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. Author manuscript; available in PMC: 2009 Dec 15.
Published in final edited form as: J Immunol. 2008 Dec 15;181(12):8670–8676. doi: 10.4049/jimmunol.181.12.8670

Chemokine Receptor CCR1 Regulates Inflammatory Cell Infiltration after Renal Ischemia-Reperfusion Injury

Kengo Furuichi *,†, Ji-Liang Gao *, Richard Horuk ‡, Takashi Wada †, Shuichi Kaneko †, Philip M Murphy *
PMCID: PMC2633769  NIHMSID: NIHMS74085  PMID: 19050287

Abstract

Neutrophils and macrophages rapidly infiltrate the kidney after renal ischemia-reperfusion injury, however specific molecular recruitment mechanisms have not been fully delineated for these cell types. Here we provide genetic and pharmacologic evidence supporting a positive role for the chemokine receptor CCR1 in macrophage and neutrophil infiltration in a 7 day mouse model of renal ischemia-reperfusion injury. By day 7, injured kidneys from mice lacking CCR1 contained 35% fewer neutrophils and 45% fewer macrophages than injured kidneys from wild type control mice. Pretreatment of WT mice with the specific CCR1 antagonist BX471 also suppressed neutrophil and macrophage infiltration in the model. Injured kidneys from mice lacking CCR1 also had reduced content of the CCR1 ligands CCL3 (MIP-1α) and CCL5 (RANTES) compared to injured kidneys from wild type controls, suggesting a leukocyte source for these inflammatory chemokines and existence of a CCR1-dependent positive feedback loop for leukocyte infiltration in the model. Local leukocyte proliferation and apoptosis were detected after injury, but were not dependent on CCR1. Also, the extent of necrotic and fibrotic damage and decline in renal function in injured kidneys was similar in wild type and CCR1-deficient mice. Thus, CCR1 appears to regulate trafficking of macrophages and neutrophils to kidney in a mouse model of renal ischemia-reperfusion injury, however this activity does not appear to affect tissue injury.

Keywords: Molecules-chemokines, Cells-monocytes/macrophages, Processes-inflammation

Introduction

Renal ischemia/reperfusion injury (IR) is a major clinical problem found in shock, renal transplantation and other clinical settings, and is associated with high morbidity and mortality. In addition to acute tubular necrosis (ATN), massive interstitial infiltration, particularly by neutrophils and macrophages and to a lesser extent by lymphocytes, is a characteristic pathologic change in IR. Although neutrophils are major mediators of tissue damage in many clinical settings (1), their specific role in IR has not been fully delineated. Evidence in support of an important role has come from loss-of-function experiments in mice targeting adhesion molecules, such as E-, P-, and L-selectin or intercellular adhesion molecule-1 (2)(3), Toll-like receptor 4, tissue factor and protease-activated receptors (4)(5). Furthermore, a recent study has shown that neutrophils (and NKT cells) initiate the immune response by production of interferon-gamma in the very early phase of IR injury (6). In contrast, neutrophil depletion studies with anti-neutrophil antibodies in mouse, rat, and rabbit models have not supported an important role for neutrophils in IR (7–9). Similarly, recombinase activating gene (RAG)-1 deficient mice, which lack both B and T cells, were not protected from renal ischemia-reperfusion injury, suggesting that lymphocytes do not drive pathogenesis in this model (10, 11). In contrast, use of clodronate to selectively deplete macrophages in vivo has supported a major role in IR pathogenesis for this cell type, which accumulates in the late phase post-injury (12, 13). The molecular basis for kidney infiltration by different types of leukocytes in IR is poorly understood. To date, osteopontin, midkine and the chemokine receptor CCR2 have been reported as partial and selective mediators of macrophage infiltration (14–16).

Chemokines are a large family of secreted proteins that together coordinate leukocyte trafficking and activation during homeostasis, development and inflammation. In many animal models of inflammatory kidney disease, it has been reported that various chemokines and chemokine receptors contribute to tissue injury. We have previously reported that CCR1+ macrophages and lymphocytes are mainly detected in interstitial, not glomerular, lesions of human renal biopsy specimens from patients with various kinds of renal diseases (17). Moreover, administration of BX471, a small-molecule nonpeptide CCR1 antagonist, markedly reduced macrophage and T cell infiltrates, and reduced tubular injury and interstitial fibrosis in diverse mouse models of kidney disease, including unilateral ureteral obstruction (18, 19), autosomal recessive Alport syndrome model (20), and murine adriamycin nephropathy (21). These data demonstrate the importance of CCR1 for the recruitment of monocytes and T cells into the renal interstitial compartment, and suggest that it may play a similar role in IR.

CCR1 binds multiple proinflammatory chemokine ligands including CCL3 (MIP-1α), CCL5 (RANTES), CCL7 (MCP-3), CCL9 (MIP-1γ), CCL14 (HCC-1), CCL15 (HCC-2), CCL16 (HCC-4) and CCL23 (MIP-3), and is expressed on T cells, neutrophils, monocyte/macrophages and dendritic cells (22). Among these ligands, CCL5 (RANTES) and CCL3 (MIP-1α) have been reported to be produced by resident renal cells under inflammatory conditions (23). Thus, we hypothesized that leukocytes may infiltrate the kidney in IR by a mechanism dependent on CCR1, and have tested this hypothesis by genetic loss of function criteria in a mouse model of disease.

Materials and Methods

Animals

Male wild type and CCR1−/− C57BL/6 mice were purchased from Taconic (Germantown, NY). CCR1−/− mice, generated as previously described (24), had been backcrossed onto the C57BL/6 background for 10 generations, and maintained under specific pathogen-free housing conditions. No significant differences in growth or weight were found between CCR1−/− and wild type C57BL/6 mice. All animals were used at 6–8 weeks of age under the auspices of a protocol approved by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee.

Renal Ischemia-Reperfusion Model

After general anesthesia was established with xylazine, ketamine and isoflurane, the left renal artery and vein of CCR1−/− and wild type control mice were exposed by flank incision and clamped for 60 min. Kidneys that did not completely recover after unclamping, as assessed by restoration of normal color, were not used for analysis. After releasing the clamp the flank incision was closed in two layers with silk sutures. The animals received warm saline instilled into the peritoneal cavity during the procedure and were allowed to recover with free access to food and water. Sham surgery was performed in a similar manner, except that the renal vessels were not clamped.

A blocking study was performed with BX471 (R-N-[5-chloro-2-[2-[4(4-fluorophenyl) methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl] urea hydrochloric acid salt; Berlex Biosciences, Richmond, CA). Male C57BL/6 mice were subcutaneously dosed with BX471 (20 mg/kg body wt) in a vehicle for 4 days at 12-hour intervals, following a previously reported protocol (18). The vehicle solution was prepared as follows: 40% cyclodextrin (#33,260-7; Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) in saline was mixed, dissolved overnight, and filtered through a 0.45µm filter. Control mice were treated subcutaneously with the same amount of vehicle for 4 days. The first dose was administered just before renal ischemia.

Immunohistochemistry

One portion of the renal tissue was fixed in 10% buffered formalin, then embedded in paraffin, sectioned and stained with periodic acid-Schiff (PAS) reagent, naphthol AS-D (25) chloroacetate esterase, Gomori’s trichrome, or indicated antibodies. Deparaffinized sections were treated with proteinase K (DAKO, California, USA) prior to staining of CCL3 (MIP-1α), F4/80, Ki67, and CD3. Endogenous peroxidase activity and nonspecific binding in the sections was blocked by Peroxidase-Blocking Reagent (DAKO), Biotin-Blocking System (DAKO) and Protein Block, Serum-Free (DAKO). Goat anti-CCL3 (MIP-1α) antibody (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-CD3 antibody (DAKO, California, USA) (25), rabbit anti-Ki67 antibody (Abcam, Cambridge, MA) and rat anti-mouse F4/80 monoclonal antibody (clone: BM8; eBioscience, San Diego, CA) were used to detect CD3, Ki67 and F4/80 antigen, respectively. Normal rabbit or rat IgG was used as a negative control. Rabbit anti-CD3 and anti Ki67 antibody were detected by EnVision+ System (peroxidase) (DAKO), and rat anti-F4/80 antibody was detected by biotinylated polyclonal rabbit anti-rat immunoglobulin antibodies (DAKO) and the LSAB+ system (peroxidase) (DAKO). To determine localization of CCL3 and F4/80 positive cells, FITC conjugated donkey anti-goat IgG antibodies (Jackson ImmunoResearch, West Grove, PA) and Cy3 conjugated donkey anti-rat IgG antibodies (Jackson ImmunoResearch) were used as second antibodies. TdT-mediated dUTP nick end labeling (TUNEL) positive cells were detected by TACS TdT DAB kit (R&D Systems). After washing with PBS containing 0.01% Tween, the sections were stained with 3, 3’-diaminobenzidine (DAB) solution and then counterstained with hematoxylin. Neutrophils were identified by naphthol AS-D chloroacetate esterase staining (Sigma, St. Louis, MO) (26)(27). Tubular necrosis was quantitated in a blinded manner as the percentage of tubules in the outer medulla in which epithelial necrosis or necrotic debris was observed in PAS-stained sections. Interstitial renal fibrosis was quantitated as the area staining blue in sections stained with Gomori’s trichrome. The area of tubular necrosis and interstitial fibrosis in the outer medulla was evaluated by NIH Image and expressed as the percentage of total area imaged. Infiltrating leukocytes, and Ki67- and TUNEL-positive cells were quantitated in the outer medulla of the injured kidney, where cell migration was maximal, and the data were expressed as leukocytes/mm2 at 320X magnification.

Cell isolation and FACS analysis

In addition to immunohistological analysis, we evaluated infiltrating cells using FACS analysis. To obtain single cell suspensions, whole injured left kidney or control right kidney from CCR1-deficient mice or wild type mice were minced and incubated with Liberase Blendzyme 3 (Roche Diagnostics, Indianapolis, IN) at 37° C for 20 min. The cells were filtered with a 40 µm cell strainer, and were washed with MACS buffer (PBS, 0.5% BSA, 2 mM EDTA, degassed) twice. Then, the cells were incubated with Fc block (BD Biosciences, Franklin Lakes, NJ) in 100 µl CD16/32 Ab (BD Biosciences) diluted 1:200 and FITC-conjugated anti–mouse CD45 for 20 min on ice, and washed three times. A portion of total kidney cells was analyzed for CD45 expression. CD45 positive cells were separated from the remainder using magnetic cell sorting (MACS; Miltenyi Biotech, Auburn, CA). The CD45 positive cells were washed three times with FACS buffer (HBSS, 0.1% BSA, 0.1% NaN3), and then incubated with Fc block and PE-conjugated F4/80, CD3, or Gr-1 Abs for 20 min on ice, washed three times, and suspended in 500 µl MACS buffer. In all cases, an isotype-matched FITC- or PE-conjugated antibody was used as the control. Cells were analyzed on a FACSCalibur (BD Biosciences) where leukocytes were further refined by gating on cells with the appropriate forward and side scatter profile. Data were analyzed using FlowJo software and are presented as the percentage of positive cells within the gated population. The final % F4/80, Gr-1, and CD3 positive cells in the total isolated cell population was calculated by (% of CD45 positive cells) X (% of F4/80, Gr-1, or CD3 positive cells) in each animal. Eight CCR1 deficient mice and 12 wild type mice were used for this FACS study. To evaluate CCR1 positive cells in injured kidney, CD45 positive cells were collected by magnetic cell sorting methods. The cells were stained by PE conjugated goat anti-CCR1 antibodies (Santa Cruz Biotechnology) and FITC(????)-conjugated anti-F4/80 antibody, anti-Gr-1 antibody and anti-CD3 antibody. PE-conjugated normal goat IgG (Santa Cruz Biotechnology) and isotype-matched FITC-conjugated antibody were used as negative controls.

Cytokine quantitation

Tissue homogenates were used to determine MIP-1α and RANTES expression using murine Quantikine Immunoassay kits (R&D Systems) following the manufacturer’s directions. All ELISA samples were run in duplicate. The amount of MIP-1α and RANTES was standardized by tissue weight.

Serum creatinine assay

Serum creatinine was measured as a marker of renal function. To test injured kidney function directly, we analyzed serum creatinine at day 7 post-injury in mice that underwent right nephrectomy at day 5 post-injury (n=12 in each group). Blood was collected from each mouse at the time of sacrifice, and stored at −80°C until use. Creatinine concentration was measured by colorimetric microplate assay (Oxford Biomedical Research, Oxford, MI).

Statistical Analysis

The mean and standard error (SEM) were calculated on all the parameters determined in this study. Statistical analyses were performed using unpaired Student's t test, Kruskal-Wallis test and ANOVA test. p<0.05 was accepted as statistically significant.

Results

Chemokine and chemokine receptor expression in the kidney after ischemia-reperfusion injury

Protein levels of CCL3 (MIP-1α) and CCL5 (RANTES) were only faintly detected in sham-operated kidney from wild type mice, however expression was significantly upregulated by ischemia-reperfusion injury, especially on days 4 and 7 post-injury (Fig. 1A, B). Expression of CCL3 (MIP-1α) was significantly lower in CCR1-deficient mice than wild type mice on both days 4 and 7 post-injury (Fig. 1A). CCL3 (MIP-1α) positive cells were mainly tubular epithelial cells (Fig. 1C). Infiltrated F4/80 positive cells were also positive for CCL3 (MIP-1α), and the cells were attracted around CCL3 (MIP-1α) positive tubular epithelial cells (Fig. 1D). Similarly, we observed significantly reduced expression of CCL5 (RANTES) in CCR1-deficient mice than in wild type mice, but the difference did not become apparent until day 7 post-injury (Fig. 1B).

Figure 1. CCL3 (MIP-1α) and CCL5 (RANTES) expression are upregulated after renal ischemia-reperfusion injury.

Figure 1

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Protein levels of CCL3 (MIP-1α) (A) and CCL5 (RANTES) (B) were detected by ELISA. −/−, CCR1 knockout mice; +/+, wild type C57BL/6 control mice; sham, sham-operated wild type C57BL/6 mice tested one day after surgery. Shown are summary data presented as mean ± SEM from 3 independent experiments with 3–5 mice in each experiment. Panel C part i and ii, ischemia-reperfusion injured kidney from wild type C57BL/6 mouse 4 days after injury stained with anti-CCL3 (MIP-1α) and control IgG, respectively. Tissue was counterstained with hematoxylin. Original magnification of all images is 320X. Panel D part i and ii, ischemia-reperfusion injured kidney from wild type C57BL/6 mouse 4 days after injury stained with anti-F4/80, and anti-CCL3 (MIP-1α), respectively. D part iii is a merged picture of part i and ii. Original magnification of all images is 400X. Scale bar indicates 50µm. * P<0.05 comparing CCR1-deficient mice versus wild type mice at the same time point.

CCR1-dependence of macrophage and neutrophil infiltration in the kidney after ischemia-reperfusion injury

F4/80 positive cells were mainly detected in the interstitial area of the outer medulla of the injured kidney in both wild type (Fig. 2Ai) and CCR1-deficient mice (Fig. 2Aii). This matched the distribution of naphthol AS-D chloroacetate esterase positive cells in these mice under these conditions (Fig. 2Bi and ii). The number of infiltrating F4/80+ cells increased in a time-dependent manner throughout the 7 day period of observation in both wild type and CCR1-deficient mice, but the increase was significantly attenuated in CCR1-deficient mice, first observed beginning at the 48 hour time point post-injury and lasting through the end of the 7 day time course (Fig. 2C). In contrast to this temporal pattern, naphthol AS-D chloroacetate esterase positive cells, which were rapidly and markedly increased within the first 24 hours post-injury in both wild type and CCR1-deficient mice, subsequently declined in number in a time-dependent manner throughout the remaining 7 day time course in both strains. However, the normal decline in esterase+ cells was significantly accelerated in CCR1-deficient mice, first observed beginning at the 4 day post-injury time point and lasting through the end of the 7 day time course (Fig. 2D). The number of CD3+ cells was increased in the injured kidney to a much lower degree compared with both F4/80+ and naphthol AS-D chloroacetate esterase+ cells (Fig. 2E). Moreover, significant differences were not observed in CD3 cell numbers between wild type and CCR1-deficient mice at any time point post-injury.

Figure 2. CCR1 regulates macrophage and neutrophil infiltration of the outer medulla after renal ischemia-reperfusion injury.

Figure 2

Figure 2

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A–E) Immunohistochemical analysis. A) Macrophages. anti-F4/80 staining of wild type C57BL/6 (i) and CCR1-deficient (ii) mouse kidney, and control IgG staining of wild C57BL/6 mouse kidney (iii). B) Neutrophils. naphthol AS-D chloroacetate esterase staining of wild type C57BL/6 (i) and CCR1-deficient (ii) mouse kidney. Original magnification of all images is 320X. Scale bar indicates 50µm. C–E, Quantitation of density of indicated leukocyte subsets in tissue sections at 320X magnification under the conditions shown. sham, sham-operated wild type mice tested 24 hours after surgery. Values are mean ± SEM and are pooled from 3 independent experiments with 3–5 animals for each condition in each experiment. * P<0.05 comparing CCR1-deficient mice versus wild type mice at the same time point. F–I, FACS analysis. Representative FACS data, in which anti-CD45 (Fi, ii) or isotype control (Fiii, iv) was used to stain cells isolated from whole left injured kidney (Fi, iii) or right control kidney (Fii, iv) 7 days post-IR. (Fv-viii) Representative FACS data for CD45+ cells purified using immunobeads and stained with anti-F4/80 (v), anti-Gr-1 (vi), anti-CD3 (vii), or isotype control (viii). G, Summary data of FACS analysis. % of total was calculated as (% of CD45+) X (% of F4/80+, Gr-1+ or CD3+ cells) in each animal. n=8 for CCR1-deficient mice, n=14 for C57BL/6 control mice. H, Representative FACS data 7 days after reperfusion, in which PE conjugated anti-CCR1 (Hii-iv) or control IgG (Hi) and FITC conjugated anti-F4/80(Hiii), anti-GR1(Hiv) or isotype control (Hii) were used to stain CD45-positive cells isolated from whole left injured kidney by magnetic cell sorting methods. I, Summary data of FACS analysis. % of F4/80- or Gr-1-positive cells in CCR1-positive cells. n=5 in each group.

Flow cytometry confirmed these immunohistochemical results. Seven days after ischemia/reperfusion, CD45+ cells represented ~10% of total cells in the injured left kidney, compared to only ~1% of total cells in the right uninjured kidney (Fig. 2F). In wild type mice, CD45+ cells in the injured kidney were ~80% F4/80+, ~20% Gr-1+, and <5% CD3+ (Fig. 2F, G). Injured kidneys of CCR1-deficient mice contained ~50% fewer F4/80+ and Gr-1+ cells on day 7 after reperfusion (Fig. 2G). In injured kidney, ~50% of infiltrated cells were CCR1 positive 7 days after reperfusion (Fig. 2Hii). Among these CCR1-positive cells, the % F4/80-positive cells gradually increased after reperfusion, whereas the % Gr1-positive cells gradually decreased.

CCR1-deficiency does not affect cell proliferation or apoptosis of interstitial cells in renal ischemia-reperfusion injury

The number of infiltrating leukocytes in the kidney after IR may be a function of influx, efflux and the local rate of leukocyte proliferation and apoptosis. To evaluate proliferation and apoptosis in the model, we measured the markers Ki67 and TUNEL in situ by immunohistochemistry. Both tubular epithelial cells and interstitial cells were positive for Ki67 and TUNEL (Figure A, B). Few weakly staining Ki67+ and TUNEL+ cells could be detected in kidneys from sham-operated mice, whereas the frequency and staining intensity of these cells were both significantly increased after injury (Fig. 3A–D). Kinetic analysis identified two peaks for both markers, one at 24 hours post-IR, the other 7 days post-IR (Fig. 3C, D). The number of Ki67+ and TUNEL+ cells did not differ significantly between wild type and CCR1-deficient mice at any time point post-IR.

Figure 3. CCR1 does not regulate cell proliferation or apoptosis in the outer medulla after renal ischemia-reperfusion injury.

Figure 3

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A and C) Proliferation. B and D) Apoptosis. Kidney sections obtained 4 days post-IR were stained with anti-Ki67 (Ai), TUNEL (Bi) or appropriate negative controls (Aii and Bii). A and B, Representative results for kidneys from injured wild type mice are depicted at 320X magnification. Scale bar indicates 50µm. C and D, Quantitation of density of Ki67 and TUNEL positive cells. Values are mean ± SEM and are from 3 independent experiments with 3–5 animals for each condition in each experiment. sham, sham-operated wild type mice tested 24 hours after surgery.

CCR1 deficiency does not affect tissue destruction or renal dysfunction after ischemia-reperfusion injury

Severe acute tubular necrosis was localized mainly to the outer medulla of the mouse kidney 24 and 48 hours after ischemia-reperfusion injury (Fig. 4Ai–iv). With time, tubular epithelial cells proliferated and tubules were regenerated in both wild type and CCR1-deficient mice, as shown for days 4 and 7 after injury in Fig. 4Av–viii. Little if any evidence of acute tubular necrosis was detectable in the kidneys of sham-operated B6 and CCR1 deficient mice at 24 hours after surgery (Fig. 4Aix, x).

Figure 4. CCR1-deficiency does not alter extent of renal dysfunction after ischemia-reperfusion injury.

Figure 4

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A, Histopathologic analysis. PAS-stained outer medulla of the left (injured) kidney is shown for wild type (Ai, iii, v, vii, ix) and CCR1-deficient (Aii, iv, vi, viii) mice at the times after ischemia-reperfusion injury indicated to the left of each panel (Ai-viii) or 24 hours after sham operation (ix). Scale bar indicates 50µm. B–E, Quantitation of the indicated measures of renal damage post-IR. sham, sham-operated wild type mice tested 24 hours after surgery. Values are mean ± SEM and are from 3 independent experiments with 3–5 animals for each condition in each experiment.

Quantitation of the area of acute tubular necrosis failed to reveal a significant difference between wild type and CCR1-deficient mice at any time point (Fig. 4B). Acute renal injury induces increased vascular permeability and renal weight gain. However, we did not observe a significant difference in kidney weight between injured wild type and CCR1-deficient mice (Fig. 4C). Fibrosis, quantitated histopathologically as area staining positively with trichrome, was present at low levels in the renal interstitium and did not increase in the first 48 hours post-injury (data not shown), however this area increased significantly by day 7, especially in the interstitium of the outer medulla. Nevertheless, the extent of fibrosis in the outer medulla of the injured kidney did not show any significant difference between CCR1-deficient and wild type mice (Fig. 4D). Renal function, measured as serum creatinine, deteriorated 7 days after ischemia-reperfusion in wild type mice, but again no significant difference was observed in the extent of decompensation compared with CCR1-deficient mice (Fig. 4E). To test renal function of the injured kidney directly, we analyzed serum creatinine at day 7 post-injury in mice that underwent right nephrectomy at day 5 post-injury. The results showed an ~200% increase in serum creatinine for injured wild type mice relative to sham-operated wild type mice. However, renal function of the injured kidney did not differ significantly for wild type and CCR1-deficient mice.

CCR1 antagonist BX471 pretreatment reduces macrophage and neutrophil infiltration in the kidney after ischemia-reperfusion injury to the same extent as CCR1 gene disruption

To test this model directly, we injected mice subcutaneously with CCR1 antagonist BX471, or vehicle, prior to ischemia of the kidney and every 12 hours after the injury. BX471 resulted in significant reduction in the number of neutrophils and macrophages infiltrating the outer medulla (Fig. 5A and B). The number of neutrophils and macrophages between untreated and vehicle-treated wild type mice, or between CCR1 gene-targeted mice and BX471-treated wild type mice were not significantly different.

Figure 5. Pretreatment witih CCR1 antagonist BX471 reduces macrophage and neutrophil accumulation in kidney after ischemia-reperfusion injury.

Figure 5

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A and B, Macrophages were immunohistochemically detected by anti-F4/80 atibodies, and neutrophils were detected by naphthol AS-D chloroacetate esterase staining. sham, sham-operated wild type mice. Values are mean ± SEM, and are pooled from 3 independent experiments with 3–5 animals for each condition in each experiment. *P<0.05 comparing to BX471 treated mice at 4 days after the injury.

Discussion

In the present study, we have used a genetic loss of function test to demonstrate that the chemokine receptor CCR1 contributes to accumulation of macrophages and neutrophils in the kidney in a mouse model of ischemia-reperfusion injury. As expected, the number of infiltrating neutrophils in the model peaked early, within 24 hours, and declined linearly with time thereafter, whereas macrophage accumulation was slow, and continued to increase linearly with time throughout the 7 day course of the experiment. Although the two cell types exhibited reciprocal kinetics of accumulation, the effect of CCR1 deficiency was similar in magnitude, direction and timing for both in the model: that is, after a time delay CCR1 deficiency attenuated both accumulation of macrophages and clearance of neutrophils.

Our data are consistent with previous reports indicating that CCR1 is expressed on mouse neutrophils, monocytes and macrophages and functions as a chemotactic receptor on these cell types in vitro. Moreover, CCR1 knockout mice or mice treated with CCR1 antagonists have been used previously in vivo to demonstrate the importance of CCR1+ leukocytes infiltrating renal interstitium in various mouse models (18–21, 28, 29). In the present study, we found that compared to untreated wild type mice pretreatment of wild type mice with the CCR1 antagonist BX471 resulted in reduction of neutrophil and macrophage infiltration after the ischemic injury to an extent similar to what was observed in CCR1 gene-targeted mice. Consistent with these animal models, we previously reported that CCR1+ monocytes and lymphocytes were predominantly detected in the interstitium of human kidney biopsy samples in various renal diseases (17). Together these data suggest that CCR1 is important for interstitial, not glomerular, macrophage infiltration in various types of renal injury.

With regard to neutrophils, we have previously reported that mature neutrophils from CCR1-deficient mice fail to chemotax in vitro and fail to mobilize into peripheral blood in vivo in response to CCL1 (MIP-1α) (24). Moreover, Ramos et al reported that MIP-1α failed to induce neutrophil migration in CCR1-deficient mice, in contrast to CCR5-deficient mice (30). Similarly, met-RANTES, an amino-terminal-modified methionylated form of CCL5 (RANTES) and antagonist of CCL5 (RANTES) receptors, has been reported to reduce the number of neutrophils, and macrophages, in the joint in an adjuvant-induced arthritis rat model (31). These data support the notion that interstitial neutrophil infiltration after ischemia-reperfusion is mediated at least in part by CCR1.

The leukocyte infiltrates that we observed are a steady state condition resulting from at least four distinct processes: leukocyte influx, egress, proliferation and death. While chemokines are best known for inducing cell migration, and probably contribute to leukocyte accumulation mainly through influx pathways, some chemokines, such as CXCL10 (IP-10), CXCL9 (Mig), CXCL13, and CCL21 (SLC), are also able to induce cell proliferation and/or deletion by apoptosis (32–34). In CCR1, activation of the receptor by CCL9 (MIP-1γ) also plays a role in the differentiation and survival of osteoclasts (35). To evaluate the contribution of proliferation and apoptosis to CCR1-dependent leukocyte infiltration, we measured staining of renal sections directly with the markers Ki67 and TUNEL. Although some infiltrating leukocytes were positive for these markers, CCR1 deficiency did not affect the number. These data support the conclusion that CCR1 promotes leukocyte influx, not proliferation or apoptosis in this model.

Despite the large attenuation in neutrophil and macrophage infiltration that may be attributed to CCR1 in the model, we did not observe any CCR1-dependent changes in tissue destruction or renal function. This may not be so surprising with respect to neutrophil depletion studies, since the experiments have previously revealed that absence of this cell type provides no protection from acute tubular necrosis or renal dysfunction in the model. However, other neutrophil blocking studies showed some protective effects fromischemic injury. Moreover, we anticipated that attenuation of macrophage infiltration would confer some protection since systemic monocyte-macrophage depletion using liposomal clodronate prevents renal tissue destruction in the model (7, 9, 10, 36), and since blocking CCR2, the receptor for the monocyte-directed CC chemokine CCL2 (MCP-1), strongly attenuated both macrophage infiltration and tissue destruction in the model (16, 37). Although the genetic background of CCR2 deficient mice was different from CCR1 deficient mice, it is possible that a critical threshold of macrophage blockade, achieved in the setting of CCR2 deficiency (on an outbred C57BL/6 × 129/Ola genetic backgrounds) but not in the setting of CCR1 deficiency (backcrossed on the C57BL/6 background for 10 generations), is required to prevent renal damage. Another possibility is that CCR1 and CCR2 or other factors, such as adhesion molecules, regulate distinct subsets of neutrophils or macrophages and/or their effector functions.

A second and related finding in our study is that CCR1 supports production of CCL5 (RANTES) and CCL3 (MIP-1α) in the model. This suggests the existence of a positive feedback loop for CCR1-dependent leukocyte recruitment to the kidney, and is consistent with previously reported data indicating that CCL5 (RANTES) and CCL3 (MIP-1α) can be produced by multiple cell types relevant to our model, including mesangial cells, fibroblasts, macrophages and T cells (23). Neutrophils also produce CCL3 (MIP-1α). In CCR1-deficient animals, reduced recruitment of macrophages would thus reduce a major potential cellular source of both CCL5 (RANTES) and CCL3 (MIP-1α), and reduced recruitment of neutrophils would reduce a second potential cellular source of CCL3 (MIP-1α). Together, this would be predicted to stall recruitment of CCR1+ cells into the model.

In conclusion, our data indicate that in a mouse model of renal ischemia-reperfusion injury CCR1 is an important factor promoting infiltration by neutrophils and macrophages as well as production of its cognate ligands CCL5 (RANTES) and CCL3 (MIP-1α). The data are consistent with a positive feedback loop involving production of these ligands by CCR1+ macrophages and neutrophils that enter the injured kidney. Although tissue destruction, fibrosis and renal function are clearly dependent on macrophages in this model, the magnitude of CCR1 control over infiltration by this cell type is evidently not sufficient to affect these parameters of disease.

Acknowledgments

We thank David McDermott (NIH), and Joan Sechler (NIH) for their excellent technical advice and support.

This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

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