Immune Monitoring of Trans-endothelial Transport by Kidney-Resident Macrophages

Abstract

Small immune complexes cause type III hypersensitivity reactions that frequently result in tissue injury. The responsible mechanisms, however, remain unclear and differ depending on target organs. Here, we identify a kidney-specific anatomical and functional unit, formed by resident macrophages and peritubular capillary endothelial cells, which monitors the transport of proteins and particles ranging from 20 to 700 kDa or 10 to 200 nm into the kidney interstitium. Kidney-resident macrophages detect and scavenge circulating immune complexes "pumped" into the interstitium via trans-endothelial transport and trigger a FcγRIV-dependent inflammatory response and the recruitment of monocytes and neutrophils. In addition, FcγRIV and TLR pathways synergistically "super-activate" kidney macrophages when immune complexes contain a nucleic acid. These data identify a physiological function of tissue-resident kidney macrophages and a basic mechanism by which they initiate the inflammatory response to small immune complexes in the kidney.

Figure 1. Uptake of immune complexes by kidney macrophages

(A) Flow cytometric analysis of BSA-alexa488 uptake by macrophages from C57BL/6 mice injected i.v. with 50 μg BSA-alexa488 or BSA-alexa488;RaBSA (50 μg BSA-alexa488 + 110 μg RaBSA) 2 hours prior to analysis. Scatter plots (top: geometric mean fluorescent intensity, MFI) and representative histograms (bottom) N = 2–6 mice per treatment. (B) Uptake of BSA-TRITC by kidneys leukocytes from Cx3cr1^gfp/+mice injected i.v. with BSA-TRITC;RaBSA (50 μg BSA-TRITC + 110 μg RaBSA) 2 hours prior to analysis. Plots are representative of 10 mice. (C) Uptake of BSA-TRITC as in (B) by kidney leukocytes in Cx3cr1^gfp/+ Rag2^−/− Il2rg^−/− mice 2 hours after i.v. injection of increasing amounts of BSA-TRITC or BSA-TRITC;RaBSA. N = 2–6 mice per treatment, per dose. (D) As in (C) for increasing amounts of OVA-alexa555 or OVA-alexa555;RaOVA. N = 2–8 mice per treatment, per dose. (E) BSA-alexa488 uptake by kidney F4/80^bright macrophages injected i.v. with PBS, 50 μg BSA-alexa488, BSA-alexa488;RaOVA or BSA-alexa488;RaBSA 2 hours prior to analysis as in (A). N = 3–6 mice per treatment. (F) Still frame from intravital imaging (IVM) of the superficial renal cortex of a Cx3cr1^gfp/+ mouse 2 hours after i.v. injection of 50 μg BSA-TRITC;RaBSA as in (C). N = 10 mice. Bar = 10 μm. (G) Flow cytometric analysis of DQ-BSA fluorescence in kidney F4/80^bright macrophages 2 hours after i.v. injection of 12.5 μg DQ-BSA or DQ-BSA;RaBSA (12.5 μg DQ-BSA + 27.5 μg RaBSA). N = 3–6 mice per treatment. Bars indicate mean value ± S.E.M. Symbols represent individual mice. *P < 0.05, **P < 0.01, ***P < 0.001 using a two-tailed, unpaired t-test. See also Figure S1.

Figure 2. Kidney F4/80^bright cells are tissue resident macrophages

(A) Immunofluorescence (IF) staining for F4/80, CD31 and rabbit IgG in kidney cryosections from a C57BL/6 mouse injected i.v. with BSA;RaBSA (50 μg BSA + 110 μg RaBSA) 2 hours prior to euthanasia. N = 3 mice. (B) IF staining for F4/80 and mouse IgG2a in kidney cryosections from a 6-month old NZBxW F1 mouse. G, glomerulus; T, tubule. Arrows indicate co-localistion of F4/80 and IgG2a staining. (C) IF staining for F4/80, CD31 and FcγRIV in kidney cryosections from C57BL/6, Fcgr4^−/− and LysM^Cre+;Fcgr4^f/f mice. (D) CD45.1 and CD45.2 congenic mice analyzed on 3, 5 or 8 weeks of parabiosis. Histograms represents percentage of partner-derived cells in the spleen, kidney and blood. N = 2–4 parabionts per time point. Bars indicate mean value ± S.E.M. Symbols represent individual mice. (E–F) Still frames from IVM of the superficial renal cortex in Cx3cr1^gfp/+ Rag2^−/− Il2rg^−/− mice. Capillaries are visualized by i.v. injection of 70 kDa dextran-TRITC. Arrow in (F) indicates macrophage filopodial extension. Time in (F) is shown in min:sec. Bars = 10 μm (A, E, F) or 50 μm (B, C). See also Figure S2 and supplementary movies S1–S3.

Figure 3. Kidney resident macrophages are located between the capillary endothelium and the basement membranes of tubules and the Bowman capsule

(A) 3D surface rendering of a still frame form IVM of the kidney in a Cx3cr1^gfp/+ Rag2^−/− Il2rg^−/− mouse. Cx3cr1^gfp macrophage is in green. Capillaries (red) are visualized in red by i.v. injection of 70 kDa dextran-TRITC. (B) Transmission Electron Microscopy (TEM) image of the kidney cortex from a C57BL/6 mouse. MΦ: macrophage, E: endothelium, T: tubule. Inset shows endothelial plasmalemmal vesicles. (C–D) TEM images of the renal cortex in a Cx3cr1^gfp/+ mouse immunostained for GFP. MΦ: macrophage, E: endothelium, T: tubule. (E–G) TEM images of the renal cortex in a C57BL/6 mouse immunostained for I-A. MΦ: macrophage, E: endothelium, PT: proximal tubule, DT: distal tubule, T: tubule, P: podocyte foot processes. Bars = 10 μm (A), 1 μm (B–H), 0.5 μm (inset in B). See also Figure S3. (H) Scheme giving the anatomical context of EM pictures. Capillaries are depicted in red, macrophage in blue, podocytes in green and epithelial structures in grey, PT: proximal tubule, DT: distal tubule. The area corresponding to EM pictures in E and F is indicated by a dashed black line.

Figure 4. Macrophages monitor transendothelial transport of circulating immune complexes

(A) Still frames from IVM of the kidney from Cx3cr1^gfp/+ mouse receiving 50 μg OVA-alexa555 i.v.. Arrows indicate OVA-positive macrophage. Time is min:sec from the time of i.v. injection. Bar = 10 μm. (B) As in (A) 10 minutes after i.v. injection of 10 μg PE-conjugated antibodies against F4/80 (top) and I-A (bottom). Bars = 20 μm. N = 3–4 mice per antibody. (C) Flow cytometry analysis of kidney leucocytes from Cx3cr1^gfp/+ mice sacrificed 5 minutes after i.v. injection of 10 μg anti-I-A. N = 6 mice. (D) TEM of the kidney cortex from mice sacrificed 30 seconds after i.v. injection of OVA;RaOVA. RaOVA is detected with gold-conjugated goat anti-rabbit. Arrow indicates gold particles in endothelial caveolae/CCV. Macrophages are identified by immunocytochemistry for I-A. MΦ: macrophage, E: endothelium, T: tubule. Bars = 0.5 μm. (E) Quantification of total (left) and endothelium-associated (right) immunogold particles in mice treated in (D). The numbers above bars indicate the absolute number of gold particles counted in total N = 3 mice. (F) Still frame from IVM of phagocytosis of 0.02 μm beads by a kidney macrophage in Cx3cr1^gfp/+ mice. Bar = 10 μm. (G) Phagocytosis of 2, 0.2 and 0.02 μm beads by splenic RPMΦs (left) and kidney macrophages (right) 2 hours after i.v. injection. N = 5–12 per treatment. (H) Still frames from IVM of the superficial renal cortex in C57BL/6 mice injected i.v. with 70 kDa dextran-TRITC to label the vascular bed (red), and 5–7 min later, with BSA, or BSA;RaBSA (50 μg/mouse). Images are shown immediately before and 10 minutes after injection of BSA, or BSA;RaBSA. Bars = 100 μm. Each symbol in (C) and (G) represents an individual mouse. See also Figure S4 and supplementary movies S4–S7.

Figure 5. FcγRIV-dependent uptake and activation of kidney, but not dermal, macrophages by small immune complexes, ‘auto’ antibodies, and NZWxBXSB serum

(A) Uptake of OVA-alexa555 by kidney macrophages from wt, Fcgr1^−/−, Fcgr4^−/− and LysM^Cre+;Fcgr4^f/f mice 2 hours after i.v. injection of 6.5 μg OVA-alexa555 or OVA-alexa555;RaOVA (6.5 μg OVA-alexa555 + 21.25 μg RaOVA). N = 3–7 mice per treatment, per genotype. (B) Uptake of BSA-TRITC by kidney F4/80^bright macrophages from wt, Csf1r^Cre+;Fcgr4^f/f and Csf1r^Cre−;Fcgr4^f/f mice 2 hours after i.v. injection of 50 μg BSA-TRITC or BSA-TRITC;RaBSA. N = 3 mice per treatment, per genotype. (C) qPCR analysis of IL-6, Il-1b, Tnf, Ccl2 transcripts in total kidney tissue from C57BL/6 mice 1, 3, and 6 hrs after i.v. injection of 50 μg BSA-TRITC or BSA-TRITC;RaBSA. N = 4–6 mice per treatment, per time point. (D) qPCR for Tnf and Il1b in FACS-sorted kidney F4/80^bright macrophages from wt (C57BL/6) and Fcgr4^−/− mice 1 hour after treatment. N = 6–7 mice per treatment, per genotype. (E–F) Representative plots (E) and quantification (F) of intracellular staining for TNF in kidney F4/80^bright macrophages from wt and Fcgr4^−/− mice 1 hour after treated as in (D). N = 5–7 mice per treatment, per genotype. (G) TNF staining in kidney and dermal F4/80^bright macrophages from wt mice 1 hour after treatment as in (D). N = 3–8 mice per treatment. (H) TNF staining in kidney F4/80^bright macrophages from wt mice 1 hour after i.v. injection of the indicated amounts of BSA, RaBSA, BSA;RaBSA, rabbit IgG (Ra-IgG), rabbit anti-mouse transferrin (Ra-mTransferrin), rabbit anti-mouse albumin (Ra-mAlbumin), rabbit anti-mouse IgG (Ra-mIgG) or Ra- mIgG (Fab′)₂. N = 4–5 mice per treatment. (I) TNF staining in kidney F4/80^bright macrophages from wt or Fcgr4^−/− mice 1 hour after i.v. injection of increasing amounts of serum from the indicated mouse strains. N = 2–5 mice per dose per genotype. Bars indicate mean value ± S.E.M. Each symbol represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001 using a two-tailed, unpaired t-test. NS, not significant. See also Figure S5.

Figure 6. FcγRIV-dependent activation of kidney macrophage by immune complexes triggers recruitment of blood leucocytes

(A) Recruitment of total monocytes, Ly6C^high monocytes and neutrophils in the kidneys of wt, Fcgr4^−/−, LysM^Cre−;Fcgr4^f/f and LysM^Cre+;Fcgr4^f/f mice, assessed by flow cytometry 6 hours after i.v. injection of 50 μg BSA-TRITC or BSA-TRITC;RaBSA. N = 3–7 mice per treatment, per genotype. (B) Intravital microscopy analysis of neutrophil recruitment in the kidneys of Cx3cr1^gfp/+ Rag2^−/− Il2rg^−/− mice (detected by i.v. injection of APC-conjugated anti-Gr1 antibody) 6 hours after i.v. injection of 50 μg BSA or BSA;RaBSA. N = 4–5 mice per treatment, per genotype. Bars = 50 μm. (C) Recruitment of CD45.2 bone marrow leucocytes transferred from wt or Fcgr4^−/− mice into wt CD45.1 recipients 1hr before or 1 hr after i.v. injection of 50 μg BSA-TRITC or BSA-TRITC;RaBSA. Recruitment is measured 6 hours after treatment. N = 7–10 mice per treatment, per genotype, per time point of transfer. (D) Recruitment of total monocytes, Ly6C^high monocytes and neutrophils (PMNs) in the kidneys of Cx3cr1^gfp/+ Rag2^−/− Il2rg^−/− mice 6 hours after treated as in (A). N = 4–10 mice per treatment. (E–F) Recruitment of total monocytes (E) and neutrophils (F) in the kidneys of Myd88^+/− or Myd88^−/− littermates treated as in (A). N = 4–7 mice per treatment, per genotype. Each symbol represents an individual mouse. **P < 0.01, ***P < 0.001 using a two-tailed, unpaired t-test. NS, not significant. See also Figure S6.

Figure 7. FcγRIV-dependent activation of macrophages by immune complexes synergizes with TLR signalling

(A) C57BL/6 or Fcgr4^−/− mice were injected i.v. with 12.5 μg OVA-GpC, 12.5 μg OVA-CpG, OVA-GpC;RaOVA (12.5 μg OVA-GpC + 42.5 μg RaOVA) or OVA-CpG;RaOVA (12.5 μg OVA-CpG + 42.5 μg RaOVA). (B–D) Flow cytometric analysis of Ly6C^high monocyte (B), Ly6C^low monocyte (C) and neutrophil (D) recruitment (per mg of tissue) in the kidneys of C57BL/6 or Fcgr4^−/− mice assessed by flow cytometry 6 hours after treatment as in (A). (E) Intracellular staining for TNF in kidney leukocytes from C57BL/6 mice 1 hour after treatment as in (A). (F–G) Flow cytometric analysis of CD86 (F) and I-A (G) expression by F4/80^bright kidney macrophages 2 hours after treatment as in (A). N = 3–9 mice per treatment, per genotype. Bars indicate mean value ± S.E.M. Symbols represent individual mice. *P < 0.05, **P < 0.01, ***P < 0.001 using a two-tailed, unpaired t-test. NS, not significant.