The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia–reperfusion injury

Abstract

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 (F4/80^low and F4/80^high) 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 F4/80^low inflamed monocytes, but not dendritic cells, into the kidney. These monocytes produced TNF-α, IL-6, IL-1 α 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 F4/80^low macrophages compared to control mice. CX3CR1 expression contributed to monocyte recruitment into inflamed kidneys, as ischemic injury in CX3CR1^−/− mice was reduced, with fewer F4/80^low 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⁺Ly6C^high 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.^1,2 Rapid accumulation of neutrophils and monocyte/macrophages in the injured kidney is an essential feature of the innate immune response in IRI.^3–6 Monocyte/macrophages are heterogeneous.^7,8 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 having a CD11b⁺ CCR2^lowGr-1⁻ Ly6C⁻CX3CR1^high phenotype, migrate to uninjured tissues rapidly after emigration from bone marrow (BM)⁷ and differentiate into resident macrophages and dendritic cells (DCs). In contrast, a distinct inflamed monocyte subset with a CD11b⁺ CCR2^highLy6C^high Gr-1^intCX3CR1^low phenotype infiltrates infected tissue^7–11 and contributes to the development of atherosclerosis and inflammation;¹² 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.^8,13 Monocyte chemoattractant protein-1 (MCP-1/CCL2) is the main ligand for CCR2,¹⁴ and the MCP-1/CCR2 signal pathway is important for monocyte recruitment to the site of inflammation.^8,13 C-X3-C motif chemokine receptor 1 (CX3CR1) and its ligand, fractalkine, are important in macrophage accumulation and inflammation¹⁵ and contribute to atherosclerosis.^12,16–18

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 F4/80^low macrophage population (derived from the blood inflamed monocytes) contributes to the innate immune response to mediate kidney IRI.

RESULTS

F4/80⁺ leukocytes are abundantly expressed in normal mouse kidneys and their numbers increase rapidly following kidney IR

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.

There are two macrophage subtypes in the kidney with distinct phenotypes

Fluorescence-activated cell sorter (FACS) analysis identified two subsets of F4/80⁺ cells in normal mouse kidneys, CD11b^lowF4/80^high (referred to as F4/80^high) and CD11b^highF4/80^low (F4/80^low) cells, based on the F4/80 and CD11b fluorescence intensity (Figure 1a). The F4/80^high 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 F4/80^low cells expressed CD62L⁺, Ly6C^high, Gr-1^int (Figure 2b), and CX3CR1^low (Figure 2c), a phenotype characteristic of the blood inflamed monocyte.^7,8 All F4/80^high cells expressed CD11c⁺, MHC class II^high (IA^high), CD86⁺, and CX3CR1^high (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, F4/80^high cells and F4/80^low cells were considered to represent kidney resident DCs and macrophages derived from the inflamed monocyte pool, respectively.

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: CD11b^highF4/80^low and CD11b^lowF4/80^high (oval circled). Further phenotyping of these two F4/80⁺ populations showed that the F4/80^low cells expressed (c) CX3CR1^low and only this population, but not the F4/80^high population, expressed (b) CD62L, Ly6C^high, and Gr-1^int (oval circled). (c) There is an F4/80^high subset (a, c) that expressed CX3CR1,^high CD11c⁺, CD86, and IA^high and is referred to as a dendritic cell (DC) population. (d) Both macrophages (F4/80^low) and DCs (F4/80^high) 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 0.35 mm intervals and (h) z-stack projection image of 5 optical slices at 0.69 µm 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 GFP^high cells that expressed CD11b⁺, CD11c⁺, and IA⁺, which are phenotypically DCs as identified in (g, h). (j) Both CX3CR1⁺ GFP^high and GFP^low cells expressed F4/80. However, compared with GFP^high cells, GFP^low cells also expressed Gr-1 and Ly6C, which represent monocytes/macrophages. Cell populations of interest are indicated within ovals or in boxed regions.

Table 1.

Phenotype of kidney F4/80^low and F4/80^high macrophages

	Antibody clone	F4/80low	F4/80high
Monocyte markers
CD11b	M1/70	High +	Low +
F4/80	BM8	Low +	High +
Ly6C	AL-21	High +	−
GR-1(Ly6G)	RB6-8C5	Intermediate +	−
CD204	2F8	−	+
CD206	MR5D3	−	−
M-CSFR	4H1	+/−	−
CX3CR1	Polyclonal	Low +	High +
DC marker
CD205	MG38	−	−
33D1	33D1	−	−
CD11c	N418	20–30% +	+
IA	M5/114.15.2	−	High +
CD80	16-10A1	−	−
CD86	GL1	−	+
CD40	HM40-3	−	−
B7-DC	TY25	−	−
B7-H1	1–111A	−	+
Adhesion marker
CD62L	MEL-14	+	−
VLA-4	HMb1-1	High +	Low +
LFA-1	M17/4	High +	Low +
TLR marker
TLR2	6C2	+	+

DC, dendritic cell; TLR, toll-like receptor.

Figure 2. Recruitment of CD11b^highF4/80^low inflamed monocyte subset following kidney IRI.

Mouse kidneys were subjected to 32 min ischemia followed by (a, c, e) 3h 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 F4/80^low, (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 (F4/80^low) and DCs (F4/80^high) 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.

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 CX3CR1^GFP/GFP circulating CD11b⁺F4/80⁺ cells.^7,19,20 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 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⁺GFP^high cells expressed CD11b, and most expressed CD11c and MHC II, markers for DCs (Figure 1i). Ly6C and Gr-1 were expressed primarily on the GFP^low population (Figure 1j), which has the same phenotype as the blood inflamed monocytes.⁷ Both GFP⁺ cell populations expressed F4/80 (Figure 1h and j). Kidney F4/80^low leukocytes had a similar phenotype to GFP^low cells in CX3CR1^+/GFP mice and represented kidney monocyte/macrophages, whereas the F4/80^high subset resemble CX3CR1⁺ GFP^high cells and represented resident kidney DCs (Figure 1b and c).

Recruitment of F4/80^low macrophage subset following kidney IRI

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 F4/80^low macrophages. However, 3 (Figure 2a and g) and 24 h (Figure 2b and h) after reperfusion there was no change in the number of F4/80^high kidney resident DCs.

Similar results were observed with Ly6C^high and Gr-1^int 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 F4/80^low macrophages were Ly6C^high (Figure 2c), which represents immature, newly arrived monocytes from BM.⁸ By 24 h, Ly6C expression was markedly downregulated in the F4/80^low 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 F4/80^lowGr-1^int 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 F4/80^lowGr-1^int decreased at 24 h compared to 3 h following kidney IRI, paralleling the findings with Ly6C. Therefore, an abundance of infiltrating macro-phages, phenotypically similar to circulating inflamed monocytes, was identified in reperfused kidneys.

Heterogeneity of proinflammatory cytokine production by CD11b^highF4/80^low and CD11b^highF4/80^high 24 h following kidney IRI

We used FACS analysis of intracellular cytokine production to determine functional differences between F4/80^low and F4/80^high populations following kidney IRI. Gating on F4/80^low and F4/80^high (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 F4/80^low macrophages producing cytokines (% of total F4/80^low population) following IRI was 4- to 8-fold greater for IRI relative to sham (Figure 3a and c). In contrast, the percentage of F4/80^high 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 F4/80^low and F4/80^high populations.

Figure 3. Heterogeneity of proinflammatory cytokine production by CD11b^high F4/80^low and CD11b^high F4/80^high macrophages 24 h 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) F4/80^low and (b) F4/80^high populations of macrophages in mouse kidney after 24 h of reperfusion (defined by gating on CD45⁺ CD11b^high F4/80^low and CD11b^lowF4/80^high macrophages, respectively, as shown in Figure 2b) for sham operation (blue line), IRI (green line), or isotype control (red line). Max (%), percentage of F4/80^low or F4/80^high macrophages that are cytokine positive. Summary of proinflammatory cytokine production by (c) F4/80^low macrophages and (d) F4/80^high macrophages. Gating on the F4/80^low 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) F4/80^high 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.

CCR2-deficient, but not MCP-1-deficient, signaling protects kidneys from IRI

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 F4/80^low macrophages infiltrated injured tissue in MCP-1^−/− mice. Although F4/80^lowLy6C⁺ cell counts were similar between sham-operated WT and MCP^−/− mice, F4/80^low 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.

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 32 min of ischemia followed by 24 h 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 F4/80^low macrophages in kidneys of CCR2^−/−, CCR2^+/+, CCR2^+/+ → CCR2^+/+, and CCR2^−/− → CCR2^+/+ chimera mice following 24 h reperfusion compared with controls. Values are mean ± s.e.; N = 4–10; *P < 0.05; **P < 0.001; NS, not significant.

We evaluated the function of CCR2 expression in mediating F4/80^low macrophage recruitment to injured kidneys. Following 24 h of reperfusion, CD11b⁺F4/80^low macrophages increased in kidneys of CCR2^+/+ mice and CCR2^+/+ → CCR2^+/+ chimeras (Figure 4g). However, the absence of CCR2 attenuated the increase in F4/80^low macrophages in CCR2^−/− mice and CCR2^−/− → CCR2^+/+ chimeras (Figure 4g). Similar results were observed with other markers of F4/80^lowLy6C⁺ macrophages and F4/80^lowGr-1^int macrophages (not shown). Interestingly, the number of F4/80^high 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 F4/80^low 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.

Deficiency of CX3CR1 reduces kidney IRI

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 CX3CR1^GFP/GFP mice (Figure 5a). Tubule necrosis produced by IRI was also attenuated in CX3CR1^GFP/GFP compared with CX3CR1^+/GFP mice (acute tubular necrosis score: 2.0 ± 0.74 vs 4.1 ± 0.22, P < 0.05) (Figure 5b).

Figure 5. Kidneys of CX3CR1-deficient mice are protected from IRI with fewer infiltrated macrophages.

Mouse kidneys were subjected to 32 min ischemia followed by 24 h 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 CX3CR1^GFP/GFP mice, resulting in a CX3CR1 knockout. (a) Plasma creatinine for CX3CR1^+/GFP mice and CX3CR1^GFP/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 CX3CR1^GFP/GFP mice. Black arrows indicate necrotic tubule cells in the outer medulla of CX3CR1^+/GFP mice after IRI. FACS analysis of kidney (c) GFP^low Gr-1⁺ and (d) GFP^lowLy6C⁺.

Monocyte infiltration of the reperfused kidney is CX3CR1 dependent

To test our hypothesis that monocyte recruitment to the reperfused kidney is CX3CR1 dependent, we examined the kidney CX3CR1⁺ GFP^lowGr-1⁺ and CX3CR1⁺ GFP^lowLy6C⁺ macrophages in CX3CR1^GFP/GFP (CX3CR1 KO) and CX3CR1^+/GFP mice⁷ following reperfusion (Figure 5c and d). We found increased CX3CR1⁺ GFP^low Gr-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 < 0.01)) but not in CX3CR1^GFP/GFP mice (1.20 ± 0.28 (n = 6) and 2.21 ± 0.29 (n = 10) for sham and IRI, respectively (P > 0.05)). Similar results were found for CX3CR1⁺ GFP^low Ly6C⁺ 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 CX3CR1^GFP/GFP (n = 9), respectively (P < 0.05). These results indicate that CX3CR1 is important in monocyte/macrophage recruitment following IRI.

Infiltrating F4/80^low macrophages in reperfused kidneys were derived from a blood CD11b^high Ly6C^high inflamed monocyte pool

Kidney-recruited F4/80^low macrophages following IRI ex-pressed Ly6C^high, Gr-1^int and CD62L, markers characteristic of blood-derived inflamed monocytes (Figure S2). The percentage of CD11b⁺Ly6C^high 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).

Because CCR2 is necessary for monocyte trafficking into reperfused kidneys, we examined the function of CCR2 on the blood content of CD11b⁺ Ly6C^high monocytes (Figure 6a) after kidney IRI. We found increased blood CD11b⁺ Ly6-C^high 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⁺ Ly6C^high 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 Ly6C^high inflamed monocytes into the reperfused kidney due to a reduced blood content of Ly6C^high inflamed monocytes in CCR2^−/− mice and CCR2^−/− → CCR2^+/+ chimeras.

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 32 min ischemia followed by 24 h reperfusion and leukocytes from (a) blood and (b) BM were quantiated by FACS. We gated on blood and BM SSC^low population and quantitated CD11b^highLy6C^high inflamed monocytes. Values show CD11b^highLy6C^high inflamed monocytes as a percentage of total leukocytes isolated after 24 h 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 (CX3CR1^GFP/GFP) (1 × 10⁷) mice were adoptively transferred separately into C57BL/6 WT mice and mice were subjected to 24 h IRI.

We next examined whether the reduction of blood CD11b⁺ Ly6C^high monocytes in CCR2^−/− mice was due to the retention of CD11b⁺ Ly6C^high monocytes in BM. After 24 h of kidney reperfusion, CCR2^+/+ and CCR2^−/− mice had a similar percentage of BM Ly6C^high monocytes, and the percentage change of BM Ly6C^high cells in CCR2^+/+ → CCR2^+/+ chimeras was similar to CCR2^−/− → CCR2^+/+ chimeras following IRI (Figure 6b). These findings demonstrated that following kidney IRI, blood CD11b⁺ Ly6C^high 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.

CCR2 and CX3CR1 are necessary for inflamed monocyte migration to the IRI kidney

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 F4/80^low 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 CX3CR1^GFP/GFP or CX3CR1^+/GFP BM monocytes into WT mice (Figure 6e and f). CX3CR1^+/GFP monocytes were detected in reperfused kidneys and com-prised primarily the F4/80^low population. There was a 3.2-fold greater influx of CX3CR1^+/GFP monocytes into the reperfused kidneys of WTmice compared to CX3CR1^GFP/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 F4/80^low 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.

DISCUSSION

Early infiltration of macrophages and neutrophils contributes to the innate immune response of kidney IRI.^4–6 Macrophages are a heterogeneous population, and two subsets of monocyte/macrophages have been identified: Ly6-C^high CCR2⁺ Gr-1⁺ CX3CR1^low and Ly6C^lowCCR2⁻Gr-1⁻ CX3CR1^high.^11,12 In this study, we made two seminal observations. First, we showed that F4/80⁺ cells in normal kidneys consist of two subsets: (1) F4/80^low macrophages derived from the blood inflamed monocyte pool and (2) F4/80^high population phenotypically representing DCs. This conclusion is supported by the following: (1) the use of multiple cell-surface markers (Table 1; Figure 1) that can distinguish between two different types of macrophages;⁷ (2) the two macrophage subsets exhibit functional differences; F4/80^high cells do not produce cytokines to a large degree with the exception of TNF-α, whereas F4/80^low cells produce an abundance of proinflammatory cytokines after IRI; and (3) the F4/80^high population possesses characteristics of a professional antigen presenting cell (L. Li and M.D. Okusa, unpublished observation, 2008). Second, increased CD11b⁺ F4/80^lowLy6C^highCCR2⁺ Gr-1⁺ CX3CR1^low cells infiltrate IRI kidneys, and CCR2 and CX3CR1 mediate trafficking of inflamed monocytes from blood to kidneys. The number of blood Ly6C^high monocytes increased dramatically after IRI suggesting that this population of circulating inflamed monocytes may be the source of macrophages infiltrating injured kidneys. We provide a comprehensive analysis of trafficking of the inflamed monocyte subset from blood to kidney, which leads to early initiation of the innate response following kidney IRI.

CCR2-dependent monocyte/macrophage trafficking following kidney IRI

In atherosclerosis, CCR2, CCR5, and CX3CR1 mediate monocyte/macrophage migration into inflamed tissue¹² and CCR2^−/− mice were protected from Listeria monocytogenes infection due to an attenuation of monocyte egress from BM.²² Further, CCR2^−/− mice were protected from kidney IRI, and this protection was associated with reduced macrophage infiltration.²³ Our studies compliment these results and demonstrate a causal relation between CCR2-expressing monocyte/macrophages and tissue injury following kidney IRI. In the current studies, CCR2^−/− mouse kidney protection was due to a reduction of blood monocytes. Although reduced infiltration of monocytes into the reperfused kidney may be due secondarily to reduction in blood levels of monocytes and not to a primary defect in CCR2^−/− monocyte response to chemokines, we do not believe that this is the case. Preventing the decrease in blood monocytes in CCR2^−/− mice by transferring CCR2^−/− monocytes did not facilitate their entry in injured tissues. However, transfer of CCR2^+/+ monocytes did facilitate macrophage infiltration and induce injury. In contrast in L. monocytogenes-infected mice, TNF-α-producing CCR2^−/− monocytes infiltrated inflamed spleen,²² a finding that differs from our study but that could be explained by differences in the model (septic vs aseptic IRI) and tissue (spleen vs kidney) examined in the two studies. Therefore, inflamed monocyte migration into the reperfused kidney is in part CCR2 dependent and contributes to early inflammation in kidney IRI.

Previous studies examining the proinflammatory cytokines in IRI kidney have used PCR or enzyme-linked immunosorbent assay of whole kidney homogenate and suggested infiltrated macrophages identified by immunohistochemistry as a likely source of cytokine production. Indeed, an increase in cytokine level in whole kidney may represent production by immune, epithelial, interstitial, or endothelial cells, and as such the cellular source of cytokines has remained unknown. Our study is the first one to determine at a single immune cell (macrophage) proinflammatory cytokine production by using FACS intracellular staining.

CX3CL1/CX3CR1-mediated monocyte/macrophage trafficking in kidney IRI

CX3CL1/CX3CR1 is another pathway mediating monocyte migration. CX3CR1^low Gr-1⁺ CCR2⁺ subsets are actively recruited to inflamed tissue and CX3CR1^highGr-1⁻ CCR2⁻ subsets migrate to uninjured tissue and differentiate into resident macrophages and DCs.⁹ CX3CR1-deficient (CX3CR1^GFP/GFP) mice are significantly protected from rejection of transplanted hearts.²⁴ We found that CX3CR1^−/− mouse kidneys were protected from IRI, confirming studies that employed a CX3CR1 blocking antibody,²⁵ and this protection was associated with less GFP^lowGr-1⁺ or GFP^lowLy6C⁺ macrophage infiltration compared to the heterogeneous CX3CR1^+/GFP mice. Thus CX3CR1 signaling mediates infiltration of inflamed monocytes into injured kidneys.

CCR2 and CX3CR1 may act cooperatively and/or independently as each may be responsible for specific mechanisms of monocyte trafficking in inflamed tissue. Tacke et al.¹² found that CCR2⁻Ly6C^low monocytes infiltrated atherosclerosis plaques less frequently compared with CCR2⁺ Ly6C^high monocytes, and CCR2⁻ Ly6C^low monocyte recruitment is CCR5 dependent and CCR2 independent, but CCR2⁺ Ly6C^high monocyte migration is CX3CR1 dependent.

DCs participate in innate and adaptive immunity, autoimmunity, allograft, and host defense.²⁶ F4/80^high resident DCs are the predominant leukocyte subset, but the function of kidney DCs in IRI is unknown completely. TNF-α production from kidney DCs at early time points following IR was involved in kidney injury.²⁷ We showed recently that kidney F4/80^high DCs mediate OT-II T-cell proliferation by presenting OVA323-339 efficiently (L. Li and M.D. Okusa, unpublished observation, 2008) and present self-glycolipid to activate natural killer T cells (NKT) by producing interferon-γ following reperfusion.²⁸ IL-12 and IL-23 are important cytokines secreted by activated DCs. IL-23 mediates neu-trophil recruitment and production of related chemokines CXCL1, 2, and 5²⁹ and may also be involved in kidney IRI. Lastly, it should be recognized that other macrophage subsets likely participate in repair of kidney following IRI.⁴ The alternative macrophage expressing the mannose receptor and activated by IL-4 and IL-13 likely participates in tissue repair.³⁰

In conclusion, this study identified the profile of leukocyte subset influx in the early and late phases following reperfusion and defined two F4/80⁺ populations in the kidney: resident DCs and macrophages. CCR2 and CX3CR1 signaling pathways play critical roles in inflamed monocyte recruitment in kidney IRI (Figure 7). Resident DCs participate in innate immunity through natural killer T-cell activation²⁸ and modulate adaptive immunity.³¹ These studies provide new insight into mechanisms of early innate immune response to acute kidney IRI, concepts that may generalize to other organs and provide the foundation for therapeutic strategies.

Figure 7. Trafficking of monocytes in ischemia/reperfusion injury (IRI) mice.

BM CD11b⁺ Ly6C^high monocyte/monocyte precursor egress to the blood circulation is CCR2-dependent. (a) Some of the Ly6C^high monocytes lose their CCR2 and Ly6C expression and are further characterized with CD62L⁻, Gr-1⁻ and CX3CR1^high. (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⁺, IA^high, CD86⁺ and F4/80^high. (c) On the other hand, some of the monocytes continue to express CCR2⁺ and Ly6C^high on the cell surface with the additional expression of CX3CR1^low, Gr-1^int, 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-1^int, Ly6C⁺, and F4/80^low expression. Infiltrated macrophages produce large amounts of proinflammatory cytokines, which are involved in tissue injury.

MATERIALS AND METHODS

Mice and surgical protocol

We adhered to the NIH Guide for the Care and Use of Laboratory Animals. The Animal Research Committee of the University of Virginia approved all procedures and protocols. We used C57BL/6 and Balb/c male background mice (~20 g, 6–8 weeks of age; Charles River Laboratories, Wilmington, MA, USA). Age-matched CCR2^−/− mice (Balb/c background), CX3CR1^+/GFP and CX3CR1^GFP/GFP mice (C57BL/6 background) were bred in our animal facility. Following anesthesia, bilateral flank incisions were performed as previously described,³² both kidney pedicles were exposed and cross-clamped for 32 min, then clamps were released (reperfusion) for different times. In sham-operated mice, kidney pedicles were exposed but not clamped.

Tissue processing and cell counting by FACS

Kidney cell pellets were prepared from sham and IRI mice and kidney leukocyte subset cell number was calculated as described.²⁸ Kidney F4/80⁺ cell subsets were identified using allophycocyanin (APC)-labeled rat anti-mouse F4/80 (BM8) and phycoerythrin (PE)-labeled rat anti-mouse CD11b (M1/70), I-A (M5/114.15.2), CD11c (N418), CD86 (GL1), TLR2 (6C2), Ly6C (AL-21; BD Bioscience, San Jose, CA, USA), Gr-1 (Ly6G) (RB6-8C5), CD62L (MEL-14), very late antigen-4 (HMb1-1), or lymphocyte function-associated antigen 1 (M17/4) antibodies, or primary rabbit anti-mouse CX3CR1 antibody followed by PE-labeled goat anti-rabbit IgG (Southern Biotech, Birmingham, AL, USA).

Fresh mouse blood and BM cells were obtained.¹⁴ Cell pellets were incubated with anti-CD11b-PE (M1/70), F4/80-APC (BM8) and Ly6C-FITC (AL-21). 7-AAD was used to exclude dead cells. Flow cytometry data acquisition was performed on BD FACSCalibur and analyzed by Flowjo software 6.4 (Tree Star Inc., Ashland, OR, USA).

Intracellular staining of proinflammatory cytokines

FACS intracellular staining of proinflammatory cytokines was performed as described.²⁸ PE-labeled anti-mouse IL-1α (ALF-161), IL-6 (MP5-20F3), IL-12p_40/70 (C17.8), TNF-α (MP6-XT22), or isotype control antibody (5 µg/ml) was added to each sample after blocking with anti-mouse CD16/32 (10 mg/ml) for 20 min on ice. All of above mAbs, unless otherwise stated, were from eBioscience (San Diego, CA, USA).

Generation of chimeric mice

Two groups of chimeric mice were generated by transferring BM cells as described.³³ (1) BM from CCR2^−/− to CCR2^+/+ mice (CCR2^−/− → CCR2^+/+) and (2) BM from CCR2^+/+ to CCR2^+/+ mice (CCR2^+/+ → CCR2^+/+). Chimeric mice were used 8 weeks after BM transfer. Reconstitution efficiency is nearly 90%.^32,33

BM monocyte isolation and adoptive transfer

BM CFSE labeling and monocyte isolation was as described before.²² The enriched population comprised 90% CD11b⁺ cells. CFSE-labeled enriched monocytes (1 × 10⁷) were injected i.v. into CCR2^−/− mice at the onset of kidney IRI.

Histochemistry

Kidneys were fixed for hematoxylin and eosin staining, and acute tubular necrosis was scored in the outer stripe of the outer medulla as described previously.³⁴ Sections were viewed by light microscopy (Zeiss AxioSkop), and photographs were taken with a SPOT-RT camera (software version 3.3; Diagnostic Instruments, Sterling Heights, MI, USA).

Immunofluorescence and confocal microscopy

Mouse kidney sections were fixed in 0.7% PLP (0.7% paraformaldehyde), 1.4% lysine, 0.2% sodium periodate in 0.1 m sodium phosphate buffer, pH7.4,³² incubated in 30% sucrose for 48 h at 4 °C, embedded and frozen in Optimal Cutting Temperature compound (Ted Pella Inc., Redding, CA, USA). Frozen sections (25 µm) were permeabilized with 0.3% Triton X-100, blocked with 10% goat serum and anti-CD16/32 antibody, and incubated for 1 h with Alexa 647-labeled F4/80 (Invitrogen, Carlsbad, CA, USA) or IA (BioLegend, San Diego, CA, USA) or PE-labeled I-A (eBioscience, Carlsbad, CA, USA) antibodies (5 µg/ml). Images were acquired using Zeiss LSM 510-UV confocal microscopy and processed using Adobe Photoshop (Adobe, San Jose, CA, USA).

Statistics

GraphPad Instat 3 (GraphPad Inc., San Diego, CA, USA) was used to analyze the data. Unpaired t-test, analysis of variance, and Tukey’s post hoc analysis were performed with P < 0.05 indicating significance.

Supplementary Material

supplement. SUPPLEMENTARY MATERIAL.

Figure S1. Normal mouse kidneys contain a high content of F4/80⁺ cells.

Figure S2. Trafficking of monocytes in ischemia/reperfusion injury (IRI) mice.

Supplementary material is linked to the online version of the paper at http://www.nature.com/ki