Potential role of fractalkine receptor expression in human renal fibrogenesis
2007; Elsevier BV; Volume: 72; Issue: 5 Linguagem: Inglês
10.1038/sj.ki.5002368
ISSN1523-1755
AutoresMichael Koziolek, Holger Schmid, Clemens D. Cohen, Sabine Blaschke, Bernhard Hemmerlein, Antonia Zapf, G.A. Müller, Frank Strutz,
Tópico(s)Complement system in diseases
ResumoThe inhibition of several chemokine/chemokine receptors has been shown to reduce progressive renal interstitial fibrosis. In this study, we examined the expression of the CX3C receptor in human renal biopsies with interstitial fibrosis and from normal kidneys by real-time polymerase chain reaction (PCR) and immunohistochemistry. The CX3C receptor was not only detected in mononuclear, tubular epithelial, and dendritic cells but also in α-smooth muscle actin and vimentin-positive interstitial myofibroblasts in fibrotic kidneys. Real-time PCR indicated a significant upregulation of CX3C receptor mRNA in fibrotic kidneys compared with non-fibrotic nephropathies or donor biopsies. In renal fibroblasts in vitro, hydrogen peroxide increased the expression of the CX3C receptor, an increase that was inhibited by N-acetylcysteine and catalase. However, neither proinflammatory nor profibrotic cytokines resulted in this upregulation. Stimulation of fibroblasts by CX3C ligand led to a significant enhancement of migration, which was abrogated by pre-incubation with a blocking anti-CX3C receptor antibody. Our studies indicate that renal fibrosis is associated with the expression of CX3C receptors on human renal fibroblasts. The expression is induced by reactive oxygen species suggesting a role of oxidative stress. The inhibition of several chemokine/chemokine receptors has been shown to reduce progressive renal interstitial fibrosis. In this study, we examined the expression of the CX3C receptor in human renal biopsies with interstitial fibrosis and from normal kidneys by real-time polymerase chain reaction (PCR) and immunohistochemistry. The CX3C receptor was not only detected in mononuclear, tubular epithelial, and dendritic cells but also in α-smooth muscle actin and vimentin-positive interstitial myofibroblasts in fibrotic kidneys. Real-time PCR indicated a significant upregulation of CX3C receptor mRNA in fibrotic kidneys compared with non-fibrotic nephropathies or donor biopsies. In renal fibroblasts in vitro, hydrogen peroxide increased the expression of the CX3C receptor, an increase that was inhibited by N-acetylcysteine and catalase. However, neither proinflammatory nor profibrotic cytokines resulted in this upregulation. Stimulation of fibroblasts by CX3C ligand led to a significant enhancement of migration, which was abrogated by pre-incubation with a blocking anti-CX3C receptor antibody. Our studies indicate that renal fibrosis is associated with the expression of CX3C receptors on human renal fibroblasts. The expression is induced by reactive oxygen species suggesting a role of oxidative stress. Tubulointerstitial fibrosis is a key component of the final common pathway in progressive chronic kidney disease and its extent is closely associated with the loss of renal function.1.Bohle A. Mackensen-Haen S. von Gise H. et al.The consequence of tubulo-interstitial changes for renal function in glomerulopathies. A morphometric and cytological analysis.Pathol Res Pract. 1990; 186: 135-144Crossref PubMed Scopus (177) Google Scholar, 2.DAmico G. Tubulointerstitium as predictor of progression of glomerular diseases.Nephron. 1999; 83: 289-295Crossref PubMed Scopus (58) Google Scholar, 3.Strutz F. Neilson E.G. New insights into mechanisms of fibrosis in immune renal injury.Semin Immunopathol. 2003; 24: 459-476Crossref PubMed Scopus (93) Google Scholar The pathogenesis of renal fibrogenesis is complex and incompletely understood.4.Strutz F. Zeisberg M. Renal fibroblasts and myofibroblasts in chronic kidney disease.J Am Soc Nephrol. 2006; 17: 2992-2998Crossref PubMed Scopus (251) Google Scholar A number of studies have demonstrated that chemokines are key mediators in various stages of that process.5.Anders H.J. Vielhauer V. Schlöndorff D. 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Guzik T.J. et al.Smooth muscle cells in human atherosclerosis plaques express the fractalkine receptor CX3CR1 and undergo chemotaxis to the chemokine fractalkine (CX3CL1).Circulation. 2003; 108: 2498-2504Crossref PubMed Scopus (133) Google Scholar, 8.Schäfer A. Schulz C. Eigenthaler M. et al.Novel role of the membrane bound chemokine fractalkine in platelet activation and adhesion.Blood. 2004; 103: 407-412Crossref PubMed Scopus (119) Google Scholar Furthermore, there is increasing evidence that certain chemokines do not only play an important role in amplification and perpetuation of interstitial inflammation, but also in the complex interactions resulting in fibroblast proliferation and matrix accumulation.5.Anders H.J. Vielhauer V. Schlöndorff D. Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.Kidney Int. 2003; 63: 401-415Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar,9.Gonzalez-Cuadrado S. Bustos C. Ruiz-Ortega M. et al.Expression of leukocyte chemoattractants by interstitial renal fibroblasts: up-regulation by drugs associated with interstitial fibrosis.Clin Exp Immunol. 1996; 106: 518-522Crossref PubMed Scopus (46) Google Scholar The sole member of the CX3C chemokine family is fractalkine (CX3C-L), which binds to the CX3C receptor (CX3C-R). In the kidney, CX3C-L expression has been described in glomerular and vascular endothelial cells, on glomerular and tubular epithelial cells, mesangial cells, and in stromal tissue including the tubulointerstitial space.7.Lucas A.D. Bursill C. Guzik T.J. et al.Smooth muscle cells in human atherosclerosis plaques express the fractalkine receptor CX3CR1 and undergo chemotaxis to the chemokine fractalkine (CX3CL1).Circulation. 2003; 108: 2498-2504Crossref PubMed Scopus (133) Google Scholar, 10.Blaschke S. Koziolek M.J. 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Li L. et al.In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar Kyoto (WKY) rats by vMIP-II.J Exp Med. 1998; 188: 193-198Crossref PubMed Scopus (230) Google Scholar, 14.Cockwell P. Chakravorty S.J. Girdlestone J. Savage C.O.S. Fractalkine expression in human renal inflammation.J Pathol. 2002; 196: 85-90Crossref PubMed Scopus (111) Google Scholar, 15.Donadelli R. Zanchi C. Morigi M. et al.Protein overload induces fractalkine upregulation in proximal tubular cells through nuclear factor kappa B- and p38 mitogen-activated protein kinase-dependent pathways.J Am Soc Nephrol. 2003; 13: 2436-2446Crossref Scopus (105) Google Scholar, 16.Garcia G.E. Xia Y. Chen S. et al.NF-κB dependent fractalkine induction in rat aortic endothelial cells stimulated by IL-1β, TNF-α and LPS.J Leukoc Biol. 2000; 67: 577-584PubMed Google Scholar, 17.Imaizumi T. Matsumiya T. Fujimoto K. et al.Interferon-γ stimulates the expression of CX3CL1/fractalkine in cultured human endothelial cells.Tohoku J Exp Med. 2000; 192: 127-139Crossref PubMed Scopus (54) Google Scholar, 18.Ludwig A. Berkhout T. Moores K. et al.Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.J Immunol. 2002; 168: 604-612Crossref PubMed Scopus (126) Google Scholar, 19.Yoshikawa M. Makajima T. Matsumoto K. et al.TNF-α and IL-4 regulate expression of fractalkine (CX3CL1) as a membrane-anchored proadhesive protein and soluble chemotactic peptide on human fibroblasts.FEBS Lett. 2004; 561: 105-111Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar CX3C-L exerts strong adhesive properties on different subsets of mononuclear cells,20.Fong A.M. Robinson L.A. Steeber D.A. et al.Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow.J Exp Med. 1998; 188: 1413-1419Crossref PubMed Scopus (577) Google Scholar, 21.Goda S. Imai T. Yoshie O. et al.CX3C-chemokine, fractalkine-enhanced adhesion of THP-1 cells to endothelial cells through integrin-dependent and -independent mechanisms.J Immunol. 2000; 164: 4313-4320Crossref PubMed Scopus (180) Google Scholar, 22.Haskell C.A. Cleary M.D. Charo I.F. Unique role of the chemokine domain of fractalkine in cell capture.J Biol Chem. 2000; 275: 34183-34189Crossref PubMed Scopus (118) Google Scholar proliferative and antiapoptotic activity on epithelial, mesenchymal, or neuronal cells,7.Lucas A.D. Bursill C. Guzik T.J. et al.Smooth muscle cells in human atherosclerosis plaques express the fractalkine receptor CX3CR1 and undergo chemotaxis to the chemokine fractalkine (CX3CL1).Circulation. 2003; 108: 2498-2504Crossref PubMed Scopus (133) Google Scholar, 23.Brand S. Sakaguchi T. Gu X. et al.Fractalkine-mediated signals regulate cell-survival and immune-modulatory responses in intestinal epithelial cells.Gastroenterology. 2002; 122: 166-177Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24.Meucci O. Fatatis A. Simen A.A. Miller R.J. Expression of CX3CR1 chemokine receptors on neurons and their role in neuronal survival.PNAS. 2000; 97: 8075-8080Crossref PubMed Scopus (310) Google Scholar upregulates expression of matrix metalloproteinases, interleukin (IL)-8, or CX3C-L itself,10.Blaschke S. Koziolek M.J. Schwarz A. et al.Proinflammatory role of fractalkine (CX3CL1) in rheumatoid arthritis.J Rheumatol. 2003; 30: 1918-1927PubMed Google Scholar,23.Brand S. Sakaguchi T. Gu X. et al.Fractalkine-mediated signals regulate cell-survival and immune-modulatory responses in intestinal epithelial cells.Gastroenterology. 2002; 122: 166-177Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar respectively, suggesting auto/paracrine properties.7.Lucas A.D. Bursill C. Guzik T.J. et al.Smooth muscle cells in human atherosclerosis plaques express the fractalkine receptor CX3CR1 and undergo chemotaxis to the chemokine fractalkine (CX3CL1).Circulation. 2003; 108: 2498-2504Crossref PubMed Scopus (133) Google Scholar,23.Brand S. Sakaguchi T. Gu X. et al.Fractalkine-mediated signals regulate cell-survival and immune-modulatory responses in intestinal epithelial cells.Gastroenterology. 2002; 122: 166-177Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Moreover, the functional significance of CX3C-L in progression of renal diseases has been demonstrated by inhibition of its receptor, CX3C-R, in crescentic anti-thy 1.1 nephritis,25.Feng L. Chen S. Garcia G.E. et al.Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1. Rapid communication.Kidney Int. 1999; 56: 612-620Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar lupus nephritis26.Inoue A. Hasegawa H. Kohno M. et al.Antagonist of fractalkine (CX3CL1) delays the initiation and ameliorates the progression of lupus nephritis in MRL/lpr mice.Arthr Rheumat. 2005; 52: 1522-1533Crossref PubMed Scopus (98) Google Scholar as well as ischemia–reperfusion injury.27.Furuichi K. Gao J.L. Murphy P.M. Chemokine receptor CX3CR1 regulates renal interstitial fibrosis after ischemia–reperfusion injury.Am J Pathol. 2006; 169: 372-387Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar CX3C-R is expressed on a number of leukocytes, including monocytes, T cells, and dendritic cells.20.Fong A.M. Robinson L.A. Steeber D.A. et al.Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow.J Exp Med. 1998; 188: 1413-1419Crossref PubMed Scopus (577) Google Scholar, 21.Goda S. Imai T. Yoshie O. et al.CX3C-chemokine, fractalkine-enhanced adhesion of THP-1 cells to endothelial cells through integrin-dependent and -independent mechanisms.J Immunol. 2000; 164: 4313-4320Crossref PubMed Scopus (180) Google Scholar, 22.Haskell C.A. Cleary M.D. Charo I.F. Unique role of the chemokine domain of fractalkine in cell capture.J Biol Chem. 2000; 275: 34183-34189Crossref PubMed Scopus (118) Google Scholar, 28.Foussat A. Coulomb-ĹHermine A. Gosling J. et al.Fractalkine receptor expression by T lymphocyte subpopulations and in vivo production of fractalkine in human.Eur J Immunol. 2000; 30: 87-97Crossref PubMed Scopus (123) Google Scholar, 29.Segerer S. Hughes E. Hudkins K.L. et al.Expression of the fractalkine receptor (CX3CR1) in human kidney diseases.Kidney Int. 2002; 62: 488-495Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 30.Soos T.J. Sims T.N. Barisoni L. et al.CX3CR1+ interstitial dendritic cells form a contiguous network throughout the entire kidney.Kidney Int. 2006; 70: 591-596Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar In contrast to most chemokine receptors, expression has been demonstrated in several intrinsic cells including vascular smooth muscle cells, synovial fibroblasts, or epithelial cells of the bowel.10.Blaschke S. Koziolek M.J. Schwarz A. et al.Proinflammatory role of fractalkine (CX3CL1) in rheumatoid arthritis.J Rheumatol. 2003; 30: 1918-1927PubMed Google Scholar, 18.Ludwig A. Berkhout T. Moores K. et al.Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.J Immunol. 2002; 168: 604-612Crossref PubMed Scopus (126) Google Scholar, 23.Brand S. Sakaguchi T. Gu X. et al.Fractalkine-mediated signals regulate cell-survival and immune-modulatory responses in intestinal epithelial cells.Gastroenterology. 2002; 122: 166-177Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar In normal and inflammatory diseases of the human kidney, CX3C-R expression was detectable in infiltrating mononuclear cells, stromal cells, smooth muscle cells of preglomerular vessels as well as within the tubulointerstitial space, though the cellular source of the latter was not further specified.12.Gröne H.J. Cohen C.D. Gröne E. et al.Spatial and temporally restricted expression of chemokines and chemokine receptors in the developing kidney.J Am Soc Nephrol. 2002; 13: 957-967PubMed Google Scholar,29.Segerer S. Hughes E. Hudkins K.L. et al.Expression of the fractalkine receptor (CX3CR1) in human kidney diseases.Kidney Int. 2002; 62: 488-495Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar The regulation of CX3C-R expression is largely unknown, although induction by the profibrotic cytokine transforming growth factor-β (TGF-β) has been described in microglia cells.31.Chen S. Luo D. Streit W.J. Harrison J.K. TGF-β1 upregulates CX3CR1 expression and inhibits fractalkine-stimulated signaling in rat microglia.J Neuroimmunol. 2002; 133: 46-55Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Studies of the expression of CX3C-R have been confined to acute inflammatory states of the kidney and the functional analyses of CX3C-L in regard to invasion of mononuclear cells. Here, we describe the expression of CX3C-R in interstitial fibroblasts in fibrotic kidneys and investigated possible mechanisms of the regulation of CX3C-R. The pattern of expression for the CX3C-R is shown in Figure 1. In normal kidneys, no expression was detectable in any compartment (Figure 1a), except for three biopsies in which a mild expression was found within tubular epithelial cells (Table 1).Table 1Results of CX3C-R expression in normal and fibrotic human kidneys after immunohistochemical stainingBowman epitheliumTubular epithelial cellsInterstitial cellsInfiltrates*Normal kidneyFibrotic kidneyNormal kidneyFibrotic kidneyNormal kidneyFibrotic kidneyNormal kidneyFibrotic kidney08/8 (100%)22/24 (92%)5/8 (62.5%)7/24 (30%)7/8 (87.5%)7/24 (30%)—2/24 (8%)+0/8 (0%)2/24 (8%)3/8 (37.5%)7/24 (30%)1/8 (12.5%)11/24 (46%)—7/24 (30%)++0/8 (0%)0/24 (0%)0/8 (0%)5/24 (20%)0/8 (0%)4/24 (17%)—4/24 (17%)+++0/8 (0%)0/24 (0%)0/8 (0%)5/24 (20%)0/8 (0%)2/24 (7%)—11/24 (45%)CX3C-R, fractalkine receptor.Sections were deparaffinized and stained for CX3C-R according to a modified sandwich technique. Tissues were evaluated semiquantitatively (0 to +++).*No infiltrates were seen in normal human kidneys. Open table in a new tab CX3C-R, fractalkine receptor. Sections were deparaffinized and stained for CX3C-R according to a modified sandwich technique. Tissues were evaluated semiquantitatively (0 to +++). *No infiltrates were seen in normal human kidneys. Evaluation of fibrotic kidneys demonstrated CX3C-R positively stained cells within the tubulointerstitium of medulla and cortex. These cells were found massed around sclerotic glomeruli (Figure 1b), but also within the tubulointerstitial space. In serial sections, these cells could only partially be colabeled by a series of antibody reagents directed against lymphocytes and monocytes/macrophage antigens (CD3 and CD68). Counting the number of positively stained cells per interstitial visual field, the number of CX3C-R-positive cells exceeded the sum of CD3- and CD68-expressing cells by the factor 4.6 in fibrotic kidneys (P=0.001 using paired Wilcoxon test) (Figure 2). Additionally, in tubular epithelial cells, CX3C-R expression was mild to strong in 70% (17 from 24 biopsies) with a representative example shown in Figure 1d. Results of semiquantitative evaluations are summarized in Table 1. In double immunofluorescence stainings, we found no constitutional expression of CX3C-R in normal human kidneys (Figure 3a and d). In fibrotic kidneys, CX3C-R was found in tubular epithelial cells (Figure 3b). Additionally, we found a positive double staining for CX3C-R, and the marker for myofibroblasts, α-smooth muscle actin (Figure 3c) or the mesenchymal marker vimentin (data not shown), in several cells within the tubulointerstitial space. Moreover, there was a cohort of interstitial cells coexpressing the dendritic cell markers, CD11c and CX3C-R (Figure 3e). The total expression of CX3C-R mRNA was robustly upregulated in biopsies from fibrotic kidneys compared with cadaveric donor biopsies (control) and non-fibrotic nephropathies (2.1- and 2.2-fold, respectively) by quantitative real-time polymerase chain reaction (PCR) (P=0.045 comparing control vs fibrosis, and P=0.045 comparing no fibrosis vs fibrosis using the t-test to the log-transferred data) (Figure 4). Even, if one takes into consideration that a decrease in tubular epithelial cells may favor an increase in expression in molecules expressed in interstitial cells, this increase remains robust. There was no correlation found between the mRNA quotient CX3C-R/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and serum creatinine (R2=0.12) and only a slight correlation with the daily amount of proteinuria (R2=0.34). Constitutive expression of CX3C-R in the human cortical fibroblast cell lines (Tk173 and Tk188) as well as in primary fibroblasts Tk461 was demonstrated by immunofluorescence and reverse-transcription PCR (Figure 5). To examine whether CX3C-R could be upregulated by proinflammatory or profibrotic cytokines, fetal calf serum (FCS)-starved Tk173 or Tk188 were treated for 24 h with 10 ng/ml of either the proinflammatory cytokines, IL-1β, or tumor necrosis factor-α, or the profibrotic cytokines PDGF, epithelial growth factor, FGF-2, or TGF-β. Under these conditions, there were no significant differences in CX3C-R/GAPDH coefficient in real-time PCR results in every investigated cytokine stimulated (Figure 6a). Shorter (6 h) or longer (48 h) stimulation did not change the CX3C-R expression level. Conversely, stimulation with non-toxic doses of the reactive oxygene species H2O2 (0.1 mM) resulted in strong upregulation in renal fibroblasts with 861±241% of controls in Tk173 (P<0.0001 using the Mann–Whitney U-rank test adjusted for multiple choice comparison) and 1027±349.1% in Tk188 (P<0.0001 also using the adjusted rank test) fibroblasts with a dose-dependent effect exemplarily demonstrated in Tk173 (Figure 6b). To further characterize these results, Tk173 were treated with H2O2 (0.1 mM) in the presence or absence or N-acetyl-L-cysteine (NAC, 1 μM) and catalase (600 U/ml) for 24 h. H2O2 upregulated the expression of CX3C-R, which was completely abrogated by pre-incubation with the radical scavengers NAC and catalase by 99.4% (P<0.0001 compared with H2O2-stimulated cells using the adjusted Mann–Whitney U-test). Figure 6c summarizes the results. Since CX3C-L is a potent chemoattractant also for intrinsic cells,7.Lucas A.D. Bursill C. Guzik T.J. et al.Smooth muscle cells in human atherosclerosis plaques express the fractalkine receptor CX3CR1 and undergo chemotaxis to the chemokine fractalkine (CX3CL1).Circulation. 2003; 108: 2498-2504Crossref PubMed Scopus (133) Google Scholar,32.Bursill C.A. Channon K.M. Greaves D.R. The role of chemokines in atherosklerosis: recent evidence from experimental models and population genetics.Curr Opin Lipidol. 2004; 15: 145-149Crossref PubMed Scopus (96) Google Scholar we investigated chemotactic effects of CX3C-L on human renal fibroblasts. CX3C-L induced cell motility in a dose-dependent manner as assessed by migration assay up to a maximum of 380±25% in Tk461, 594±228% in Tk173, and 745±187% of controls in Tk188 (all n=6, P<0.0001 using the Mann–Whitney U-rank test adjusted for multiple comparison) under stimulation with 20 ng/ml CX3C-L. Pre-incubation of fibroblasts with a blocking anti-CX3C-R antibody resulted in a significant reduction of migrated cells under a stimulation with 20 ng/ml CX3C-L by a rate of 57.9% in Tk461, 57.1% in Tk173, and 53.8% in Tk188 (all P<0.0001 compared to stimulation with 20 ng/ml CX3C-L using the adjusted rank test). These results are summarized in Figure 7. In the past, there have been several reports on the role of chemokines and their receptors in progressive renal failure. After inhibition of CCL-2/MCP-1, CCL-5/Rantes, CX3C-L or their corresponding receptors, respectively, a significant reduction of infiltrating sites and a reduction of irreversible, chronic tubulointerstitial damages has been achieved in several models of progressive glomerular and tubulointerstitial diseases.5.Anders H.J. Vielhauer V. Schlöndorff D. Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.Kidney Int. 2003; 63: 401-415Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 13.Chen S. Bacon K.B. Li L. et al.In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar Kyoto (WKY) rats by vMIP-II.J Exp Med. 1998; 188: 193-198Crossref PubMed Scopus (230) Google Scholar, 25.Feng L. Chen S. Garcia G.E. et al.Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1. Rapid communication.Kidney Int. 1999; 56: 612-620Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar The reduction of inflammation is thought to be the main cause underlying this amelioration. Recent data suggested additional mechanisms of CX3C-L or CX3C-R in extrarenal tissues.7.Lucas A.D. Bursill C. Guzik T.J. et al.Smooth muscle cells in human atherosclerosis plaques express the fractalkine receptor CX3CR1 and undergo chemotaxis to the chemokine fractalkine (CX3CL1).Circulation. 2003; 108: 2498-2504Crossref PubMed Scopus (133) Google Scholar, 23.Brand S. Sakaguchi T. Gu X. et al.Fractalkine-mediated signals regulate cell-survival and immune-modulatory responses in intestinal epithelial cells.Gastroenterology. 2002; 122: 166-177Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24.Meucci O. Fatatis A. Simen A.A. Miller R.J. Expression of CX3CR1 chemokine receptors on neurons and their role in neuronal survival.PNAS. 2000; 97: 8075-8080Crossref PubMed Scopus (310) Google Scholar We used immunohistochemical detection of CX3C-R, double immunofluorescence staining for CX3C-R and several epithelial and mesenchymal markers along with real-time PCR and, additionally, in vitro analyses for the localization and quantification of the CX3C-R. The main finding in our study was that CX3C-R is not only expressed in CD3- and CD68-positive infiltrating cells but also in resident interstitial cells with a CD3- and CD68-negative staining pattern, and in tubular epithelial cells in advanced stages of renal disease. Segerer et al.29.Segerer S. Hughes E. Hudkins K.L. et al.Expression of the fractalkine receptor (CX3CR1) in human kidney diseases.Kidney Int. 2002; 62: 488-495Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar described CX3C-R-positive cells in the two most common groups of infiltrating cells, T cells and monocytes/macrophages, in human inflammatory kidney diseases or renal allograft rejection, although the authors could not exclude an expression in stromal cells. Another CX3C-R-positive interstitial cell type are dendritic cells, which have been shown to form a heterogenous pervasive network in normal kidney of the mouse,30.Soos T.J. Sims T.N. Barisoni L. et al.CX3CR1+ interstitial dendritic cells form a contiguous network throughout the entire kidney.Kidney Int. 2006; 70: 591-596Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar though these findings have not been confirmed in humans. Our knowledge of human renal dendritic cells is still limited due to technical difficulties in detecting and isolating these cells.33.Kurts C. Dendritic cells: not just another cell type in the kidney, but a complex immune sentinel network.Kidney Int. 2006; 70: 412-414Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar In our analysis, we found a group of interstitial cells colabeling for the dendritic cell markers, CD11c and CX3C-R, suggesting analogy to data obtained in mouse kidneys.30.Soos T.J. Sims T.N. Barisoni L. et al.CX3CR1+ interstitial dendritic cells form a contiguous network throughout the entire kidney.Kidney Int. 2006; 70: 591-596Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar Gröne et al.12.Gröne H.J. Cohen C.D. Gröne E. et al.Spatial and temporally restricted expression of chemokines and chemokine receptors in the developing kidney.J Am Soc Nephrol. 2002; 13: 957-967PubMed Google Scholar described CX3C-R-positive cells within the stroma of medulla and cortex in developing human kidneys, which could not be colabeled by a series of antibody reagents directed against monocyte/macrophage antigens (CD68, MAC387, MRP8, and MRP14), or the stem cell antigen (CD34). This is not the first study which described a CX3C-R expression in mesenchymal cells, since the receptor has been detected in synovial fibroblasts,10.Blaschke S. Koziolek M.J. Schwarz A. et al.Proinflammatory role of fractalkine (CX3CL1) in rheumatoid arthritis.J Rheumatol. 2003; 30: 1918-1927PubMed Google Scholar,34.Ruth J.H. Volin M.V. Haines III, G.K. et al.Fractalkine, a novel chemokine in rheumatoid arthritis an in adjuvant-induced arthritis.Arthritis Rheum. 2001; 44: 1568-1581Crossref PubMed Scopus (214) Google Scholar in vascular smooth muscle cells7.Lucas A.D. Bursill C. Guzik T.J. et al.Smooth muscle cells in human atherosclerosis plaques express the fractalkine receptor CX3CR1 and undergo chemotaxis to the chemokine fractalkine (CX3CL1).Circulation. 2003; 108: 2498-2504Crossref PubMed Scopus (133) Google Scholar,32.Bursill C.A. Channon K.M. Greaves D.R. The role of chemokines in atherosklerosis: recent evidence from experimental models and population genetics.Curr Opin Lipidol. 2004; 15: 145-149Crossref PubMed Scopus (96) Google Scholar and recently in mesenchymal stem cells in the brain.35.Ji J.F. He B.P. Dheen S.T. Tay S.S. Interacion of chemokines and chemokine receptors mediated migrationb of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury.Stem cells. 2004; 22: 415-427Crossref PubMed Scopus (389) Google Scholar As there is no specific antibody available for the detection of human renal fibroblasts in vivo,3.Strutz F. Neilson E.G. New insights into mechanisms of fibrosis in immune renal injury.Semin Immunopathol. 2003; 24: 459-476Crossref PubMed Scopus (93) Google Scholar we ascertained CX3C-R expression by double immunofluorescence staining using the myofibroblast marker α-smooth muscle actin or the mesenchymal marker vimentin, respectively, in vivo and, additionally, in well-characterized human fibroblast cell lines Tk173 and Tk188 as well as in primary cortical fibroblasts Tk461 in vitro.36.Heeg M.H.J. Koziolek M.J. Vasko R. et al.The anti-fibrotic effects of relaxin in human renal fibroblasts are in part mediated by inhibition of the Smad2 pathway.Kidney Int. 2005; 68: 96-109Abstract Full
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