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-alpha, IL-6, IL-1alpha 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.

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.

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.

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.

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.

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⁺.

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.

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.