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BASIC SCIENCE |
*Nephrological Center, Medical Policlinic, University of Munich, Munich, Germany; Department of Immunology, Berlex Biosciences, Richmond, California; Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; Department of Pathology, University of Washington Medical Center, Seattle, Washington; ¶Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany.
Correspondence to Dr. Hans-Joachim Anders, Medizinische Poliklinik LMU, Pettenkoferstr. 8a, 80336 München, Germany. Phone: 49-89-5996856; Fax: 49-89-5996860; E-mail: hjanders{at}med.uni-muenchen.de
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Abstract |
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1 mRNA expression, interstitial volume, and collagen I deposits were all significantly reduced in CCR1-deficient mice. In contrast, renal leukocytes and fibrosis were unaffected in CCR5-deficient mice with UUO. However, if treated with the CCR1 antagonist BX471, CCR5-deficient mice showed a similar reduction of renal leukocytes and fibrosis as CCR1-deficient mice. To determine the underlying mechanism labeled macrophages and T cells isolated from either wild-type, CCR1-deficient, or CCR5-deficient mice were injected into wild-type mice with UUO. Three hours later, renal cell recruitment was reduced for CCR1-deficient cells or cells pretreated with BX471 compared with CCR5-deficient or wild-type cells. Thus, CCR1 but not CCR5 is required for leukocyte recruitment and fibrosis after UUO in mice. Therefore, CCR1 is a promising target for therapeutic intervention in leukocyte-mediated fibrotic tissue injury, e.g. progressive renal fibrosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Infiltration of circulating leukocytes is triggered by locally secreted chemokines (4), and chemokine-mediated leukocyte infiltration is thought to contribute to the initiation and progression of renal disease (5). Studies on progressive renal fibrosis using the model of unilateral ureteral ligation (UUO) in the mouse have demonstrated that increasing amounts of the chemokine receptors CCR1 (ligands CCL3 and CCL4) and CCR5 (ligands CCL5 and CCL4) were expressed on infiltrating macrophages and T cells in parallel to the development of renal fibrosis (6). CCR1 and CCR5 are thought to mediate the migration of T cells and macrophages into inflamed tissues (4,7). Evidence for the role of CCR1 in renal disease comes from a study that showed that the CCR1 antagonist BX471 was similar to cyclosporin in its ability to prevent renal allograft rejection and to improve survival in rabbits (8). Furthermore, we have recently shown that BX471 reduced leukocyte accumulation and renal fibrosis after UUO in mice (9). However, when CCR1-deficient mice were injected with nephrotoxic serum they showed enhanced glomerular T cell infiltration, crescent formation, and proteinuria in association with an enhanced Th1-response, indicating CCR1 is also involved in the regulation of systemic immune responses (10). Conflicting results have also been found with Met-RANTES or AOP-RANTES, two antagonists that can block CCR5. AOP-RANTES reduced macrophage infiltration in Thy1.1 antibody-induced glomerulonephritis in rats (11), and Met-RANTES improved rat renal allograft rejection (12). However, both antagonists aggravated immune complex glomerulonephritis in mice, which was associated with antagonist-induced modulation of macrophage function toward a proinflammatory phenotype (13).
Although the multiple roles of CCR1 and CCR5 activation on leukocytes may show different outcomes in systemic immune responses, their specific roles for localized leukocyte recruitment in vivo at sites of restricted inflammation or tissue injury remain do be determined. In vitro studies have suggested different roles of CCR1 and CCR5 for leukocyte recruitment. CCR1 but not CCR5 was found to be required for the initial adhesion of human monocytes and T cells to activated endothelium, whereas CCR5 seemed to be involved in the subsequent transendothelial cell migration (14).
We therefore hypothesized that both CCR1 and CCR5 might be involved in macrophage and T cell infiltration and the development of renal fibrosis after UUO in mice. Using a multipronged approach involving combinations of CCR1-deficient and CCR5-deficient mice, transfer of leukocytes from these animals into wild-type mice with UUO, and a specific CCR1 antagonist, we could clearly demonstrate that CCR1 but not CCR5 is required for leukocyte recruitment in this model. We conclude that CCR1 but not CCR5 may be a valuable target for therapeutic intervention for chronic nephropathies accompanied by leukocyte-mediated progressive interstitial fibrosis.
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Materials and Methods |
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Morphologic Evaluation
Tissue sections from the obstructed and the contralateral kidneys of each mouse were fixed in 4% formalin in PBS and embedded in paraffin. Two-micrometer sections were stained with periodic acid-Schiff reagent and silver following the instructions of the supplier (Bio-Optica, Milano, Italy). To count interstitial cells, 15 high power fields (hpf, x400) were analyzed by a blinded observer. Positive cells were counted per hpf omitting positive cells in glomerular fields. Quantitation of the interstitial volume (IVol), interstitial collagen deposition (Icol), and tubular dilatation (ITdil) was performed as described previously (9).
Immunohistochemistry
All immunohistologic studies were performed on paraffin-embedded sections as described (6). The following rat and rabbit antibodies were used as primary antibodies: rat anti-F4/80 (1:50; Serotec, Oxford, UK), rat anti-CD3 (1:50; Serotec), rabbit anti-human TGF- crossreacting with mouse TGF- (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-FSP1 (1:500; kindly provided by Dr. E. G. Neilson, Vanderbilt University, Nashville, TE).
In Situ Hybridization
In situ hybridization for murine TGF-1 was performed as described previously (16). The TGF-1 probe was a gift from H.L. Moses (Department of Cell Biology, Vanderbilt University, Nashville, TN) as described. Negative controls included hybridization performed on replicate tissue sections using the sense riboprobe.
Isolation of Renal Cells for FACS Analysis
A preparation of isolated renal cells including infiltrating leukocytes was obtained as described previously (6) from obstructed and contralateral kidneys. For flow cytometry, the resulting cell suspensions were incubated with 5 µg/ml monoclonal antibodies against murine CCR5 or the isotype control rat IgG2b (Pharmingen, Hamburg, Germany), as described (17). To identify CD8, T cells samples were incubated with a Cy-chrome-labeled anti-mCD8 antibody (Pharmingen).
In Vivo Assay of Renal Leukocyte Infiltration
F4/80 macrophages and CD8 T cells were prepared from spleens of CCR1 -/-, CCR5 -/-, and wild-type mice by a previously described isolation and labeling method (18). In brief, spleen T cells and F4/80 macrophages were isolated by immunomagnetic selection using the following antibodies: CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), FITC rat anti-mouse F4/80 (Serotec, Düsseldorf, Germany), and Anti-FITC MicroBeads (Miltenyi). Purity of isolated cells was verified by flow cytometry. Separated cells were labeled with PKH26 (Red Fluorescence Cell Linker Kit, Sigma-Aldrich Chemicals, Steinheim, Germany), and labeling efficacy was assessed by flow cytometry to be >97%. Viability assessed by trypan blue exclusion was >90%. Wild-type mice underwent surgery for UUO and were injected with either 7.5 x 105 F4/80 positive cells or CD8 positive T cells in 200 µl of isotonic saline into a tail vein 10 d after surgery. One group of mice was injected with CCR5-deficient cells that were preincubated with 600 µM CCR1 antagonist BX471 for 30 min (9). Mice of this group received a single subcutaneous injection of BX471 (50 mg/kg) at the time of injection. Renal tissue was obtained after 3 h, snap frozen, and prepared for microscopy. Interstitial cells from UUO and contralateral kidneys were analyzed as above.
Isolation of Cells for Real-Time RT-PCR
To assess CCR1 and CCR5 mRNA expression in renal fibroblasts and tubular epithelial cells, renal tissue samples were obtained 10 d after UUO. Tubular segments were microdissected from RNase inhibitor treated tissue in ice-cold PBS, as described previously for human renal biopsies (19). For isolation of primary renal fibroblasts, small pieces of renal tissue were incubated in DMEM (Invitrogen Corporation, Karlsruhe, Germany) supplemented with 10% FCS (Invitrogen), penicillin, and streptomycin for 21 d. Adherent cells were lifted with 1.5 mM EDTA (Calbiochem-Novabiochem, San Diego, CA) and were depleted for leukocytes by immunomagnetic selection using FITC anti-mCD45 (Pharmingen) and anti-FITC MicroBeads as described (Miltenyi). F4/80-positive macrophages and CD8-positive T cells were obtained from wild-type mice as described above. mRNA of isolated cells was prepared by standard methods as described (9)
Real-Time Quantitative RT-PCR
Pieces of kidney from each animal were snap frozen in liquid nitrogen and stored at -80°C. RNA preparation and real-time RT-PCR on a TaqMan ABI 7700 Sequence Detection System (PE Biosystems, Weiterstadt, Germany) were performed as described (9). Controls consisting of ddH2O were negative for target genes and the housekeeper gene GAPDH. The following oligonucleotide primers (300 nM) and probes (100 nM) were used. Murine CCR1: forward 5'-TTAGCTTCCATGCCTGCCTTATA-3', reverse 5'-TCCACTGCTTCAGGCTCTTGT-3', internal fluorescence labeled probe (FAM) 5'-ACTCACCGTACCTGTAGCCCTCATTTCCC-3', the probe is located in the deleted region of the CCR1 gene in CCR1-deficient mice; murine CCR5: forward 5'-CAAGACAATCCTGATCGTGCAA-3', reverse 5'-TCCTACTCCCAAGC-TGCATAGAA-3'; FAM 5'-TCTATACCCGATCCACAGGAG-AACATG-AAGTTT-3', the probe is located in the deleted region of the CCR5 gene in CCR5-deficient mice, CCR5 specificity of primers and probe were tested on CCR plasmids; murine TGF-1: forward 5'-CACAGTACAGCAAGGTCCTTGC-3', reverse 5'-AGTAGACGATGGG-CAGTGGCT-3', FAM 5'-GCTTCGGCGTCACCGTGCT-3'; murine GAPDH: forward 5'-CATGGCCTTCCGTGTTCCTA-3', reverse 5'-ATGCCTGCTTCACCACCTTCT-3', internal fluorescence labeled probe (VIC) 5'-CCCAATGTGTCCGTCGTGGATCTGA-3'. All primers and probes were obtained from PE Biosystems. For the expression, CCR levels in isolated cells values are expressed as ratio of respective CCR mRNA to GAPDH mRNA expression. Expression levels of CCR in the UUO model were analyzed accordingly, showing the aforementioned ratio in each animal separately for UUO and contralateral kidneys (CLK). For comparison of mRNA expression, levels between different groups the ratio of UUO to CLK is shown.
RNase Protection Assay
Total spleen RNA was isolated from three C57BL/6 wild-type mice, five CCR1-/- mice, and four CCR5-/- mice. Multiprobe RNase protection assay was performed with the RiboQuant multiprobe template set for murine CC chemokine receptors (mCR-5) obtained from Pharmingen using 10 µg of total spleen RNA per lane as described (6). Unfortunately, the CCR1-specific RPA probe from the multiprobe template set is located outside of the deleted region of the CCR1 gene in CCR1-deficient mice and therefore not suited to differentiate between wild-type and knockout transcripts.
Statistical Analyses
Data are presented as mean ± SD. Comparison of groups was performed using univariant ANOVA and post-hoc Bonferroni correction was used for multiple comparisons. Paired t test was used for the comparison of single groups (FACS data). A value of P < 0.05 was considered to indicate statistical significance.
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Results |
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Reduced Interstitial Leukocyte Infiltration in CCR1-Deficient Mice Is Associated with a Decrease of Renal TGF-1 mRNA Expression
We were intrigued by the finding that the extent of renal fibrosis after UUO in CCR1-deficient and wild-type mice directly paralleled the amount of interstitial leukocytes. As renal fibroblasts were negative for CCR1, the lack or blockade of CCR1 is unlikely to directly affect fibroblast activation. In contrast, the observed reduction of renal fibrosis could be secondary to reduced secretion of profibrotic cytokines. We therefore determined the expression of TGF-1 mRNA in total renal RNA by real-time RT-PCR. UUO kidneys from mice that lack CCR1 had a marked reduction of TGF-1 mRNA expression compared with wild-type mice (Figure 6A). In contrast, lack of CCR5 did not affect renal TGF-1 mRNA expression. However, if treated with BX471, CCR5-deficient mice revealed a significant reduction of renal TGF-1 mRNA expression compared with untreated CCR5-deficient mice (Figure 6A). To determine the source of renal TGF-1, we performed in situ hybridization and immunostaining for TGF-. In unobstructed contralateral kidneys of wild-type mice, the in situ hybridization yielded only a weak diffuse deposition of silver grains not different from the sense controls (Figure 6B). The strongest signal for TGF- mRNA was found in areas of tubulointerstitial infiltrates (Figure 6B). The resolution of the in situ hybridization did not allow assignment of the signal in the infiltrate to specific cells. In areas without prominent cell infiltration, only the background signal was present, similar to unobstructed control kidneys. No clear tubular expression of TGF- mRNA was apparent. Immunostaining for TGF- protein was also restricted to the interstitial cell infiltrates of UUO kidneys from wild-type mice (Figure 6B). In unobstructed contralateral kidneys, only a weak background immunostaining for TGF- was detected. UUO kidneys of CCR1-deficient mice had fewer interstitial TGF- positive cells as detected by immunostaining, which correlated with less tubulointerstitial deposition of silver grains by in situ hybridization for TGF- mRNA. (Figure 6B) and with the lower degree of cell infiltrate in these mice (Figure 2). In contrast, lack of CCR5 did not affect tubulointerstitial expression of TGF- mRNA and protein (Figure 6B).
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is in the interstitial infiltrate and not the tubular cells. Furthermore, the reduction of interstitial leukocyte infiltration observed in CCR1-deficient mice is associated with a decrease of TGF-1 mRNA and protein, a cytokine that can stimulate epithelial-mesenchymal transformation, apoptosis, and collagen secretion by renal fibroblasts. |
Discussion |
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CCR1 Is Required for Leukocyte Infiltration after UUO
Here we show that CCR1 is involved in renal macrophage and T cell recruitment after UUO in mice. Other studies using CCR1-deficient mice have documented its role for neutrophil migration in the defense of certain infectious organisms such as Toxoplasma gondii, Paramyxovirus, and Aspergillus fumigatus (15,20,21). In the context of progressive fibrotic disease states, a recent study reported the effects of a neutralizing antibody against murine CCR1 in the bleomycin-induced pulmonary fibrosis model (22). Antibodies against CCR1 but not against CCR2 reduced pulmonary mononuclear cell infiltration 10 d after induction of disease. We have recently shown in the UUO model in mice that the small molecule CCR1 antagonist BX471 reduces the amount of interstitial leukocytes (9). Previous in vitro studies demonstrated that CCR1 mediates leukocyte adhesion to activated endothelium of human T cells and monocytes under conditions of shear stress and flow (8,17). In addition, our studies with labeled leukocyte subsets in vivo clearly confirmed a role of CCR1 for macrophage and T cell recruitment into the UUO kidney in mice. As the UUO model does not involve a systemic immune response, our data are not in conflict with a report that lack of CCR1 enhances Th1 responses and glomerular injury during nephrotoxic serum nephritis (10). The data on nephrotoxic nephritis would indicate that CCR1 is also involved in systemic immune responses. The fact that genetically generated CCR1 deficiency and intermittent CCR1 blockade with a specific antagonist had the same effect on renal inflammatory infiltrates and fibrosis after UUO provides powerful evidence for the role of CCR1 for macrophage and T cell recruitment after UUO and the concomitant fibrosis. Our results are consistent with the beneficial effects of CCR1 blockade on the functional outcome in renal and heart transplantation, pulmonary fibrosis, or experimental encephalomyelitis and argue for an important role of CCR1-mediated leukocyte recruitment in these models (8,23,24).
Lack of CCR5 Expression Does Not Affect Leukocyte Infiltration and Renal Fibrosis after UUO
CCR1 and CCR5 are coexpressed to different extents on macrophages and T cells. Although T cells expressed only low levels of CCR1 mRNA, the absence of CCR1 or its blockade resulted in significant reduction of renal CCR5-positive T cells in the UUO kidneys, arguing for a role of CCR1 in the recruitment of CCR5-positive cells. In contrast, despite expression of high CCR5 mRNA levels on T cells in wild-type mice, the lack of CCR5 itself did not influence renal leukocyte recruitment after UUO. Interestingly, CCR5 is involved in recruitment of leukocyte subsets in other disease models. For example, lack of CCR5 has been shown to attenuate disease activity in a model of Aspergillus fumigatus conidia-induced asthma, which was associated with a decrease of T cells but not of macrophages in bronchial lavage fluids (25). In a model of dextran sodium sulfate-mediated colitis, improvement of colitis in CCR5-deficient mice was also associated with impaired intestinal lymphocyte recruitment compared with wild-type controls, whereas macrophage recruitment did not depend on CCR5 expression (26). Furthermore, lack of CCR5 improved outcome after cardiac transplantation in fully MHC-mismatched mice, which was associated with reduced host T cell and macrophage recruitment to the rejected allograft (27). Interestingly, in a model of pulmonary Cryptococcus neoformans-infection, 12-wk survival was markedly reduced in CCR5-deficient mice, although no defects in lung leukocyte recruitment were observed (28). However, in this model, CCR5-deficient mice showed impaired leukocyte recruitment into the brain, leading to a reduction in cerebral elimination of cryptococcal polysaccharide (28). It was concluded that CCR5-mediated leukocyte trafficking is organ-specific during host defense against C. neoformans. In contrast, macrophage recruitment did not depend on CCR5 in experimental encephalomyelitis (29) and a model of peripheral nerve injury induced by sciatic nerve axotomy (30). The lack of an effect of CCR5 elimination on inflammatory infiltration after UUO cannot be explained by lack of the chemokine ligand CCL5 in UUO kidneys. The chemokine ligand CCL5 for CCR5 is markedly expressed in UUO kidneys (6). Thus the interaction of CCL5-CCR5 may have functions other than cell recruitment in UUO. In contrast, our data clearly demonstrate the nonredundant role of CCR1 (ligands CCL3 and CCL4) for renal leukocyte recruitment after UUO in mice. The finding that CCR1 is important for renal leukocyte recruitment in vivo is consistent with in vitro data showing that CCR1 but not CCR5 is essential for the initial leukocyte attachment to endothelium (14). Initial attachment mediated by CCR1 is therefore critical for renal leukocyte recruitment. We therefore conclude that the role of CCR5 for leukocyte recruitment may vary in different disease models, which may relate differential chemokine expression patterns or to additional functions of CCR5 in these models.
Lack of CCR1 on Leukocytes Reduces Renal Fibrosis after UUO
How could the reduction in mononuclear leukocyte infiltration in CCR1-deficient mice or with CCR1 blockade relate to the concomitant reduction in interstitial fibroblasts and fibrosis? The infiltrating leukocytes could via secretion of, e.g. cytokines such as TGF-, contribute to epithelial-mesenchymal transformation, fibroblast proliferation, and collagen production (2). In fact, CCR1-deficient mice showed a marked reduction of renal TGF-1 mRNA expression, a key cytokine for the induction of fibroblast proliferation and the development of renal fibrosis (31). To localize the site of TGF- production, we therefore performed both in situ hybridization and immunostaining for TGF-. Both methods localized the TGF- production to the interstitial cell infiltrate. Compared with real-time RT-PCR data, both in situ hybridization and immunostaining signals for TGF- were reduced in UUO kidneys of CCR1-deficient mice and in mice treated with the CCR1 antagonist. The resolution of both methods did not allow assignment of the signal to specific cells in the infiltrate. Clearly, however, the TGF- is not coming from the tubular epithelial cells or from the interstitium in areas without inflammatory cell infiltrate. It therefore appears reasonable to assign the TGF- signals to inflammatory cells, i.e., the infiltrating mononuclear leukocytes. This interpretation is also consistent with another report that localized TGF- to infiltrating leukocytes in the same model using the same TGF- antibody (32). It appears that the reduction in TGF- mRNA and immunostaining in the UUO kidney from CCR1-deficient mice or with CCR1 receptor blockade relates directly to the reduced number of infiltrating cells observed under these conditions and is less likely a result of TGF- production per infiltrating cell. In fact, in vitro studies with peritoneal macrophages from mice of all three strains expressed comparable amounts of TGF- mRNA after stimulation with LPS (unpublished data).
In summary, CCR1 appears to play an important role in mediating the infiltration of mononuclear cells after UUO. Lack of CCR1 reduced the infiltration of macrophages and lymphocytes in the obstructed kidney and the resultant interstitial fibrosis. In contrast, lack of CCR5 had no effect on leukocyte infiltration in the UUO model. Previous studies have demonstrated a similar effect with a CCR1 antagonist; we therefore propose that CCR1 blockade may offer a new therapeutic strategy to reduce renal leukocyte infiltration and resulting fibrosis in chronic nephropathies leading to end-stage renal disease or even leukocyte-mediated progressive tissue fibrosis in general.
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(CCR1+/+) or thick black line (CCR1-/-) and IgG2b isotype control as 
(CCR1+/+) and thin black line (CCR1-/-), respectively. Data shown are from obstructed kidneys of one animal of each group and are representative of four independent samples. (B) Real-time RT-PCR was performed using total renal RNA of UUO kidneys of 4 to 5 mice 10 d after UUO. Levels of mRNA expression for CCR1 (gray) and CCR5 (black) in UUO kidneys are expressed in relation to renal GAPDH mRNA expression and to the respective ratio of the unobstructed contralateral kidney. The expression of control mice is set as 1. Primers for CCR1 were located in the deleted region of the CCR1 gene. Compared with kidneys of wild-type (WT) mice, CCR1-deficient mice revealed a significant reduction of CCR5 mRNA expression (P = 0.03). As to be expected, CCR1 mRNA expression was not detectable in CCR1-deficient mice. Renal CCR1 mRNA expression in CCR5-deficient mice was not reduced compared with wild-type controls. CCR5-deficient mice treated with BX471 revealed a significant reduction of CCR1 mRNA expression (* P < 0.04). BX471 was administered in CCR5-deficient mice as described in Materials and Methods. As to be expected, CCR5 mRNA was not detectable in CCR5-deficient mice. WT, wild-type; CCR -/-, mice negative for respective chemokine receptor expression; n.d., not detected; * P < 0.05.
