The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia–reperfusion injury
2008; Elsevier BV; Volume: 74; Issue: 12 Linguagem: Inglês
10.1038/ki.2008.500
ISSN1523-1755
AutoresLi L, Liping Huang, Sun‐Sang J. Sung, Amy L. Vergis, Diane L. Rosin, C. Edward Rose, Peter I. Lobo, Mark D. Okusa,
Tópico(s)Adenosine and Purinergic Signaling
ResumoChemokines and their receptors such as CCR2 and CX3CR1 mediate leukocyte adhesion and migration into injured tissue. To further define mechanisms of monocyte trafficking during kidney injury we identified two groups of F4/80-positive cells (F4/80low and F4/80high) in the normal mouse kidney that phenotypically correspond to macrophages and dendritic cells, respectively. Following ischemia and 3 h of reperfusion, there was a large influx of F4/80low inflamed monocytes, but not dendritic cells, into the kidney. These monocytes produced TNF-α, IL-6, IL-1α and IL-12. Ischemic injury induced in CCR2−/− mice or in CCR2+/+ mice, made chimeric with CCR2−/− bone marrow, resulted in lower plasma creatinine levels and their kidneys had fewer infiltrated F4/80low macrophages compared to control mice. CX3CR1 expression contributed to monocyte recruitment into inflamed kidneys, as ischemic injury in CX3CR1−/− mice was reduced, with fewer F4/80low macrophages than controls. Monocytes transferred from CCR2+/+ or CX3CR1+/− mice migrated into reperfused kidneys better than monocytes from either CCR2−/− or CX3CR1−/− mice. Adoptive transfer of monocytes from CCR2+/+ mice, but not CCR2−/− mice, reversed the protective effect in CCR2−/− mice following ischemia-reperfusion. Egress of CD11b+Ly6Chigh monocytes from blood into inflamed kidneys was CCR2- and CX3CR1-dependent. Our study shows that inflamed monocyte migration, through CCR2- and CX3CR1-dependent mechanisms, plays a critical role in kidney injury following ischemia reperfusion. Chemokines and their receptors such as CCR2 and CX3CR1 mediate leukocyte adhesion and migration into injured tissue. To further define mechanisms of monocyte trafficking during kidney injury we identified two groups of F4/80-positive cells (F4/80low and F4/80high) in the normal mouse kidney that phenotypically correspond to macrophages and dendritic cells, respectively. Following ischemia and 3 h of reperfusion, there was a large influx of F4/80low inflamed monocytes, but not dendritic cells, into the kidney. These monocytes produced TNF-α, IL-6, IL-1α and IL-12. Ischemic injury induced in CCR2−/− mice or in CCR2+/+ mice, made chimeric with CCR2−/− bone marrow, resulted in lower plasma creatinine levels and their kidneys had fewer infiltrated F4/80low macrophages compared to control mice. CX3CR1 expression contributed to monocyte recruitment into inflamed kidneys, as ischemic injury in CX3CR1−/− mice was reduced, with fewer F4/80low macrophages than controls. Monocytes transferred from CCR2+/+ or CX3CR1+/− mice migrated into reperfused kidneys better than monocytes from either CCR2−/− or CX3CR1−/− mice. Adoptive transfer of monocytes from CCR2+/+ mice, but not CCR2−/− mice, reversed the protective effect in CCR2−/− mice following ischemia-reperfusion. Egress of CD11b+Ly6Chigh monocytes from blood into inflamed kidneys was CCR2- and CX3CR1-dependent. Our study shows that inflamed monocyte migration, through CCR2- and CX3CR1-dependent mechanisms, plays a critical role in kidney injury following ischemia reperfusion. Acute kidney injury induced by ischemia–reperfusion injury (IRI) is associated with high morbidity and mortality and remains a critical clinical issue.1.Thadhani R. Pascual M. Bonventre J.V. Acute renal failure.N Engl J Med. 1996; 334: 1448-1460Crossref PubMed Scopus (1434) Google Scholar,2.Jo S.K. Rosner M.H. Okusa M.D. Pharmacologic treatment of acute kidney injury: why drugs haven't worked and what is on the horizon.Clin J Am Soc Nephrol. 2007; 2: 356-365Crossref PubMed Scopus (193) Google Scholar Rapid accumulation of neutrophils and monocyte/macrophages in the injured kidney is an essential feature of the innate immune response in IRI.3.Bonventre J.V. Weinberg J.M. Recent advances in the pathophysiology of ischemic acute renal failure.J Am Soc Nephrol. 2003; 14: 2199-2210Crossref PubMed Scopus (608) Google Scholar,4.Li L. Okusa M.D. Blocking the immune response in ischemic acute kidney injury: the role of adenosine 2A agonists.Nat Clin Pract Nephrol. 2006; 2: 432-444Crossref PubMed Scopus (50) Google Scholar,5.Jo S.K. Sung S.A. Cho W.Y. et al.Macrophages contribute to the initiation of ischaemic acute renal failure in rats.Nephrol Dial Transplant. 2006; 21: 1231-1239Crossref PubMed Scopus (236) Google Scholar,6.Day Y.J. Huang L. Ye H. et al.Renal ischemia–reperfusion injury and adenosine 2A receptor-mediated tissue protection: role of macrophages.Am J Physiol Renal Physiol. 2005; 288: F722-F731Crossref PubMed Scopus (210) Google Scholar Monocyte/macrophages are heterogeneous.7.Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar,8.Sunderkotter C. Nikolic T. Dillon M.J. et al.Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417Crossref PubMed Scopus (810) Google Scholar Neither the blood monocyte subset that mobilizes rapidly in response to IRI nor the mechanism of monocyte/macrophage trafficking is known. Two types of blood monocytes were recently identified in mice. Resident monocytes,7.Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar having a CD11b+CCR2lowGr-1−Ly6C−CX3CR1high phenotype, migrate to uninjured tissues rapidly after emigration from bone marrow (BM)7.Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar and differentiate into resident macrophages and dendritic cells (DCs). In contrast, a distinct inflamed monocyte subset with a CD11b+CCR2highLy6ChighGr-1intCX3CR1low phenotype infiltrates infected tissue7.Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar,8.Sunderkotter C. Nikolic T. Dillon M.J. et al.Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417Crossref PubMed Scopus (810) Google Scholar,9.Gordon S. Taylor P.R. Monocyte and macrophage heterogeneity.Nat Rev Immunol. 2005; 5: 953-964Crossref PubMed Scopus (3528) Google Scholar,10.Qu C. Edwards E.W. Tacke F. et al.Role of CCR8 and other chemokine pathways in the migration of monocyte-derived dendritic cells to lymph nodes.J Exp Med. 2004; 200: 1231-1241Crossref PubMed Scopus (241) Google Scholar,11.Tacke F. Randolph G.J. Migratory fate and differentiation of blood monocyte subsets.Immunobiology. 2006; 211: 609-618Crossref PubMed Scopus (386) Google Scholar and contributes to the development of atherosclerosis and inflammation;12.Tacke F. Alvarez D. Kaplan T.J. et al.Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques.J Clin Invest. 2007; 117: 185-194Crossref PubMed Scopus (942) Google Scholar its function in kidney injury has not been investigated. Chemokines are potent mediators of leukocyte cell adhesion and migration. C–C motif chemokine receptor 2 (CCR2) is expressed on a subset of monocytes that participates in defense against infection and chronic inflammation.8.Sunderkotter C. Nikolic T. Dillon M.J. et al.Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417Crossref PubMed Scopus (810) Google Scholar,13.Charo I.F. Ransohoff R.M. The many roles of chemokines and chemokine receptors in inflammation.N Engl J Med. 2006; 354: 610-621Crossref PubMed Scopus (1874) Google Scholar Monocyte chemoattractant protein-1 (MCP-1/CCL2) is the main ligand for CCR2,14.Charo I.F. Taubman M.B. Chemokines in the pathogenesis of vascular disease.Circ Res. 2004; 95: 858-866Crossref PubMed Scopus (622) Google Scholar and the MCP-1/CCR2 signal pathway is important for monocyte recruitment to the site of inflammation.8.Sunderkotter C. Nikolic T. Dillon M.J. et al.Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417Crossref PubMed Scopus (810) Google Scholar,13.Charo I.F. Ransohoff R.M. The many roles of chemokines and chemokine receptors in inflammation.N Engl J Med. 2006; 354: 610-621Crossref PubMed Scopus (1874) Google Scholar C-X3-C motif chemokine receptor 1 (CX3CR1) and its ligand, fractalkine, are important in macrophage accumulation and inflammation15.Damas J.K. Boullier A. Waehre T. et al.Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, is elevated in coronary artery disease and is reduced during statin therapy.Arterioscler Thromb Vasc Biol. 2005; 25: 2567-2572Crossref PubMed Scopus (101) Google Scholar and contribute to atherosclerosis.12.Tacke F. Alvarez D. Kaplan T.J. et al.Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques.J Clin Invest. 2007; 117: 185-194Crossref PubMed Scopus (942) Google Scholar,16.Combadiere C. Potteaux S. Gao J.L. et al.Decreased atherosclerotic lesion formation in CX3CR1/apolipoprotein E double knockout mice.Circulation. 2003; 107: 1009-1016Crossref PubMed Scopus (401) Google Scholar,17.McDermott D.H. Fong A.M. Yang Q. et al.Chemokine receptor mutant CX3CR1-M280 has impaired adhesive function and correlates with protection from cardiovascular disease in humans.J Clin Invest. 2003; 111: 1241-1250Crossref PubMed Scopus (235) Google Scholar,18.Wallace G.R. Vaughan R.W. Kondeatis E. et al.A CX3CR1 genotype associated with retinal vasculitis in patients in the United Kingdom.Invest Ophthalmol Vis Sci. 2006; 47: 2966-2970Crossref PubMed Scopus (10) Google Scholar In the current study we test the hypothesis that trafficking of the blood inflamed monocyte subset to injured kidneys requires MCP-1/CCR2 and fractalkine/CX3CR1 and that the F4/80low macrophage population (derived from the blood inflamed monocytes) contributes to the innate immune response to mediate kidney IRI. Flow cytometry and immunohistochemistry revealed that F4/80-positive (F4/80+) cells were the most abundant resting leukocyte population, constituting ~50% of the CD45+ population in normal kidneys (Figure S1a and b). We next examined the effect of kidney IR on the time course of F4/80+ infiltration relative to other leukocytes (Figure S1c). Although at baseline the F4/80+ cell population was the most abundant leukocyte subset, at 30 min of reperfusion there was no increase in infiltration of F4/80+ cells above sham. At 1 h of reperfusion, the number of F4/80+ cells (macrophages) increased significantly, peaking at 24 h, and persisting for at least 7 days following reperfusion. Download .jpg (.07 MB) Help with files Figure S1 Fluorescence-activated cell sorter (FACS) analysis identified two subsets of F4/80+ cells in normal mouse kidneys, CD11blowF4/80high (referred to as F4/80high) and CD11bhighF4/80low (F4/80low) cells, based on the F4/80 and CD11b fluorescence intensity (Figure 1a). The F4/80high cells comprised ~40% of the total leukocyte (CD45+) population and represented the major leukocyte population within normal mouse kidneys. Using monocyte, DC, chemokine receptor, adhesion and toll-like receptor (TLR) markers these two F4/80+ subsets had distinct phenotypes (Figure 1b–d; Table 1). All F4/80low cells expressed CD62L+, Ly6Chigh, Gr-1int (Figure 2b), and CX3CR1low (Figure 2c), a phenotype characteristic of the blood inflamed monocyte.7.Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar,8.Sunderkotter C. Nikolic T. Dillon M.J. et al.Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417Crossref PubMed Scopus (810) Google Scholar All F4/80high cells expressed CD11c+, MHC class IIhigh (IAhigh), CD86+, and CX3CR1high (Figure 1c), a phenotype characteristic of DCs. Both subsets expressed lymphocyte function-associated antigen 1, very late antigen-4, TLR2, MD-1 (Figure 1d), and RP-105 (not shown). Therefore, F4/80high cells and F4/80low cells were considered to represent kidney resident DCs and macrophages derived from the inflamed monocyte pool, respectively.Table 1Phenotype of kidney F4/80low and F4/80high macrophagesAntibody cloneF4/80lowF4/80highMonocyte markers CD11bM1/70High +Low + F4/80BM8Low +High + Ly6CAL-21High +- GR-1(Ly6G)RB6-8C5Intermediate +- CD2042F8-+ CD206MR5D3-- M-CSFR4H1+/-- CX3CR1PolyclonalLow +High +DC marker CD205MG38-- 33D133D1-- CD11cN41820–30% ++ IAM5/114.15.2-High + CD8016-10A1-- CD86GL1-+ CD40HM40-3-- B7-DCTY25-- B7-H11-111A-+Adhesion marker CD62LMEL-14+- VLA-4HMb1-1High +Low + LFA-1M17/4High +Low +TLR marker TLR26C2++DC, dendritic cell; TLR, toll-like receptor. Open table in a new tab Figure 2Recruitment of CD11bhighF4/80low inflamed monocyte subset following kidney IRI. Mouse kidneys were subjected to 32 min ischemia followed by (a, c, e) 3 h or (b, d, f) 24 h of reperfusion. All leukocytes from sham and IRI kidneys of equal weight were counted and evaluated by FACS (see ‘Materials and Methods’). Results clearly indicated that there was an increase in total number of CD45+ leukocytes in IRI kidneys at (a, right) 3 h and (b, right) 24 h when compared to respective sham-operated mice. The increase in leukocytes was mostly from F4/80low, (a, b) CD11b+, (c, d) Ly6C+, (e, f) Gr-1+ cells, consistent with macrophages derived from the inflamed monocytes subset. Summary of the cell numbers of macrophages (F4/80low) and DCs (F4/80high) in sham and IRI kidneys after (g) 3 h and (h) 24 h of reperfusion. Values are means±s.e.; N=7–10; **P<0.01, ***P<0.001; NS, not significant.View Large Image Figure ViewerDownload (PPT) DC, dendritic cell; TLR, toll-like receptor. As both kidney F4/80+ subsets express CX3CR1, we used CX3CR1+/GFP mice to track macrophages and DCs in normal kidney. Targeted deletion of CX3CR1 and replacement with the gene encoding green fluorescent protein (GFP) provides a useful means for following labeled cells and for studying the function of CX3CR1. GFP is expressed in all CX3CR1+/GFP and homozygous CX3CR1GFP/GFP circulating CD11b+F4/80+ cells.7.Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar,19.Jung S. Aliberti J. Graemmel P. et al.Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.Mol Cell Biol. 2000; 20: 4106-4114Crossref PubMed Scopus (1682) Google Scholar,20.Palframan R.T. Jung S. Cheng G. et al.Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues.J Exp Med. 2001; 194: 1361-1373Crossref PubMed Scopus (411) Google Scholar Most of the kidney CX3CR1 GFP+ DCs are resident DCs and are distributed throughout the cortex (Figure 1e) and outer medulla (Figure 1f), consistent with a recent report.21.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 MHC II (IA) distribution was mainly in the kidney medulla (Figure 1f), compared to cortex (Figure 1e), and most GFP+ cells expressed IA (Figure 1e–g) and F4/80 on the cell surface (Figure 1h). DCs comprised 85% of the GFP+ cells (Figure 1i). All of the CX3CR1+GFPhigh cells expressed CD11b, and most expressed CD11c and MHC II, markers for DCs (Figure 1i). Ly6C and Gr-1 were expressed primarily on the GFPlow population (Figure 1j), which has the same phenotype as the blood inflamed monocytes.7.Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar Both GFP+ cell populations expressed F4/80 (Figure 1h and j). Kidney F4/80low leukocytes had a similar phenotype to GFPlow cells in CX3CR1+/GFP mice and represented kidney monocyte/macrophages, whereas the F4/80high subset resemble CX3CR1+GFPhigh cells and represented resident kidney DCs (Figure 1b and c). Following 3 (Figure 2a and g) and 24 h (Figure 2b and h) of reperfusion FACS analysis showed an increase in the cell number of F4/80low macrophages. However, 3 (Figure 2a and g) and 24 h (Figure 2b and h) after reperfusion there was no change in the number of F4/80high kidney resident DCs. Similar results were observed with Ly6Chigh and Gr-1int markers expressed on the blood inflamed monocytes, at 3 (Figure 2c and e) and 24 h (Figure 2d and f). At 3 h following reperfusion, recruited F4/80low macrophages were Ly6Chigh (Figure 2c), which represents immature, newly arrived monocytes from BM.8.Sunderkotter C. Nikolic T. Dillon M.J. et al.Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417Crossref PubMed Scopus (810) Google Scholar By 24 h, Ly6C expression was markedly downregulated in the F4/80low monocytes (Figure 2d); the values for mean fluorescence index were 2243.3±134.2 (n=3) and 768.0±47.4 (n=3) for 3 and 24 h IRI, respectively (P<0.001). This finding suggests that either macrophages mature after migration to the inflamed kidney or mature macrophages infiltrate IRI kidneys. Similarly, the number of F4/80lowGr-1int macrophages in kidneys increased at 3 h (Figure 2e) and was greater (relative to sham) by 24 h of reperfusion (Figure 2f, right). The mean fluorescence index of F4/80lowGr-1int decreased at 24 h compared to 3 h following kidney IRI, paralleling the findings with Ly6C. Therefore, an abundance of infiltrating macrophages, phenotypically similar to circulating inflamed monocytes, was identified in reperfused kidneys. We used FACS analysis of intracellular cytokine production to determine functional differences between F4/80low and F4/80high populations following kidney IRI. Gating on F4/80low and F4/80high (Figure 2b) macrophages from 24 h sham and IRI kidney, we determined expression of intracellular interleukin (IL)-1α, IL-6, IL-12p40/70, and tumor-necrosis factor (TNF)-α (Figure 3a and b). The proportion of F4/80low macrophages producing cytokines (% of total F4/80low population) following IRI was 4- to 8-fold greater for IRI relative to sham (Figure 3a and c). In contrast, the percentage of F4/80high cells (Figure 3b and d) that produced IL-1α, IL-6, IL-12p40/70 was unchanged but TNF-α-producing cells increased significantly following IRI compared to sham. Therefore, 24 h following reperfusion, FACS analysis reveals functional differences between F4/80low and F4/80high populations. Chemokine production attracts monocytes, T cells, and neutrophils to the reperfused kidney. Compared with sham mouse kidneys, expression of RANTES, MIP-2, IP-10, and MCP-1 mRNA increased at 24 h after IRI (data not shown). As CCR2/MCP-1 is an important signaling pathway in mediating monocyte/macrophage migration, we performed kidney IRI in mice deficient in CCR2 (CCR2−/−) or its ligand, MCP-1 (MCP-1−/−). Following 24 h of reperfusion, plasma creatinine levels increased significantly in CCR2+/+but not in CCR2−/− mice (Figure 4a). To determine the contribution of CCR2 expressed on bone marrow (BM)-derived cells to tissue injury following reperfusion, we generated BM chimeras by transferring BM-derived cells from CCR2−/− mice into BM-depleted CCR2+/+ mice (CCR2−/− → CCR2+/+). IRI led to an increase in plasma creatinine in CCR2+/+ → CCR2+/+ but not in CCR2−/− → CCR2+/+ chimeras (Figure 4b). Tubule cell necrosis in the kidney outer medulla was lower in CCR2−/− mice following IRI compared to CCR2+/+ mice (acute tubular necrosis score: 0.5±0.1 vs 4.5±0.3, P<0.0001) (Figure 4c and d) and in CCR2−/− → CCR2+/+ compared with CCR2+/+ → CCR2+/+ chimeras (acute tubular necrosis score: 0.6±0.4 vs 4.4±0.3, P<0.0001) (Figure 4e and f). In contrast, the kidneys of MCP-1−/− mice were not protected from IRI; plasma creatinine levels were 2.20±0.13 (n=5) and 0.27±0.02 (n=4) mg/100 ml for IRI and sham, respectively (P<0.001) and more F4/80low macrophages infiltrated injured tissue in MCP-1−/− mice. Although F4/80lowLy6C+ cell counts were similar between sham-operated WT and MCP−/− mice, F4/80low Ly6C+ cell counts ( × 105 cells per g kidney) increased to a similar degree in WT (n=3) and KO (n=3) mice following IRI (2.42±0.31 and 2.57±0.12, respectively, P=NS). This is probably due to the contribution of other MCP family members to kidney IRI. We evaluated the function of CCR2 expression in mediating F4/80low macrophage recruitment to injured kidneys. Following 24 h of reperfusion, CD11b+F4/80low macrophages increased in kidneys of CCR2+/+ mice and CCR2+/+ → CCR2+/+ chimeras (Figure 4g). However, the absence of CCR2 attenuated the increase in F4/80low macrophages in CCR2−/− mice and CCR2−/− → CCR2+/+ chimeras (Figure 4g). Similar results were observed with other markers of F4/80lowLy6C+ macrophages and F4/80lowGr-1int macrophages (not shown). Interestingly, the number of F4/80high kidney resident DCs did not increase in CCR2+/+, CCR2−/−, CCR2+/+ → CCR2+/+, or CCR2−/− → CCR2+/+ mice following 24 h of reperfusion compared to sham (data not shown). Thus, CCR2 expressed on BM-derived cells mediates F4/80low monocyte/macrophage infiltration into reperfused kidneys. The infiltration of neutrophils in kidneys subjected to IRI in relation to macrophage recruitment was also examined. In CCR2−/− and CCR2−/− → CCR2+/+ mice, neutrophils comigrated with macrophages into injured kidneys. Kidney neutrophil cell number ( × 105 per g kidney) following IRI was 2.79±0.23 (n=4) and 10.87±1.78 (n=4) (P<0.005) for CCR2−/− and CCR2+/+, respectively, and 3.46±1.55 (n=8) and 13.28±2.31 (n=10) (P<0.005) for CCR2−/− → CCR2+/+ and CCR2+/+ → CCR2+/+, respectively. These results suggested that CCR2 also mediated neutrophil recruitment to inflamed kidneys. CX3CR1 is another chemokine receptor mediating monocyte migration. Following kidney IRI, an increase in plasma creatinine levels was observed in CX3CR1+/GFP mice, but levels were significantly lower in CX3CR1-deficient CX3CR1GFP/GFP mice (Figure 5a). Tubule necrosis produced by IRI was also attenuated in CX3CR1GFP/GFP compared with CX3CR1+/GFP mice (acute tubular necrosis score: 2.0±0.74 vs 4.1±0.22, P<0.05) (Figure 5b). To test our hypothesis that monocyte recruitment to the reperfused kidney is CX3CR1 dependent, we examined the kidney CX3CR1+GFPlowGr-1+ and CX3CR1+GFPlowLy6C+ macrophages in CX3CR1GFP/GFP (CX3CR1 KO) and CX3CR1+/GFP mice7.Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar following reperfusion (Figure 5c and d). We found increased CX3CR1+GFPlowGr-1+ macrophage infiltration (cell number, × 105 per g kidney) into reperfused kidneys of CX3CR1+/GFP mice (1.64±0.51 (n=7) and 6.86±1.35 (n=11) for sham and IRI, respectively (P 0.05)). Similar results were found for CX3CR1+GFPlowLy6C+ macrophages (Figure 5d). The increase from sham following IRI (%) was 316.9±117.5 (n=5) and 78.8±39.5 for CX3CR1+/GFP and CX3CR1GFP/GFP (n=9), respectively (P<0.05). These results indicate that CX3CR1 is important in monocyte/macrophage recruitment following IRI. Kidney-recruited F4/80low macrophages following IRI expressed Ly6Chigh, Gr-1int and CD62L, markers characteristic of blood-derived inflamed monocytes (Figure S2). The percentage of CD11b+Ly6Chigh monocytes in blood increased as early as 3 h after kidney IRI (4.45±0.85; n=12) compared with sham (1.72±0.28; n=12) (P<0.01). Download .jpg (.06 MB) Help with files Figure S2 Because CCR2 is necessary for monocyte trafficking into reperfused kidneys, we examined the function of CCR2 on the blood content of CD11b+Ly6Chigh monocytes (Figure 6a) after kidney IRI. We found increased blood CD11b+Ly6Chigh cells in 24 h IRI CCR2+/+ mice compared with sham but not in CCR2−/− mice. We also found that following IRI the percentage (and absolute cell counts; not shown) of blood CD11b+Ly6Chigh monocytes increased in the CCR2+/+ → CCR2+/+ but not in the CCR2−/− → CCR2+/+ chimeras (Figure 6a). These results suggest that CCR2 deficiency leads to reduced influx of Ly6Chigh inflamed monocytes into the reperfused kidney due to a reduced blood content of Ly6Chigh inflamed monocytes in CCR2−/− mice and CCR2−/− → CCR2+/+ chimeras. We next examined whether the reduction of blood CD11b+Ly6Chigh monocytes in CCR2−/− mice was due to the retention of CD11b+Ly6Chigh monocytes in BM. After 24 h of kidney reperfusion, CCR2+/+ and CCR2−/− mice had a similar percentage of BM Ly6Chigh monocytes, and the percentage change of BM Ly6Chigh cells in CCR2+/+ → CCR2+/+ chimeras was similar to CCR2−/− → CCR2+/+ chimeras following IRI (Figure 6b). These findings demonstrated that following kidney IRI, blood CD11b+Ly6Chigh monocytes are reduced in CCR2−/− mice and could contribute to a diminished blood to kidney tissue gradient. This could contribute to reduced monocyte/macrophage infiltration and tissue protection observed in CCR2−/− mice following IRI. The observed decrease in kidney infiltration of monocytes into reperfused kidneys may be due to: (1) the reduced blood content of monocytes in the CCR2−/− mice (hence reduced gradient from blood to kidney) or (2) the necessity of CCR2 expression on monocytes to facilitate migration from blood to the injured kidney. To distinguish between these two possibilities we transferred the same number (1 × 107) of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled BM monocytes either from CCR2+/+ or CCR2−/− mice to CCR2−/− mice at the onset of kidney IRI and tracked their infiltration into CCR2−/− kidneys subjected to IR (Figure 6c and d). CFSE-labeled CCR2+/+ monocytes were detected in reperfused kidneys and comprised primarily the F4/80low population. There was a threefold greater influx of CFSE-labeled CCR2+/+ monocytes in CCR2−/− IRI kidney compared to CFSE-labeled CCR2−/− monocytes. Similarly, we adoptively transferred CX3CR1GFP/GFP or CX3CR1+/GFP BM monocytes into WT mice (Figure 6e and f). CX3CR1+/GFP monocytes were detected in reperfused kidneys and comprised primarily the F4/80low population. There was a 3.2-fold greater influx of CX3CR1+/GFP monocytes into the reperfused kidneys of WT mice compared to CX3CR1GFP/GFP monocytes. The protected kidney function in CCR2−/− mice was reversed following adoptive transfer of CCR2+/+ (1 × 107) but not CCR2−/− monocytes. Creatinine level following IRI was 0.81±0.11 (n=4) and 0.51±0.03 (n=6) mg/100 ml for CCR2−/− mice that received CCR2+/+ and CCR2−/− monocytes, respectively (P<0.05). These data demonstrated that monocytes expressing CCR2 represent inflamed monocytes that migrate to the IRI kidney and contribute to tissue injury in a CCR2-dependent manner. The data from these two experiments provide strong evidence that the protection observed following reperfusion in CCR2- and CX3CR1-deficient mice is due to a reduced CCR2- and CX3CR1-dependant infiltration of F4/80low macrophages and that the reduced infiltration into reperfused kidneys is due to the combined effect of reduced emigration of monocytes from BM (CCR2−/− mice) and reduced migration of monocytes into injured kidneys. Early infiltration of macrophages and neutrophils contributes to the innate immune response of kidney IRI.4.Li L. Okusa M.D. Blocking the immune response in ischemic acute kidney injury: the role of adenosine 2A agonists.Nat Clin Pract Nephrol. 2006; 2: 432-444Crossref PubMed Scopus (50) Google Scholar,5.Jo S.K. Sung S.A. Cho W.Y. et al.Macrophages contribute to the initiation of ischaemic acute renal failure in rats.Nephrol Dial Transplant. 2006; 21: 1231-1239Crossref PubMed Scopus (236) Google Scholar,6.Day Y.J. Huang L. Ye H. et al.Renal ischemia–reperfusion injury and adenosine 2A receptor-mediated tissue protection: role of macrophages.Am J Physiol Renal Physiol. 2005; 288: F722-F731Crossref PubMed Scopus (210) Google Scholar Macrophages are a heterogeneous population, and two subsets of monocyte/macrophages have been identified: Ly6ChighCCR2+Gr-1+CX3CR1low and Ly6ClowCCR2−Gr-1−CX3CR1high.11.Tacke F. Randolph G.J. Migratory fate and differentiation of blood monocyte subsets.Immunobiology. 2006; 211: 609-618Crossref PubMed Scopus (386) Google Scholar,12.Tacke F. Alvarez D. Kaplan T.J. et al.Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques.J Clin Invest. 2007; 117: 185-194Crossref PubMed Scopus (942) Google Scholar In this study, we made two seminal observations. First,
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