Volume 74, Issue 12, Pages 1526-1537 (December 2008) The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia–reperfusion injury  Li Li, Liping Huang, Sun-Sang J. Sung, Amy L. Vergis, Diane L. Rosin, C. Edward Rose, Peter I. Lobo, Mark D. Okusa  Kidney International  Volume 74, Issue 12, Pages 1526-1537 (December 2008) DOI: 10.1038/ki.2008.500 Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 1 There are two F4/80+ macrophage subtypes in the kidney with distinct phenotypic characteristics (see also Table 1). (a) Four-color FACS analysis identified two F4/80+ populations in the normal C57BL/6 mouse kidney: CD11bhighF4/80low and CD11blowF4/80high (oval circled). Further phenotyping of these two F4/80+ populations showed that the F4/80low cells expressed (c) CX3CR1low and only this population, but not the F4/80high population, expressed (b) CD62L, Ly6Chigh, and Gr-1int (oval circled). (c) There is an F4/80high subset (a, c) that expressed CX3CR1high, CD11c+, CD86, and IAhigh and is referred to as a dendritic cell (DC) population. (d) Both macrophages (F4/80low) and DCs (F4/80high) expressed LFA-1, VLA-4, TLR2, and MD-1. (e, f) In kidney sections from C57BL/6 background CX3CR1+/GFP mice, GFP is expressed mainly on monocyte/macrophages and DCs, and many CX3CR1+GFP+ green fluorescing cells were seen in the cortex (e) and medulla (f). (g, h) Higher magnification z-stack projection images of the kidney medulla viewed under a Zeiss LSM-510 confocal microscope showed CX3CR1-GFP-positive cells in green, PE-tagged IA-positive cells in red (g), Alexa 647-tagged F4/80 positive cells in red (h), and colocalization of the two fluorophores in CX3CR1+GFP+IA+ or CX3CR1+GFP+F4/80+ DCs in yellow, respectively; (g) z-stack projection image of 14 optical slices at 0.35μm intervals and (h) z-stack projection image of 5 optical slices at 0.69μm intervals. IA and F4/80 expression appears on the DC cell surface (arrowheads in g and h, respectively). Tubule lumen is represented by *. (i) Depiction of GFPhigh cells that expressed CD11b+, CD11c+, and IA+, which are phenotypically DCs as identified in (g, h). (j) Both CX3CR1+ GFPhigh and GFPlow cells expressed F4/80. However, compared with GFPhigh cells, GFPlow cells also expressed Gr-1 and Ly6C, which represent monocytes/macrophages. Cell populations of interest are indicated within ovals or in boxed regions. Kidney International 2008 74, 1526-1537DOI: (10.1038/ki.2008.500) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 2 Recruitment of CD11bhighF4/80low inflamed monocyte subset following kidney IRI. Mouse kidneys were subjected to 32min ischemia followed by (a, c, e) 3h or (b, d, f) 24h 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) 3h and (b, right) 24h 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) 3h and (h) 24h of reperfusion. Values are means±s.e.; N=7–10; **P<0.01, ***P<0.001; NS, not significant. Kidney International 2008 74, 1526-1537DOI: (10.1038/ki.2008.500) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 3 Heterogeneity of proinflammatory cytokine production by CD11bhighF4/80low and CD11bhighF4/80high macrophages 24h following kidney IRI. Representative tracings from FACS analysis of expression of intracellular cytokines, including IL-1α, IL-6, IL-12p40/70, and TNF-α, in the (a) F4/80low and (b) F4/80high populations of macrophages in mouse kidney after 24h of reperfusion (defined by gating on CD45+CD11bhighF4/80low and CD11blowF4/80high macrophages, respectively, as shown in Figure 2b) for sham operation (blue line), IRI (green line), or isotype control (red line). Max (%), percentage of F4/80low or F4/80high macrophages that are cytokine positive. Summary of proinflammatory cytokine production by (c) F4/80low macrophages and (d) F4/80high macrophages. Gating on the F4/80low population (c), the percentage of cells that produced IL-1α, IL-6, IL-12p40/70, and TNF-α increased significantly following IRI compared to sham (P<0.05 for all IRI compared to sham groups). Values are mean±s.e.; N=3–6. Similarly, gating on the (d) F4/80high population, the percentage of cells that produced IL-1α, IL-6, IL-12p40/70 was unchanged following IRI compared to sham, but TNF-α increased significantly following IRI compared to sham (P<0.05). Values are mean±s.e.; N=3–6. Kidney International 2008 74, 1526-1537DOI: (10.1038/ki.2008.500) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 4 CCR2-deficient signaling protects kidneys from IRI. (a, c, d) WT mice (CCR2+/+) and age-, weight-, and gender-matched CCR2−/− mice and (b, e, f) CCR2+/+ → CCR2+/+, CCR2−/− → CCR2+/+ BM chimera mice were subjected to 32min of ischemia followed by 24h of reperfusion. Plasma creatinine is shown in (a, b). Values are mean±s.e.; N=4–11; **P<0.001. (c–f) H&E staining of kidney outer medulla is shown. There was more tubule cell injury and necrosis after IRI in WT (CCR2+/+) and CCR2+/+ → CCR2+/+ BM chimera mice (c, e, right). However, IRI kidneys from CCR2−/− mice and CCR2−/− → CCR2+/+ BM chimera mice were protected from IRI (d, f, right). The arrows are pointing to the necrotic tubules in (c, e, right). Magnification, × 200. (g) Recruitment of F4/80low macrophages in kidneys of CCR2−/−, CCR2+/+, CCR2+/+ → CCR2+/+, and CCR2−/− → CCR2+/+ chimera mice following 24h reperfusion compared with controls. Values are mean±s.e.; N=4–10; *P<0.05; **P<0.001; NS, not significant. Kidney International 2008 74, 1526-1537DOI: (10.1038/ki.2008.500) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 5 Kidneys of CX3CR1-deficient mice are protected from IRI with fewer infiltrated macrophages. Mouse kidneys were subjected to 32min ischemia followed by 24h of reperfusion. In C57BL/6 background CX3CR1+/GFP mice, one CX3CR1 allele was replaced by GFP, which has no effect on CX3CR1 gene function, whereas two CX3CR1 alleles were replaced with GFP in CX3CR1GFP/GFP mice, resulting in a CX3CR1 knockout. (a) Plasma creatinine for CX3CR1+/GFP mice and CX3CR1GFP/GFP mice. Values are mean±s.e.; N=6–11; **P<0.001. (b) H&E staining of kidney sections in sham and IRI for CX3CR1+/GFP and CX3CR1GFP/GFP mice. Black arrows indicate necrotic tubule cells in the outer medulla of CX3CR1+/GFP mice after IRI. FACS analysis of kidney (c) GFPlowGr-1+ and (d) GFPlowLy6C+. Kidney International 2008 74, 1526-1537DOI: (10.1038/ki.2008.500) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 6 CCR2 and CX3CR1 are necessary for monocyte egress from BM (to blood) and from blood (to tissue) following kidney IRI. Mouse kidneys were subjected to 32min ischemia followed by 24h reperfusion and leukocytes from (a) blood and (b) BM were quantiated by FACS. We gated on blood and BM SSClow population and quantitated CD11bhighLy6Chigh ‘inflamed monocytes. Values show CD11bhighLy6Chigh inflamed monocytes as a percentage of total leukocytes isolated after 24h IRI or sham operation from blood of CCR2+/+ (n=9 and 6), CCR2−/− (n=4 and 4), CCR2−/− → CCR2+/+ (n=4 and 4) and CCR2+/+ → CCR2+/+ (n=4 and 3) mice and from BM of CCR2+/+ (n=9 and 7), CCR2−/− (n=8 and 4), CCR2−/− → CCR2+/+ (n=7 and 4) and CCR2+/+ → CCR2+/+ (n=8 and 6) mice. *P<0.05; **P<0.001. (c, d) CCR2−/− monocytes (Mo) and CCR2+/+ Mo were isolated from BM and labeled with CFSE. 1 × 107 CFSE-labeled BM monocytes were adoptively transferred separately to the CCR2−/− mice at the onset of the surgery. Infiltrated labeled cells are denoted by oval. (e, f) GFP-positive monocytes from CX3CR1+/GFP and CX3CR1 KO (CX3CR1GFP/GFP) (1 × 107) mice were adoptively transferred separately into C57BL/6 WT mice and mice were subjected to 24h IRI. Kidney International 2008 74, 1526-1537DOI: (10.1038/ki.2008.500) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 7 Trafficking of monocytes in ischemia/reperfusion injury (IRI) mice. BM CD11b+Ly6Chigh monocyte/monocyte precursor egress to the blood circulation is CCR2-dependent. (a) Some of the Ly6Chigh monocytes lose their CCR2 and Ly6C expression and are further characterized with CD62L−, Gr-1− and CX3CR1high. (b) These cells migrate to normal noninflamed tissue rapidly after they are released in the blood and differentiate into tissue dendritic cells (DCs), which express CD11b+, CD11c+, IAhigh, CD86+ and F4/80high. (c) On the other hand, some of the monocytes continue to express CCR2+ and Ly6Chigh on the cell surface with the additional expression of CX3CR1low, Gr-1int, and CD62L+. (d) These inflamed monocytes respond to the gradient of chemokines (e) released from IRI kidneys. In the injured tissue, these macrophages derived from inflamed monocytes are characterized by CD62L+, Gr-1int, Ly6C+, and F4/80low expression. Infiltrated macrophages produce large amounts of proinflammatory cytokines, which are involved in tissue injury. Kidney International 2008 74, 1526-1537DOI: (10.1038/ki.2008.500) Copyright © 2008 International Society of Nephrology Terms and Conditions