/L Hepes, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 2 mmol/L dithiothreitol [DTT]) supplemented with proteinase and phosphatase inhibitors (5 µg/ml E-64, 1 mmol/L NaF, 0.2 mmol/L Na₃VO₄, 0.5 mg/ml Pefabloc), incubated for 15 min on ice; thereafter, 25 µl of 10% NP-40 was added and the tubes were vigorously vortexed for 10 s. The nuclei were recovered by centrifugation (14,000 rpm; 1 min; 4°C). The nuclear pellets were resuspended in 50 µl of cold buffer C (20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 2 mmol/L DTT supplemented with 5 µg/ml E-64, 1 mmol/L NaF, 0.2 mmol/L Na₃VO₄, 0.5 mg/ml Pefabloc), and the tubes were vortexed for 15 min at 4°C. After centrifugation (14,000 rpm; 5 min; 4°C), the supernatants containing nuclear protein were collected and stored at -80°C until used.

AP-1 Gel Mobility Shift Assay
Nuclear extracts (2 µg each) were incubated with labeled oligonucleotide probes and 2 µg of poly(deoxyinosine-deoxycytidine)-poly(deoxyinosine-deoxycytidine) in 20 µl of binding buffer (60 mmol/L HEPES, pH 7.9, 50% glycerol, 20 mmol/L Tris-HCl, pH 8.0, 300 mmol/L KCl, 5 mmol/L EDTA, 100 µg/ml BSA, 2.5 mg/ml Pefabloc, 25 µg/mL E-64, 5 mmol/L NaF, 1 mmol/L Na₃VO3, 5 mmol/L DTT for 5 min at RT). The sequence of the double-stranded oligonucleotide used was as follows: consensus AP-1, 5'-CGCTTGATGACTCAGCCGGAA-3'. The oligonucleotides were labeled with [³²P]-ATP by using T4 polynucleotide kinase. Binding reactions were resolved on a 4% native polyacrylamide gel containing 1 x TAE buffer (25 mmol/L Tris, 25 mmol/L boric acid, 0.5 mmol/L EDTA). Gels were run at 150 V in a cold room (4°C) for 2 to 3 h, dried, and exposed to x-ray film for 12 to 24 h. In addition, a supershift assay for AP-1 was carried out using rabbit polyclonal antibodies against c-Jun and c-Fos. The specific antibodies were incubated with samples after the initial binding reaction between nuclear protein extracts and ³²P-labeled consensus oligonucleotide (1 h at RT).

NF-B Electrophoretic Mobility Shift Assay
Protein extracts from TECs were prepared as follows. After MCP-1 stimulation, 2 x 10⁶ cells were washed twice with PBS (4°C), scraped off the tissue culture dish, and sedimented by centrifugation. The cell pellet was lysed in lysis-buffer (20 mmol/L HEPES-KOH (pH 7.9), 0.35 mol/L NaCl, 20% glycerol, 1% NP-40, 1 mmol/L MgCl₂, 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 10 µg/ml leupeptin, 0.5 mmol/L DTT, 0.2 mmol/L PMSF) during an incubation for 30 min on ice. After centrifugation, the supernatants containing the protein fraction were collected and stored at -80°C until used.

For electrophoretic mobility shift assay, protein extracts (10 µg each) and labeled oligonucleotides (50,000 to 70,000 cpm) were incubated for binding of active NF-B for 20 min at RT in a buffer containing 20 µg poly(deoxyinosine-deoxycytidine)-poly(deoxyinosine-deoxycytidine), 8% Ficoll 400, 44 mmol/L HEPES-KOH (pH 7.9), 140 mmol/L KCl, 4% glycerol, 0.05% NP-40, 0.1 mmol/L EDTA, 4.4 mmol/L DTT, and 0.06 mmol/L PMSF. Immediately after binding, the protein/DNA complexes were separated from unbound oligonucleotides by electrophoresis on a native 5% polyacrylamide gel in 1 x TAE buffer. Autoradiography was performed with the dried gels. For testing of NF-B/DNA binding specificity, (1) antibodies against p65 or p50 subunits of NF-B were added to the proteins for supershift assay, and (2) a 20-fold molar excess of unlabeled oligonucleotide was added to the binding reaction, leading to a decrease in NF-B–bound radioactivity and ³²P-labeled consensus oligonucleotide (1 h at RT).

Western Blot Analyses for I-B
Quiescent TECs were stimulated with MCP-1. After stimulation, cells were washed twice with ice-cold PBS (4°C), lysed with lysis buffer (4°C; 20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L Na₃VO₄, 1 µg/ml leupeptin, 1 mmol/L PMSF), scraped off the dish, sonicated, and centrifuged (13,000 rpm; 4°C; 10 min). Protein concentrations were determined by using a bicinchoninic acid protein assay kit from Pierce (Rockford, IL), according to the manufacturer’s protocol. The TEC lysates (10 µg per lane) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). The membranes were blocked (RT; 1 h; TBST: 20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, and 0.05% Tween 20, 5% BSA) and then incubated with primary antibodies (rabbit anti-I-B; 1 h; RT) followed by incubation with the appropriate secondary peroxidase-conjugated antibodies (1 h; RT). The proteins were detected using an enhanced chemiluminescence detection system (ECL; Amersham, Buckinghamshire, UK) according to the manufacturer’s instructions. Exposures were recorded on Hyperfilm (Amersham, UK) for different time points.

Decoy Oligodeoxynucleotide (ODN) Technique
NF-B decoy, AP-1 decoy, and mutated controls used were double-stranded phosphorothioate-oligonucleotides. Their sequences were as follows: NF-B consensus sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3'), mutated control (5'-AGTTGAGGCGACTTTCCCAGGC-3'), AP-1 consensus sequence(5'-CGCTTGATGACTCAGCCGGAA-3'), and mutated control (5'-CGCTTGATGACTTGGCCGGAA-3'). Double-stranded (ds) ODN were prepared from complementary single-stranded phosphorothioate-bonded oligonucleotides, by melting at 95°C for 5 min followed by 3- to 4-h reconstitution period at RT. TECs were preincubated with 10 µM ds ODN for 6 h. Thereafter, the oligonucleotide-containing medium was removed, cells were washed twice with medium and then incubated in fresh medium containing the stimuli for the indicated time.

Statistical Analyses
Multiple comparisons were evaluated with ANOVA, followed by Fisher‘s protected least significant difference method. Data are presented as mean ± SD, and P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of IL-6 by MCP-1 in Human TECs
To determine the inflammatory capacity of MCP-1 and other chemokines in tubulointerstitial disorders, we measured the IL-6 release of human TECs in the cell supernatant after stimulation with MCP-1, RANTES, or IL-8, respectively. Only stimulation with recombinant MCP-1 (10 ng/ml) resulted in a time-dependent IL-6 secretion that was significantly increased after 24 h (Figure 1A), peaking with a 3.8-fold increase over control cells after 48 h. To investigate whether the effect of MCP-1 was dose-dependent, human TECs were exposed to increasing concentrations of MCP-1 (0.01 to 100 ng/ml). After 48 h, the supernatants were collected and IL-6 concentrations were assessed (Figure 1B). In contrast to 10 ng/ml MCP-1, 100 ng/ml MCP-1 induced only a slight further increase. Therefore, cells in all subsequent experiments were treated with 10 ng/ml MCP-1 for 48 h. Six experiments with cells from different donors yielded similar results.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Monocyte chemoattractant protein–1 (MCP-1) induces interleukin-6 (IL-6) secretion of human tubular epithelial cells (TECs). (A) TECs (105 cells/ml) were stimulated with MCP-1 (10 ng/ml), RANTES, or IL-8, respectively. The supernatants were collected after the indicated periods of time and assayed for IL-6 concentration by enzyme-linked immunosorbent assay (ELISA). IL-6 release is shown as mean ± SD from four independent experiments; *P < 0.05. (B) Dose effect of MCP-1 on IL-6 secretion into the supernatant. TECs (105 cells/ml) were exposed to increasing doses of MCP-1 (0.01 to 100 ng/ml) for 48 h. Values are mean ± SD (n = 6); *P < 0.05.

Spontaneous Release of MCP-1 by TECs
We had found that stimulation of human TECs with MCP-1 increases IL-6 secretion into the supernatant; we therefore investigated the spontaneous release of MCP-1 by TECs into the supernatant. This addresses the question of whether TECs are able to produce sufficient amounts of MCP-1 to activate themselves in an autocrine fashion. Table 1 shows that unstimulated TECs secreted significant amounts of MCP-1 into the supernatant.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. MCP-1 induces intercellular adhesion molecule-1 (ICAM-1) expression by human TECs. (A) TECs were stimulated with MCP-1 (10 ng/ml), and ICAM-1 expression was assessed by flow cytometry after the indicated periods of time. ICAM-1 expression is shown as mean ± SD–fold change from six independent experiments compared with unstimulated controls (1.0); *P < 0.05. (B) Dose effect of MCP-1 on ICAM-1 synthesis. TECs were exposed to increasing doses of MCP-1 (0.01 to 100 ng/ml) for 24 h. Values are mean ± SD–fold change compared with unstimulated controls (1.0) from six independent experiments; *P < 0.05.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. IL-6 release and ICAM-1 expression by human TECs is MCP-1-dependent. (A) TECs were exposed to MCP-1 (10 ng/ml) and neutralizing MCP-1 antibody or control IgG (10 µg/ml each). IL-6 release was determined by ELISA. Values are mean ± SD (n = 4); *P < 0.05 compared with unstimulated controls or **P < 0.05 compared with MCP-1. (B) TECs were exposed to MCP-1 (10 ng/ml) and neutralizing MCP-1 antibody, IL-6 antibody, or control IgG (10 µg/ml each). ICAM-1 expression was assessed by flow cytometry after 24 h. Values are mean ± SD–fold change compared with unstimulated controls (n = 4); *P < 0.05 compared with unstimulated controls or **P < 0.05 compared with MCP-1.

View larger version (55K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. MCP-1 increases mRNA for IL-6 and ICAM-1 in human TECs. Cells were stimulated with MCP-1 (10 ng/ml) for the indicated time periods. Total RNA was isolated from cultured TECs, reverse transcribed, and PCR for IL-6, ICAM-1, and GAPDH were performed. Representative ethidium bromide–stained agarose gels of the RT-PCR products for IL-6 mRNA (A, top panel), ICAM-1 (B, top panel), and GAPDH (A and B, bottom panels) are shown. Results are representative for three independent experiments with cells from different donors.

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Analyses of MCP-1 receptor in TECs. (A) RT-PCR was performed using reverse-transcribed cDNA isolated from human TECs (lane 2) and monocytes (lane 3, as positive control). Using specific primers for CC chemokine receptor 2 (CCR2), PCR products were analyzed by agarose gel electrophoresis. Results are representative for three independent experiments with cells from different donors. (B) Flow cytometry analyses for the CCR2 receptor on TECs and monocytes; a, IgG control; b, CCR2 antibody; c, IgG control; d, CCR2 antibody. Results are representative for three independent experiments with cells from different donors.

An additional experiment was performed to exclude that MCP-1–induced IL-6 secretion from human TECs is mediated via the CCR2 receptor. As shown in Figure 6A, MCP-1–dependent IL-6 release from TECs was not inhibited by preincubation with the CCR2 antibody (20 µg/mL; 15 min). To further exclude that IL-6 release depends on MCP-1–binding to the CCR1, CCR3, or CCR5, respectively, we also performed preincubation experiments using specific antibodies against these CC chemokine receptors. Again, none of these antibodies blocked the effect of MCP-1 on IL-6 release from human TECs. In contrast, preincubation with the CCR2 antibody inhibited IL-6–release from human monocytes. As in TECs, the antibodies against CCR1, CCR3, or CCR5 had no effect on MCP-1–induced IL-6 release from monocytes (Figure 6B).

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. IL-6 release by human TECs is independent of CCR2. (A) TECs were preincubated with CCR1, CCR2, CCR3, or CCR5 antibody (20 µg/mL; 15 min), respectively. Therafter, cells were stimulated with MCP-1 (10 ng/mL) for 48 h. (B) Human monocytes were preincubated with CCR1, CCR2, CCR3, or CCR5 antibody (20 µg/mL, 15 min), respectively. Therafter, cells were stimulated with MCP-1 (10 ng/mL) for 24 h. IL-6 release was determined by ELISA. Values are mean ± SD (n = 4); *P < 0.05 compared with unstimulated controls or **P < 0.05 compared with MCP-1.

View larger version (32K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Flow cytometric analyses of MCP-1 binding to human TECs. Cells were incubated at 4°C with MCP-1–biotin or a biotinylated control protein, followed by avidin-FITC. Washed cells were analyzed by flow cytometry in which accumulated events were gated against the biotinylated negative control. Binding was displaced by preincubation with anti–MCP-1 antibody or coincubation with nonbiotinylated MCP-1. Results are representative for three independent experiments with cells from different donors.

View larger version (26K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. MCP-1–induced IL-6 release and ICAM-1 expression involves Gi-proteins, protein kinase C (PKC), Ca2+, and nuclear factor–B (NF-B). TECs were preincubated with pertussis toxin (PTX; 100 ng/ml; 16 h), GF109203X (2 x 10-6 mol/L; 60 min), EGTA (4 mmol/L), BAPTA-AM (10 µmol/L), or PDTC (20 µmol/L; 2 h) respectively, followed by stimulation with MCP-1 (10 ng/ml). (A) The supernatants were collected after 48 h and assayed for IL-6 concentration by ELISA. Values are mean ± SD (n = 6); *P < 0.05 compared with unstimulated controls or **P < 0.05 compared with MCP-1. (B) ICAM-1 expression was assessed by flow cytometry after 24 h. Values are mean ± SD–fold change compared with unstimulated controls (1.0) from 6 independent experiments; *P < 0.05 compared with unstimulated controls or **P < 0.05 compared with MCP-1.

MCP-1 Activates Transcription Factors, NF-B and AP-1

View larger version (64K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Effect of MCP-1 on the DNA-binding activity of NF-B and activator protein-1 (AP-1). (A) TECs were stimulated with MCP-1 (10 ng/ml) for the indicated periods of time, and electrophoretic mobility shift assay (EMSA) for NF-B was performed as described in Materials and Methods. (B) Supershift experiments with antibodies against the subunits of NF-B. EMSA for NF-B after treatment of TECs with MCP-1 (10 ng/ml) for 1 h was performed. Cellular extracts were incubated with labeled oligonucleotides and polyclonal antibodies against the p65 and p50 subunit of NF-B as described in Materials and Methods. Competition of MCP-1 induced NF-B binding activity with a 20-fold excess of unlabeled consensus NF-B oligonucleotides demonstrates specificity of MCP-1–induced DNA binding complexes (lane 2). (C) TECs were stimulated with MCP-1 (10 ng/ml) for the indicated periods of time, and EMSA for AP-1 was performed as described in Materials and Methods. Competition of MCP-1 induced AP-1 binding activity with a 20-fold excess of unlabeled consensus AP-1 or NF-B oligonucleotides demonstrates specificity of MCP-1–induced DNA binding complexes (lanes 8 and 9). (D) Supershift experiments with antibodies against subunits of AP-1. EMSA for AP-1 after treatment of TECs with MCP-1 for 2 h was performed. Cellular extracts were incubated with labeled oligonucleotides, and polyclonal antibodies against c-Jun and c-Fos as described in Materials and Methods. Representative autoradiograms of three independent experiments are shown.

Degradation of Cytoplasmic I-B
To further understand the mechanism of NF-B activation in TECs stimulated with MCP-1, the levels of I-B was determined with Western blot of cytoplasmic extracts (Figure 10). Stimulation of cells with MCP-1 led to a transient decrease of I-B within 1 h, which recovered after 2 h.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. Expression of I-B in TECs stimulated by MCP-1. TECs were stimulated with MCP-1 (10 ng/ml) for the indicated time periods, and cytoplasmic I-B levels were assayed by Western blot. A representative Western blot of three independent experiments is shown.

Significance of NF-B and AP-1 Activation for MCP-1–Induced IL-6 Synthesis

View larger version (38K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 11. NF-B and AP-1 decoy ODN technique. (A) Human TECs were preincubated with NF-B decoy ODN (10 µM) or control decoy ODN (10 µM) for 6 h, followed by stimulation with MCP-1 (10 ng/ml) for 1 h. EMSA for NF-B was performed. (B) TECs were preincubated with AP-1 decoy ODN (10 µM) or control decoy ODN (10 µM) for 6 h, followed by stimulation with MCP-1 (10 ng/ml) for 2 h. EMSA for AP-1 was performed. Representative autoradiograms of three independent experiments are shown.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 12. Transcription factor–dependent IL-6 and ICAM-1 expression of human TECs. Human TECs were preincubated with NF-B decoy ODN (10 µM), NF-B control decoy ODN (10 µM), AP-1 decoy ODN (10 µM), or AP-1 control decoy ODN (10 µM), respectively, for 6 h, followed by stimulation with MCP-1 (10 ng/ml). (A) The medium was collected after 48 h and assayed for IL-6 concentration by ELISA. Values are mean ± SD (n = 5); *P < 0.05 compared with unstimulated controls or **P < 0.05 compared with MCP-1. (B) ICAM-1 expression was assessed by flow cytometry after 24 h. Values are mean ± SD–fold change compared with unstimulated controls (1.0) from six independent experiments; *P < 0.05 compared with unstimulated controls or **P < 0.05 compared with MCP-1.

Significance of NF-B and AP-1 Activation for MCP-1-Induced ICAM-1 Expression

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we demonstrate for the first time that MCP-1 activates human TECs, leading to a time- and dose-dependent increase in IL-6 secretion and ICAM-1 expression via Gi-protein-, PKC-, and intracellular Ca²⁺-dependent mechanisms. MCP-1 activates (1) NF-B, a transcription factor commonly involved in inflammatory and immune responses, and (2) AP-1, a transcription factor involved in inflammatory and growth responses. This suggests that in addition to acting as a chemoattractant, MCP-1 may further increase the inflammatory response by inducing cytokine and adhesion molecule expression in human TECs.

The CC chemokine MCP-1 appears to play a predominant role in the pathogenesis of renal diseases. Wada et al. (22) demonstrated that the expression of MCP-1 is upregulated in TECs, endothelial cells, and mononuclear cell infiltrates in human diabetic nephropathy. In addition, urinary MCP-1 levels are significantly elevated in patients with nephrotic proteinuria due to diabetic nephropathy with advanced tubulointerstitial lesions (22). Renal proximal TECs have been reported to produce MCP-1 in response to cytokines (7,23) and urinary proteins (24). Thus TECs, in addition to mesangial cells and infiltrating mononuclear cells (25), might contribute to the increased urinary excretion of MCP-1. Urinary MCP-1 levels reflect the disease activity of lupus nephritis (26) and correlate with the extent of proteinuria and the number of glomerular macrophages in various glomerular diseases in humans (27). Glomerular expression of MCP-1 has been documented in experimental and human glomerulopathies (28,29). Administration of antibodies to MCP-1 decreases the extent of proteinuria, reduces glomerulosclerosis, and improves renal dysfunction in experimental crescentic glomerulonephritis (30). Furthermore, MCP-1 plays a role in hypertensive renal damage in the two-kidney one-clip rat model (31), which is possibly mediated by angiotensin II (32). Interestingly, MCP-1–deficient mice compared with wild-type mice exhibit no differences in glomerular lesions in nephrotic serum-induced nephritis, but exhibit less tubulointerstitial lesions (33). These reports support an important role of locally produced MCP-1 on the initiation and progression of renal damage, particularly tubulointerstitial damage.

However, besides induction of monocyte recruitment, no study demonstrated that MCP-1 directly induces proinflammatory responses in resident renal cells such as TECs.

Tubulointerstitial inflammation, often followed by fibrosis, has been proposed as a final common pathway for progressive renal injury in most renal diseases. There is a strict correlation between tubular atrophy, interstitial fibrosis, the extent of interstitial infiltrates, and renal dysfunction (1). TECs, once considered passive bystanders in the disease process, have been shown to be actively involved being a rich source of cytokines, chemokines, and others inflammatory mediators (3,7,9,22,23). For the following reasons, we investigated the pro-inflammatory cytokine IL-6 and the adhesion molecule ICAM-1 as relevant markers of inflammatory activation in human TECs. Within the kidney, elevated levels of IL-6 have been demonstrated in both resident and infiltrating cells in various forms of glomerulonephritis and tubulointerstitial nephritis. IL-6 has been suggested to contribute to the pathogenesis and progression of renal diseases (4,34). The degree of mesangial hyperproliferation, tubular atrophy, and the intensity of interstitial infiltrates correlate to the renal expression of IL-6 (35,36). Furthermore, urinary IL-6 excretion correlates to disease progression and increases with the degree of tubular dysfunction (37). De novo expression of the adhesion molecule ICAM-1 by TECs and increased expression by interstitial and glomerular cells has been observed in different forms of glomerulonephritis, tubulointerstitial inflammation, and renal allograft rejection (8).

This study shows that MCP-1 was able to induce IL-6 release and the synthesis of ICAM-1 by human TECs. These effects were specific for MCP-1 and could be induced by neither the CC chemokine, RANTES, nor the CXC chemokine, IL-8. Consistent with previous reports (6,7), unstimulated human TECs release significant amounts of MCP-1 into the supernatant. Stimulation of TECs with MCP-1 from the apical and the basolateral compartment was equally effective in stimulating IL-6 secretion. Thus, both urinary MCP-1 as well as MCP-1 generating from the tubulointerstitial compartment may be able to activate TECs in vivo. TECs secreted approximately fourfold higher amounts of IL-6 into the basolateral compartment as compared with the apical compartment. This implicates that MCP-1–induced IL-6 secretion into the tubulointerstitium may be a player in the genesis of tubulointerstitial inflammation in vivo. IL-6 modulates the expression of ICAM-1 in other culture systems (38); therefore, the role of IL-6 as a mediator of ICAM-1 expression was examined. Although MCP-1 induced a significant increase of IL-6 release into the culture medium, blockade of the effects of IL-6 by a monoclonal antibody did not modify the increase of MCP-1–induced ICAM-1 expression. Therefore, the stimulation of the expression of ICAM-1 by human TECs in vitro does not depend on the autocrine secretion of IL-6. Furthermore, pharmacologic doses of recombinant IL-6 had no effect on the expression of ICAM-1 by human TECs (data not shown).

The MCP-1 receptors belong to the family of heptahelical, pertussis-sensitive G-protein–coupled receptors (39). The MCP-1 receptor on TECs appears to be coupled to Gi-protein activation, because the induction of IL-6 and ICAM-1 was inhibited by PTX. These findings are in accordance with our observations in VSMC (Viedt et al., unpublished observation) and the data of Myers et al. (39) and Schecter et al. (12). The latter have shown that signal transduction of the human MCP-1 receptor could be blocked by PTX in VSMC and embryonic kidney cells transfected with the MCP-1 receptor, respectively. To investigate the role of PKC in MCP-1–induced upregulation of IL-6 and ICAM-1, we preincubated TECs with the PKC antagonist, G109203X, which resulted in inhibition of MCP-1–induced IL-6 secretion and ICAM-1 expression. MCP-1 has been shown to mobilize Ca²⁺ in a number of studies (12,16,39). Schecter et al. (12) demonstrated that the induction of tissue factor by MCP-1 in human VSMC required intracellular Ca²⁺ (Ca²⁺_i) mobilization and that it was PKC-dependent. Similarly, the present study demonstrates that MCP-1–induced IL-6 and ICAM-1 expression is Ca²⁺_i-dependent. Therefore, although currently unproven, it is likely that MCP-1 signaling is mediated by the classical PKC subgroups , , or .

Due to our above observations, we anticipated that cultured human TECs possess a functionally coupled MCP-1 receptor. To date, expression of CCR1–5 mRNA transcripts in renal tissue of humans and experimental animals were detected only in infiltrating mononuclear cells (9,40,41). Two MCP-1 receptors, generated by alternative splicing and designated as CCR2A and CCR2B, have been cloned in human monocytes (17). On the basis of our PCR studies, the MCP-1 receptor on human TECs is distinct from these two receptors. In addition, it is unlikely that the MCP-1 receptor on human TECs is generated by alternative splicing of the same gene, because no signal was seen on RNA blot analyses at moderate stringency washings using a random-primed probe encompassing a substantial part of the coding region (data not shown). Additional PCR studies employing primers from other cloned human CC chemokine receptors also failed to produce a signal. These negative results were confirmed by using flow cytometry analyses for the detection of the CCR2 on TECs. In contrast, the CCR2 could be detected on monocytes using PCR and flow cytometry analyses, respectively. Preincubation with a specific CCR2 antibody inhibited MCP-1–induced expression of IL-6 and ICAM-1 in monocytes, but not in TECs. Antibodies against CCR1, CCR3 and CCR5 failed to inhibit the effects of MCP-1 in both monocytes and TECs. Therefore, we performed binding studies for MCP-1. These studies clearly show that cultured human TECs express a MCP-1 binding protein on the cell surface. The data obtained after incubation of human TECs with biotinylated MCP-1 in the presence of a neutralizing antibody against MCP-1 or competition with 100-fold excess of nonbiotinylated MCP-1 support the notion that the effects observed are specific for MCP-1. Since we were not able to characterize a classic receptor for MCP-1 on TECs, but could prove binding of MCP-1, we investigated whether the effects of MCP-1 are mediated via an endosome-lysosomal pathway. The latter could be ruled out by the negative results of pharmacologic blockade of the endosome-lysosomal pathways on MCP-1-induced IL-6 mRNA expression in TECs (data not shown). Taken together, our results implicate that it is likely that the MCP-1 receptor on human TECs is different from previously cloned CC chemokine receptors. The nature of the MCP-1 receptor(s) on human TECs remains to be determined.

Previous studies focused on induction of MCP-1 by inflammatory agents in renal diseases and TECs in vivo and in vitro (9). The present data provide evidence that MCP-1 itself—through production of IL-6 in response to NF-B and AP-1 activation—acts as an inflammatory mediator for TECs. Several studies have shown that NF-B plays an important role in renal damage (42). Ruiz-Ortega et al. (43) found that activation of NF-B and MCP-1 in the renal cortex is reduced by angiotensin-converting enzyme inhibition in experimental immune complex nephritis. Renal damage in rats harboring both human renin and angiotensinogen genes is accompanied by the activation of NF-B and AP-1 in the kidney (44). Filtered albumin is able to activate NF-B in the kidney in vivo (45) and TECs in vitro (24,46).

IL-6 and ICAM-1 syntheses are regulated at the transcriptional level. Previous reports indicated that the NF-B binding site located between positions -72 and -63 on the IL-6 gene is important for the induction of IL-6 (20). AP-1 is another important transcription factor that is involved in regulation of IL-6 transcription. AP-1 has a consensus binding sequence found in position -283 to -277 in the IL-6 promoter (19). NF-B dimers do not promote gene transcription by themselves, but as a part of a complex of several coactivators (47). Moreover, NF-B interacts with a variety of other transcription factors in a positive or negative manner. One of the factors most commonly involved in the activation of NF-B target genes is AP-1. Both NF-B and AP-1 are activated in response to some proinflammatory stimuli, but they differ in their response to oxidative stress (48).

We demonstrated in this study that MCP-1 (1) increased IL-6 and ICAM-1 expression and (2) stimulated NF-B (mediated, at least partially, by degradation of I-B) and AP-1 activation. Therefore, we reasoned that MCP-1 stimulates IL-6 gene expression through the NF-B and AP-1 complexes. To prove this hypothesis, we employed the decoy approach against NF-B and AP-1 binding sites, respectively. Gel mobility shift assay showed that decoy against NF-B or AP-1 binding sites specifically competed, whereas control decoy ODN did not. Next, we examined the functional coupling between NF-B and AP-1 activation and MCP-1–induced IL-6 synthesis. Our results show that NF-B, and to a lesser extent AP-1 decoy ODN, effectively inhibit IL-6 production in response to MCP-1. On the other hand, control decoy had no effect. This clearly shows that NF-B and AP-1 play an important role in MCP-1–induced IL-6 secretion. In contrast, MCP-1–induced ICAM-1 expression was only dependent on NF-B activation, whereas AP-1 decoy activation had no significant effect. These data suggest that, despite MCP-1–induced AP-1 binding, AP-1 activation is not required for MCP-1–dependent induction of ICAM-1 expression. These results are in agreement with a report from Papi and Johnston (49), which shows that rhinovirus infection of airway epithelium results in AP-1 and NF-B activation. Despite activation of both transcription factors, only the NF-B binding site was required for rhinovirus-dependent ICAM-1 expression (49).

In conclusion, our data demonstrate that besides its chemotactic property, MCP-1 promotes proinflammatory responses in human TECs. MCP-1 is able to directly upregulate the proinflammatory cytokine IL-6 and the adhesions molecule ICAM-1. The MCP-1–induced intracellular signaling mechanism involves Gi-protein, PKC, intracellular Ca²⁺, and classical inflammatory pathways, including sequence-specific DNA binding of the nuclear factors, NF-B and AP-1. Thus, MCP-1 must be regarded as more than just a "chemokine," but as a proinflammatory mediator inducing production of proinflammatory molecules.

	Acknowledgments

 
We thank Ms. Katharina Hanna for excellent technical assistance.

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