Expression of CX3CL1/fractalkine by mesangial cells in vitro and in acute anti-Thy1 glomerulonephritis in rats

Yung-Ming Chen, Mi-I Hu-Tsai, Shuei-Liong Lin, Tun-Jun Tsai and Bor-Shen Hsieh

Department of Internal Medicine, National Taiwan University Hospital and College of Medicine National Taiwan University, Taipei, Taiwan

Correspondence and offprint requests to: Dr Tun-Jun Tsai, Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, 10016, Taiwan. Email: paul{at}ha.mc.ntu.edu.tw



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Mesangial cells (MCs) can promote glomerular macrophage accumulation in glomerulonephritis through production of a variety of chemokines. This study investigated the potential of MCs to synthesize CX3CL1/fractalkine, a CX3C chemokine, both in vitro and in acute anti-Thy1 glomerulonephritis in rats.

Methods. Anti-Thy1 glomerulonephritis was induced in Wistar rats by a single injection of mouse anti-rat Thy1.1 antibody intravenously. Glomerular mRNAs for CX3CL1/fractalkine, CCL2/monocyte chemoattractant protein (MCP)-1, and their cognate receptors, CX3CR1 and CCR2, were determined by northern blot analysis or reverse-transcription polymerase chain reaction. CX3CL1/fractalkine mRNA and protein expression in vivo was localized by in situ hybridization and immunohistochemistry. Monocytes/macrophages and activated MCs were detected by immunohistochemistry. Regulation of CX3CL1/fractalkine expression in cultured MCs was determined by northern and western blot analysis.

Results. After induction of anti-Thy1 disease, glomerular CX3CL1/fractalkine mRNA was significantly up-regulated, peaking at 2 h and sustaining into day 5 of the nephritis. A corresponding increase in urinary CX3CL1/fractalkine protein was evident after day 1 of the nephritis, but became more prominent during the MC proliferative phase (days 3–5). Meanwhile, induction of glomerular CCL2/MCP-1 mRNA and urinary CCL2/MCP-1 protein occurred within 24 h, and was barely detectable after day 3 of the nephritis. Urinary CCL2/MCP-1, but not CX3CL1/fractalkine, correlated with glomerular macrophage accumulation (r = 0.936, P<0.01) and glomerular CCR2 mRNA expression (r = 0.965, P<0.01). In contrast, only urinary CX3CL1/fractalkine coincided temporally to glomerular mRNA for CX3CR1 (r = 0.809, P < 0.01). Combined in situ hybridization and immunohistochemistry revealed that activated MCs were a major source for CX3CL1/fractalkine mRNA and protein during days 3–5 of the nephritis. Incubation of cultured MCs with tumour necrosis factor (TNF)-{alpha}, interleukin (IL)-1ß, platelet-derived growth factor (PDGF)-AB or basic fibroblast growth factor (bFGF) significantly up-regulated CX3CL1/fractalkine mRNA and protein expression. This cytokine- and growth factor-stimulated CX3CL1/fractalkine expression could be abolished by the nuclear factor-{kappa}B inhibitors, curcumin and MG132.

Conclusions. Our data demonstrate that activated MCs are a source for the augmented glomerular CX3CL1/fractalkine expression during the proliferative phase of acute anti-Thy1 glomerulonephritis. Up-regulation of MC CX3CL1/fractalkine by TNF-{alpha}, IL-1ß, PDGF-AB and bFGF is mediated, at least in part, via the nuclear factor-{kappa}B signalling pathway. The differential expression of CCL2/MCP-1 and CX3CL1/fractalkine may sequentially recruit distinct subsets of monocytes to the glomerulus during acute anti-Thy1 glomerulonephritis.

Keywords: anti-Thy1 glomerulonephritis; CCL2/MCP-1, CCR2; CX3CL1/fractalkine; CX3CR1; mesangial cells



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Glomerular monocyte/macrophage infiltration is an early pathobiologic event in various forms of human and experimental glomerulonephritis [1]. The migration of monocytes into renal glomerulus involves a cascade of molecular and cellular events including elaboration of adhesion molecules and chemokines, and interactions of monocytes with intrinsic glomerular cells [2,3]. Among the chemokine superfamily, CX3CL1/fractalkine is unique in that it exists as two distinct forms. The cell-bound protein contains a N-terminal CX3C chemokine domain attached to a mucin-like transmembrane stalk, and displays adhesive properties [4]. The soluble molecule, in contrast, is cleaved from the cell membrane and functions as a chemoattractant [4]. CX3CL1/fractalkine acts by binding to the G-protein-coupled receptor, CX3CR1, in receptive mononuclear leukocytes [5]. In addition to vascular endothelium, a number of non-endothelial tissue cells also express CX3CL1/fractalkine, which includes rat mesangial cells (MCs) [611]. Thus far, it is not known whether MC can produce CX3CL1/fractalkine in vivo. Previously, Ito et al. [12] have reported the presence of CX3CL1/fractalkine protein within the glomerular mesangium in an uninephrectomized prolonged model of anti-Thy1 glomerulonephritis. However, they did not provide additional information regarding the cellular source for the mesangial CX3CL1/fractalkine expression. Because of the perceived importance of MCs in the development of glomerular diseases, this study investigated: (i) the potential of MCs to synthesize CX3CL1/fractalkine in vivo in rat anti-Thy1 disease—an experimental model of acute mesangial proliferative glomerulonephritis characterized by intense mesangiolysis followed by rebound MC proliferation and (ii) the regulation of MC CX3CL1/fractalkine expression in vitro in response to various cytokines and growth factors.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents
RPMI 1640 media, fetal calf serum (FCS) and other tissue culture reagents were obtained from Gibco BRL (Rockville, MD, USA). Culture flasks and plates were purchased from Costa Corning (Cambridge, MA, USA). Mouse anti-rat Thy1.1 (CD90) monoclonal antibody was obtained as lyophilized ascites from Cedarlane (Ontario, Canada). Mouse anti-rat ED-1 (monocytes/macrophages) monoclonal antibody was purchased from Chemicon (CA, USA), mouse anti-rat {alpha}-smooth muscle actin (SMA) monoclonal antibody was purchased from Sigma (MO, USA), and goat anti-rat CX3CL1/fractalkine polyclonal antibody was obtained from R & D Systems (MN, USA). Mouse anti-c-Jun, anti-phospho-c-Jun and anti-ß-actin monoclonal antibodies, and rabbit anti-inhibitory protein of nuclear factor (NF)-{kappa}B{alpha} (I-{kappa}B{alpha}) polyclonal antibody were obtained from Santa Cruz Biotechnologies (CA, USA). Rabbit anti-rat CCL2/monocyte chemoattractant protein (MCP)-1 was purchased from PetroTech EC LTD (London, UK). The avidin–biotin–peroxidase reagent, 3-amino-9-ethylcarbazole chromogen (AEC) and diaminobenzidine (DAB) tablets for immunological colour development were purchased from Dakopatts (Glostrup, Denmark). Reagents for immunological detection in in situ hybridization (NBT [nitroblue tetrazolium] and BCIP [5-bromo-4-chloro-3-indolyl-phosphate]), in northern blot analysis (CSPD®), and in western blot analysis (Renaissance®) were obtained from Boehringer Mannheim (Mannheim, Germany) and NEN® Life Science (MA, USA). Rat tumour necrosis factor (TNF)-{alpha}, interleukin (IL)-1ß and human platelet-derived growth factor (PDGF)-AB, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-ß1 were purchased from R & D Systems. Curcumin and MG132 were obtained from Calbiochem (La Jolla, CA, USA). All chemicals used for total RNA isolation, reverse transcription-polymerase chain reaction (RT–PCR), and northern blot analysis were of molecular grade and were obtained from Sigma or Boehringer Mannheim unless otherwise specified.

Animals
Male Wistar rats weighing 180–220 g were obtained from the animal centre of the College of Medicine of National Taiwan University. The anti-Thy1 glomerulonephritis was induced as described previously [13]. Briefly, the control rats (group A, n = 10) received only 0.2 ml of 1x phosphate-buffered saline (PBS, pH 7.4) intravenously at the beginning of experiment. The nephritic rats (group B, n = 40) received 250 µg of a mouse anti-rat Thy1.1 antibody in 0.2 ml of 1x PBS intravenously at the beginning of experiment. Animals were killed at various time points (2 h, and days 1, 3 and 5) after induction of the nephritis. Renal tissues were prepared for isolation of glomerular total RNA, immunohistochemical staining, or in situ hybridization. The investigation complied with the standards delineated in the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (Bethesda, MD, USA).

Cell cultures
Primary culturing of rat MCs was performed as described previously [14]. Rat MCs were characterized on the basis of the presence of {alpha}-SMA and Thy1.1 antigen with the avidin–biotin–peroxidase using DAB as the chromogen. Rat MCs between 10 and 20 passages were used and grown in RPMI 1640 media containing 10% FCS and 15 µg/ml of insulin.

Northern blot analysis and RT–PCR
Total RNA were extracted from isolated glomeruli and cultured cells by the acid guanidinium thiocyanate phenol–chloroform method as described previously [13,14]. Ten to twenty micrograms of total RNA were electrophoresed on formaldehyde-denatured 1% agarose gels and subsequently transferred to nylon membranes according to standard protocols. To synthesize riboprobes, cDNA fragments of rat CX3CL1/fractalkine, rat CCL2/MCP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were first amplified by RT–PCR from glomerular RNA of nephritic rats, as described previously [13,14], using the following specific primer pairs. Rat CX3CL1/fractalkine, upstream 5'-ATTTTCCAAGACAGAGGACC-3', downstream 5'-GAAGAGTAGACCAAGAAAGG-3' (GenBank accession number AF030358); rat CCL2/MCP-1, upstream 5'-TCAGCCAGATGCAGTTAATG-3', downstream 5'-TTCTCTGTCATACTGGTCAC-3' (GenBank accession number M57441); and rat GAPDH, upstream 5'-TCATTGACCTCAACTACATG-3', downstream 5'-CAAAGTTGTCATGGATGACC-3' (GenBank accession number NM_017008). The PCR products were then eluted from polyacrylamide gels, and subcloned into pGEM-T vectors (Promega). The accuracy of the inserts was confirmed by DNA sequence analysis. The cloned cDNAs were then linearized and used as templates for in vitro transcription of antisense digoxigenin-conjugated riboprobes, following the manufacturer’s instructions (Boehringer Mannheim). After hybridization, the blots were developed using CSPD® as the substrate for alkaline phosphatase. The intensity of the signal was then quantified with computerized densitometry, and normalized against the signal of GAPDH messages. Separate experiments were performed to detect glomerular mRNAs for CX3CR1 and CCR2 (receptor for CCL2/MCP-1), using the following primers: rat CX3CR1, upstream 5'-GCTGGAAGGCATAATTACAT-3', downstream 5'-TTGGGACTCATAATGGAAAG-3' (GenBank accession number U04808); rat CCR2, upstream 5'-AGATGATCAGCATACTTGTG-3', downstream 5'-AATGATAGGATTAACGCAGC-3' (GenBank accession number U77349).

Immunohistochemical staining of macrophages
Immediately after the rats were killed, 150 ml of ice-cold 0.9% saline was perfused into the heart followed by 150 ml of ice-cold 4% paraformaldehyde (PFA) in 1x PBS. The kidneys were then removed and cut coronally into 9 µm-thick slices, and immersed into 4% PFA at 4°C overnight. The next day, PFA solutions were decanted and tissues immersed in 30% sucrose in 1x PBS at 4°C overnight. Tissues were then mounted in Tissue-Tek® O.C.T. compound (Miles, IN, USA) in liquid nitrogen and stored at -70°C until cryostat sectioning. Immunohistochemical staining was performed using the avidin–biotin–peroxidase method as described previously [13]. The sections were stained with a mouse anti-rat ED-1 monoclonal antibody and detected with peroxidase substrate containing DAB.

In situ hybridization for CX3CL1/fractalkine mRNA
Rat CX3CL1/fractalkine anti-sense and sense riboprobes were synthesized from cloned rat CX3CL1/fractalkine cDNA fragment in pGEM-T vector and filtered through Chroma Spin®-100 columns (Clontech, CA, USA) to separate unincorporated digoxigenin-11-UTPs from labelled riboprobes. The in situ hybridization was then performed as described previously with minor modifications [15]. Briefly, after permeabilization with proteinase K, sections were treated with 0.2 M HCl and acetylated with 0.1 M triethanolamine containing 0.25% acetic anhydride. Sections were then incubated with pre-hybridization buffer at 42°C for 2 h, followed by hybridization buffer containing 5 µg/ml of digoxigenin-labelled riboprobe at 4°C overnight in a humid chamber. The following day, sections were washed for 15 min twice in 2x SSC and 15 min twice in 0.1x SSC at 50°C, blocked with 10% skimmed milk, and then incubated with anti-digoxigenin-alkaline phosphatase solution at room temperature for 1 h. Sections were finally visualized with NBT and BCIP as the manufacturer’s instructions.

Identification of activated MCs expressing CX3CL1/fractalkine mRNA and protein in vivo
Separate experiments were performed to demonstrate cellular source of CX3CL1/fractalkine mRNA by combined in situ hybridization and immunohistochemistry. After in situ detection of CX3CL1/fractalkine mRNA, sections were stained with a mouse anti-{alpha}-SMA monoclonal antibody, and detected by the avidin–biotin–peroxidase method using AEC as substrate. For double immunohistochemistry, the sections were first stained with a goat anti-rat CX3CL1/fractalkine polyclonal antibody and detected with AEC, followed by staining with a mouse anti-{alpha}-SMA monoclonal antibody and detected with DAB enhanced with 1% ammonium nickel sulfate.

Western blot analysis
Immunoblot analysis was performed to detect CX3CL1/fractalkine protein in glomeruli isolated from control and anti-Thy1 nephritic rats [13], and in MCs treated with TNF-{alpha} (5 ng/ml), IL-1ß (10 ng/ml), PDGF-AB (50 ng/ml), bFGF (100 ng/ml) or TGF-ß1 (5 ng/ml), with or without pre-incubation with the NF-{kappa}B inhibitors, curcumin (40 µM) and MG132 (10 µM). Briefly, glomeruli or cells were first washed with ice-cold 1x PBS and lysed at 4°C for 15 min in lysis buffer (pH 7.4) containing 50 mM Tris–HCl, 150 mM NaCl, 1% Igepal CA-630 (Sigma), 0.25% sodium deoxycholate (Sigma), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate and 1 µg/ml each for aprotinin, leupeptin and pepstatin. Forty micrograms of protein extracts were heated at 100°C for 10 min, applied to SDS–polyacrylamide gels, and electrophoresed. For detection of urinary CCL2/MCP-1, and soluble CX3CL1/fractalkine in the urine and conditioned media, samples were first concentrated with Centricon-10® (Millipore, Bedford, MA, USA), and 50 µg of protein were electrophoresed. A pre-stained standard was also electrophoresed as a molecular weight marker. After electrophoresis, proteins were transferred onto a PVDF membrane (Millipore) using a transblot chamber with Tris buffer at 4°C. The membranes were incubated with goat anti-CX3CL1/fractalkine, rabbit anti-CCL2/MCP-1, anti-I-{kappa}B{alpha} or mouse anti-c-Jun, anti-phospho-c-Jun, anti-{alpha}-SMA, anti-ß-actin at 4°C overnight. The following day, membranes were washed with 1x PBS/0.5% Tween-20 for 10 min twice, 1x PBS/0.5%Tween-20/5% skimmed milk for 10 min twice, and incubated with peroxidase-conjugated anti-goat IgG (for CX3CL1/fractalkine), anti-rabbit IgG (for CCL2/MCP-1 and I-{kappa}B{alpha}), or anti-mouse IgG (for c-Jun, phospho-c-Jun, {alpha}-SMA and ß-actin) at room temperature for 1 h. After washing, the membranes were incubated with enhanced chemiluminescent reagent (Renaissance®) according to the manufacturer’s instructions.

Statistics
Data are expressed as mean ± SEM. All comparisons were done by analysis of variance or by the Spearman’s rank correlation analysis using the StatView’s package for the Macintosh computer (Abacus Concepts, CA, USA). A P-value of <0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of CX3CL1/fractalkine and CCL2/MCP-1 in anti-Thy1 glomerulonephritis
Northern blot analysis demonstrated that an ~3.5 kb CX3CL1/fractalkine mRNA was up-regulated in the glomerulus of nephritic rats, which peaked (a mean of 4.6-fold over control) at 2 h and sustained into day 5 of the nephritis (Figure 1A). Surprisingly, the augmented glomerular CX3CL1/fractalkine mRNA expression was not paralleled by a corresponding increase in glomerular CX3CL1/fractalkine protein (~90 kDa) until 72 h after induction of the nephritis (Figure 1B). Similarly, induction of urinary CX3CL1/fractalkine (~75 kDa) was most prominent during days 3–5 (the proliferative phase) of the nephritis (Figure 1C). For comparison, we examined glomerular mRNA expression of the CC chemokine, CCL2/MCP-1. The glomerular CCL2/MCP-1 mRNA was maximally induced between 2 and 24 h, and was barely detectable after day 3 of the nephritis (Figure 1A). The increase in glomerular mRNA for CCL2/MCP-1 was confirmed at the urinary protein level (Figure 1C). We also examined {alpha}-SMA and ß-actin protein expression in the glomerulus, and the results showed that the former was significantly up-regulated during the proliferative phase, whereas the latter was markedly reduced during 2–24 h (the mesangiolytic phase) (Figure 1B).



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Fig. 1. Expression of CCL2/MCP-1 and CX3CL1/fractalkine mRNA and protein during anti-Thy1 glomerulonephritis. (A) Twenty micrograms of total RNA extracted from glomeruli pooled from two rats were used in each lane for detection of mRNAs. GAPDH mRNA was examined to correct any difference of loading or transfer of RNA. (B) Fifty micrograms of protein isolated from glomeruli pooled from two rats were used in each lane for detection of CX3CL1/fractalkine, SMA and ß-actin. (C) Fifty micrograms of proteins concentrated from urine by Centricon-10® as the supplier’s instructions were used in each lane for detection of urinary proteins. Pictures are representative from four separate experiments with similar results.

 
Urinary CCL2/MCP-1 and glomerular/urinary CX3CL1/fractalkine correlated with glomerular mRNAs for CCR2 and CX3CR1, respectively, in anti-Thy1 glomerulonephritis
The immunohistochemical staining showed that accumulation of glomerular ED-1-positive monocytes/macrophages began at 2 h, peaked at day 1, and sustained into day 5 of the nephritis (Figure 2A). The extent of glomerular macrophage accumulation at days 1, 3 and 5 correlated well to the levels of urinary CCL2/MCP-1 (Spearman’s coefficient r = 0.936, P < 0.01), but not glomerular or urinary CX3CL1/fractalkine (Figure 2B–D). Our RT–PCR results further revealed differential expression of glomerular mRNAs for CCR2 and CX3CR1, with the mRNA levels of CCR2 peaking around 24 h, while that of CX3CR1 reaching a maximum during the proliferative phase (Figure 3). The normalized glomerular mRNAs for CCR2 and CX3CR1 coincided temporally to the induction of urinary CCL2/MCP-1 protein, and up-regulation of glomerular/urinary CX3CL1/fractalkine, respectively (Spearman’s coefficient r = 0.965, and r = 0.766/0.809, respectively, P < 0.01).



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Fig. 2. Relationships between accumulation of glomerular macrophages and up-regulation of CCL2/MCP-1 and CX3CL1/fractalkine proteins during anti-Thy1 glomerulonephritis. (A) Accumulation of ED-1-positive monocytes/macrophages per glomerular cross-section (gcs). Each point is mean ± SEM of 90 glomeruli with similar cross-sectional diameters from six rats (15 glomeruli per animal). (BD) Arbitrary quantification of urinary CCL2/MCP-1 protein, glomerular CX3CL1/fractalkine protein (G-FKN), and urinary CX3CL1/fractalkine protein relative to that of controls at zero time, respectively. ND, not done. Values are mean ± SEM based on four experiments. *P < 0.05 vs control rats.

 


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Fig. 3. RT–PCR for glomerular CX3CR1 and CCR2 mRNA expression in anti-Thy1 glomerulonephritis. (A) Representative RT–PCR pictures from four separate experiments with similar results. CX3CR1: receptor for CX3CL1/fractalkine; CCR2: receptor for CCL2/MCP-1. (B) Arbitrary quantification of CX3CR1 and CCR2 mRNAs corrected for GAPDH, and relative to that of control. Values are mean ± SEM based on four experiments. *P < 0.05 vs control.

 
Activated MCs express CX3CL1/fractalkine mRNA and protein in anti-Thy1 glomerulonephritis
To examine whether MCs are a source for glomerular CX3CL1/fractalkine mRNA, in situ hybridization was performed in kidney sections using cRNA probes. Before induction of the nephritis, no signals could be detected within the glomeruli of the control rats (Figure 4A). Two hours after induction of the nephritis, CX3CL1/fractalkine mRNA was markedly up-regulated, mainly in the non-mesangial areas of the glomerulus (Figure 4B). At day 1 of the nephritis, CX3CL1/fractalkine mRNA signals were still present, although less prominent than at 2 h (Figure 4D). Beginning from day 3 of the nephritis, CX3CL1/fractalkine mRNA was detected with a predominant mesangial distribution (Figure 4F and H). Combined in situ hybridization and immunohistochemistry, and double immunohistochemistry showed that almost all CX3CL1/fractalkine mRNA and protein signals at day 5 were expressed by {alpha}-SMA-positive MCs (Figure 4J and L). As a negative control, sense probes showed no signals in the corresponding renal tissues (Figure 4C, E, G, I and K).



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Fig. 4. Glomerular CX3CL1/fractalkine expression in anti-Thy1 glomerulonephritis assessed by in situ hybridization and immunihistochemistry. (A) Before induction of the nephritis, no CX3CL1/fractalkine mRNA signal was detected within the glomerulus by in situ hybridization. (B, D, F and H) Prominent up-regulation of CX3CL1/fractalkine mRNAs (purple) at 2 h, days 1, 3, and 5, respectively, of the nephritis. (C, E, G and I) Adjacent nephritic sections at 2 h, days 1, 3 and 5, respectively, hybridized with the sense probe. (J) Combined in situ hybridization and immunohistochemistry showing co-localization of SMA staining (arrowheads, red) and CX3CL1/fractalkine mRNAs (arrows, purple) at day 5 of the nephritis. (K) Combined in situ hybridization with the sense riboprobe and immunohistochemistry showing only SMA staining in MCs (arrowheads, red) and vessel walls (arrows, red) at day 5 of the nephritis. (L) Double immunohistochemistry showing co-localization of SMA staining (black) and CX3CL1/fractalkine protein (orange-red) at day 5 of the nephritis. Original magnification x200.

 
Cytokine and growth factor regulation of CX3CL1/fractalkine expression by cultured MCs
To examine the regulation of CX3CL1/fractalkine expression by MCs, TNF-{alpha}, IL-1ß, PDGF, bFGF and TGF-ß1 were used in the present study. These molecules are potent inducers for chemokines, and are activated in various glomerular diseases including the anti-Thy1 model [3]. Furthermore, because CX3CL1/fractalkine can exist as either cell-bound or cleaved soluble forms [5], it was of interest to determine which form of CX3CL1/fractalkine protein was induced in MCs. As shown in Figure 5, MCs grown in vitro were found to constitutively express a low level of CX3CL1/fractalkine mRNA and protein. After stimulation with TNF-{alpha}, MC CX3CL1/fractalkine mRNA and protein expression was markedly up-regulated in a time-dependent manner. IL-1ß, PDGF-AB and bFGF also activated CX3CL1/fractalkine mRNA and protein expression by MCs, although to a lesser extent than TNF-{alpha}. In contrast, TGF-ß1 had no discernible effect on MC CX3CL1/fractalkine expression. In addition to cell-bound CX3CL1/fractalkine, MC also released an ~75 kDa soluble form of CX3CL1/fractalkine into the culture medium following stimulation with TNF-{alpha}, IL-1ß, PDGF-AB and bFGF (Figure 6).



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Fig. 5. Effects of cytokines and growth factors on the expression of CX3CL1/fractalkine mRNA and protein. (A) Left panel shows representative northern blotting of CX3CL1/fractalkine and GAPDH mRNA expression by rat MCs in response to TNF-{alpha} (5 ng/ml), IL-1ß (10 ng/ml), PDGF-AB (PDGF, 50 ng/ml), bFGF (100 ng/ml) or TGF-ß1 (5 ng/ml) for indicated times. Ten micrograms of total RNA were used in each lane. Right panel shows arbitrary quantification of CX3CL1/fractalkine mRNA corrected for GAPDH, and relative to that of control at zero time. (B). Left panel shows representative western blots of protein extracts from rat MCs treated similarly, and detected with an anti-CX3CL1/fractalkine or anti-ß-actin antibody. Forty micrograms were used in each lane. Right panel shows arbitrary quantification of cell-bound CX3CL1/fractalkine protein corrected for ß-actin, and relative to that of control at zero time. Values are mean ± SEM based on four experiments. *P < 0.05 vs control.

 


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Fig. 6. Effects of cytokines and growth factors on the expression of soluble CX3CL1/fractalkine protein. MCs were incubated with TNF-{alpha} (5 ng/ml), IL-1ß (10 ng/ml), PDGF-AB (PDGF, 50 ng/ml) or bFGF (100 ng/ml) for 24 h. The media were removed and concentrated by Centricon-10® as the supplier’s instructions. Fifty micrograms of the concentrates were used in each lane and detected with an anti-CX3CL1/fractalkine antibody. Upper panel shows representative western blots of soluble CX3CL1/fractalkine (sFKN) expression, middle panel shows corresponding Coomassie Blue staining to verify equal loading of proteins in each lane (FCSA: fetal calf serum albumin), and lower graph denotes arbitrary quantitation of sFKN relative to controls (C). Values are mean ± SEM based on four experiments. *P < 0.05 vs control.

 
To investigate further whether the NF-{kappa}B and AP-1 pathways were involved in MC CX3CL1/fractalkine expression induced by these cytokines and growth factors, two NF-{kappa}B inhibitors, curcumin and MG132 were used [11]. When MCs was pre-incubated with curcumin (40 µM) or MG132 (10 µM) for 1.5 h, both basal and cytokine-/growth factor-stimulated CX3CL1/fractalkine mRNA and protein expression were inhibited (Figure 7). At these concentrations, curcumin and MG132 were found to suppress cytokine- and growth factor-activated degradation of I-{kappa}B{alpha} (Figure 8A). The present study also demonstrated that curcumin attenuated cytokine- and growth factor-augmented phosphorylation of c-Jun, consistent with its role as an inhibitor for AP-1 activation [16]. In contrast, MG132 significantly increased the levels of phosphorylated c-Jun by these cytokines and growth factors (Figure 8B), in accord with its role as an activator for c-Jun/AP-1 pathway [17].



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Fig. 7. Effects of curcumin and MG132 on CX3CL1/fractalkine mRNA and protein expression stimulated by TNF-{alpha}, PDGF-AB, IL-1ß or bFGF. MCs were stimulated with the given cytokine or growth factor for 4 or 24 h, or they were pre-incubated with curcumin (40 µM) or MG132 (10 µM) for 1.5 h prior to stimulation. The controls were cells treated with vehicles alone. (A) Representative northern and western blots of CX3CL1/fractalkine (FKN) mRNA and cell-bound CX3CL1/fractalkine (cFKN) expression in cells stimulated with TNF-{alpha} (5 ng/ml) or PDGF-AB (50 ng/ml). (B) Representative northern and western blots of CX3CL1/fractalkine (FKN) mRNA and cell-bound CX3CL1/fractalkine (cFKN) expression in cells stimulated with bFGF (100 ng/ml) or IL-1ß (10 ng/ml). Bar graphs show arbitrary quantitation of cFKN corrected for ß-actin, and relative to that of control. Values are mean ± SEM based on four experiments. Star, P < 0.05 vs control; filled circle, clear diamond, clear circle and filled diamond, P < 0.05 vs TNF-{alpha}-, IL-1ß-, PDGF-AB- and bFGF-treated cells, respectively.

 


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Fig. 8. Effects of curcumin and MG132 on cytokine- and growth factor-stimulated I-{kappa}B{alpha} degradation and c-Jun phosphorylation. MCs were pre-treated with or without curcumin (40 µM) or MG132 (10 µM) for 1.5 h, followed by stimulation with TNF-{alpha} (5 ng/ml), IL-1ß (10 ng/ml), PDGF-AB (50 ng/ml) or bFGF (100 ng/ml) for 15 min. The controls were cells treated with vehicles alone for 15 min. Twenty micrograms of protein extracts were used in each lane. Bands corresponding to I-{kappa}B{alpha}, phosphorylated and nonphosphorylated c-Jun, and ß-actin were identified by immunoblotting using specific antibodies as described in Materials and methods. All western blots are representative of three separate experiments with similar results.

 


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CX3CL1/fractalkine was originally thought to be produced mainly by endothelial cells [5]. However, it is now known that CX3CL1/fractalkine can be synthesized by a variety of non-endothelial tissue cells, including MC in culture [611]. This study further demonstrated that activated MCs during the proliferative phase (days 3–5) of acute anti-Thy1 glomerulonephritis can synthesize CX3CL1/fractalkine. The evidence was based on combined in situ hybridization and immunohistochemistry, and double immunohistochemistry, which showed that almost all glomerular CX3CL1/fractalkine mRNA and protein detected during this period were expressed by {alpha}-SMA-positive MCs. Consistent with the morphologic findings, our immunoblotting results showed a parallel increase in the expression of glomerular CX3CL1/fractalkine and {alpha}-SMA proteins during days 3–5 of the nephritis. On the other hand, the augmented expression of glomerular CX3CL1/fractalkine mRNA during the initial 24 h was not paralleled by a corresponding increase in glomerular CX3CL1/fractalkine protein expression. The reasons for this paradox are unknown, but a generalized reduction in glomerular protein synthesis due to mesangiolysis-related glomerular injury may be a partial explanation. Indeed, our immunoblotting results showed that expression of glomerular ß-actin between 2 and 24 h was markedly reduced when compared with the proliferative phase of the nephritis. In contrast to CX3CL1/fractalkine, urinary CCL2/MCP-1 protein induction corresponded closely to glomerular CCL2/MCP-1 mRNA expression during the mesangiolytic phase of the nephritis. This can be explained, at least in part, by the fact that CCL2/MCP-1 is produced mainly by infiltrating immune cells as a secretory peptide and therefore its induction, as reflected by urinary CCL2/MCP-1, may be less affected by mesangiolysis.

Our results demonstrate that incubation of primary cultures of rat MCs with TNF-{alpha} and IL-1ß up-regulated CX3CL1/fractalkine expression in a time-dependent manner. In contrast, Zernecke et al. [18] reported that CX3CL1/fractalkine expression in human MCs was not clearly up-regulated by the stimulation of TNF-{alpha} at 100 U/ml, a dose equivalent to 0.37 ng/ml. The reasons for this discrepancy are not clear, but may be attributed in part to differences in cell species and/or TNF-{alpha} concentrations. Additionally, this study shows that bFGF and PDGF, both being implicated in migration and reparative growth of MC during anti-Thy1 glomerulonephritis [19,20], also stimulated CX3CL1/fractalkine expression in MCs. To our knowledge, this is the first demonstration that bFGF and PDGF can modulate CX3CL1/fractalkine expression. Our results further reveal that blockade of I-{kappa}B{alpha} degradation by the pharmacological NF-{kappa}B inhibitors, curcumin and MG132, suppressed the stimulatory effects of cytokines and growth factors on CX3CL1/fractalkine mRNA and protein expression. This suggests that NF-{kappa}B activation is crucial for cytokine- and growth factor-stimulated CX3CL1/fractalkine gene transcription in rat MCs. Our results are in agreement with a recent report that shows that cytokine-stimulated CX3CL1/fractalkine production by rat aortic endothelial cells is NF-{kappa}B dependent [21]. The c-Jun/AP-1 pathway, which has also been implicated in the transcription of chemokine genes [22], does not appear to play a major role in MC CX3CL1/fractalkine expression because MG132 abolished both basal and cytokine-/growth factor-stimulated CX3CL1/fractalkine production in the presence of augmented levels of phosphorylated c-Jun.

This study shows that MCs can produce soluble CX3CL1/fractalkine in response to cytokines and growth factors, and are a likely source for urinary CX3CL1/fractalkine during the proliferative phase of anti-Thy1 glomerulonephritis. Soluble CX3CL1/fractalkine is believed to be shed from its cell-bound form at the syndecan-like motif near the transmembranous domain via metalloproteinase-dependent cleavage [5]. Because the chemokine domain remains intact, soluble CX3CL1/fractalkine may possess chemotactic ability. Indeed, we have demonstrated that MC-derived soluble CX3CL1/fractalkine modulates transmigration of monocytes by using an in vitro chemotaxis assay [11]. Thus, MC-derived soluble CX3CL1/fractalkine may act as a potential chemoattractant in the kidney. In this study, urinary CX3CL1/fractalkine was found to coincide temporally with glomerular mRNA levels for CX3CR1 during days 1–5 of the nephritis. This suggests that CX3CL1/fractalkine may have a role in the recruitment of CX3CR1-positive leukocytes, including monocytes, T cells and natural killer cells, to the glomerulus of the nephritis. Consistent with this notion, a recent report by Ito et al. [12] also implicates CX3CL1/fractalkine in the recruitment of CX3CR1-positive inflammatory leukocytes in a prolonged model of anti-Thy1 glomerulonephritis. This study also shows that urinary CCL2/MCP-1 correlated with glomerular macrophage accumulation during the course of the nephritis. Further analysis indicates that urinary CCL2/MCP-1 correlated with glomerular mRNAs for CCR2, but not CX3CR1, suggesting that monocyte recruitment during the mesangiolytic phase was mediated preferentially via CCL2/MCP-1- CCR2 interaction. This is in contrast with the CX3CL1/fractalkine-CX3CR1 system, which appeared to prevail during especially the proliferative phase. We surmise that this differential expression of CCL2/MCP-1 and CX3CL1/fractalkine may also explain partially why blockade of either CCL2/MCP-1 or CX3CL1/fractalkine in previous neutralization studies fails to prevent macrophage infiltration completely [3,23]. Because of the pleiotropy and redundancy of the chemokine system, it is evident that antichemokine therapeutic measures for glomerulonephritis will require not only broad-spectrum but also sequential blockade to control inflammation more effectively.

In summary, our data demonstrate that MC is a source of CX3CL1/fractalkine mRNA and protein expression during immune-mediated glomerular injury. MC CX3CL1/fractalkine expression is modulated by various cytokines and growth factors through at least in part the NF-{kappa}B signalling pathway. The sequential expression of CCL2/MCP-1 and CX3CL1/fractalkine may recruit distinct subsets of monocytes to the glomerulus at different time points during acute anti-Thy1 glomerulonephritis.



   Acknowledgments
 
This work was supported by grants from the National Science Council, 88-2314-B002-242 and 89-2314-B002-068, the Ta-Tung Kidney Foundation, and the Mrs Hsiu-Chin Lee Kidney Research Fund, Taipei, Taiwan. We are grateful to Mr Kuo-Tong Huang and Mr Tsao-Wei Liu for excellent technical assistance.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 14.10.02
Accepted in revised form: 18. 7.03