Fc receptor-mediated accumulation of macrophages in crescentic glomerulonephritis induced by anti-glomerular basement membrane antibody administration in WKY rats
Pavel Kovalenko1,
Hidehiko Fujinaka1,
Yutaka Yoshida1,
Hiroki Kawamura2,
Zhenyun Qu1,
Adel Galal Ahmed El-Shemi1,
Huiping Li1,
Asako Matsuki1,
Vladimir Bilim1,
Eishin Yaoita1,
Toru Abo2,
Makoto Uchiyama3 and
Tadashi Yamamoto1
1 Department of Structural Pathology, Institute of Nephrology, 2 Division of Immunology and Zoology, Department of Infectious Disease Control, Course for Community Disease Control and 3 Division of Pediatrics, Department of Homeostatic Regulation and Development, Course for Biological Functions and Medical Control, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8510, Japan
Correspondence to: T. Yamamoto; E-mail: tdsymmt{at}med.niigata-u.ac.jp
Transmitting editor: K. Okumura
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Abstract
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Anti-glomerular basement membrane (GBM) glomerulonephritis induced in WKY rats is characterized by glomerular accumulation of CD8+ T cells and monocytes/macrophages, followed by crescent formation. The mechanism of leukocyte accumulation after antibody binding to GBM is still unclear. To unveil an involvement of Fc
receptors (Fc
R) in leukocytes recruitment we examined the expression of Fc
R in glomeruli and the effects of the administration of F(ab')2 fragment of anti-GBM antibody or Fc
R blocking on the initiation and progression of this model. A gradual increase of Fc
R mRNA expression in glomeruli during the time course of disease suggested their significance in the development of glomerulonephritis. Glomerular lesions and proteinuria were induced only in rats injected with intact IgG of anti-GBM antibody, but not with the F(ab')2 fragment. In vivo blocking of Fc
R by administering heat-aggregated IgG led to the decrease of mRNA expression for all types of Fc
R (types 1, 2 and 3) and a significant amelioration of glomerulonephritis manifestations. By flow cytometry and immunohistochemistry Fc
R2-expressing cells in glomeruli were identified as macrophages, but not CD8+ T cells. The expression of Fc
R1 and 3 was significantly decreased, and that of Fc
R2 became undetectable in CD8+ T cell-depleted rats. Thus, CD8+ T cells may stimulate Fc
R expression on macrophages, contributing to their glomerular accumulation and injury. These studies provide direct evidence for a crucial involvement of IgG FcFc
R interaction in glomerular recruitment of macrophages and following induction of anti-GBM glomerulonephritis in WKY rats.
Keywords: anti-glomerular basement membrane antibody, CD8+ T cell, crescentic glomerulonephritis, Fc
R, macrophage
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Introduction
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Anti-glomerular basement membrane (GBM) glomerulonephritis induced by the administration of a small dose (25 µl/ 100 g body wt) of anti-GBM antibody is characterized by glomerular accumulation of CD8+ T cells and macrophages, followed by crescentic formation (1). This model is comparable with anti-GBM antibody-mediated crescentic glomerulonephritis in humans, which progresses to renal failure rapidly. To develop therapeutic methods for the crescentic glomerulonephritis, its pathogenesis needs to be understood. Our previous studies provided evidence for crucial roles of CD8+ T cells in the glomerular accumulation of macrophages, which were then presumed to injure glomerular structure and functions (1,2). Later on, roles of leukocyte adhesion molecules and chemokines in the development of this model were demonstrated (210). Although the glomerulonephritis is initiated by the binding of anti-GBM antibody to GBM, it is unclear how the infiltrates are accumulated in the glomeruli after anti-GBM antibody binding. One of the possible mechanisms is immune adherence of leukocytes through interaction between the Fc portion of anti-GBM antibody and Fc
receptors (Fc
R) expressed on the surface of the infiltrating cells.
Three distinct types of receptors for IgG have been defined: Fc
R1 (CD64) is the high-affinity receptor for IgG and Fc
R2 (CD32)/Fc
R3 (CD16) are the low-affinity receptors. These Fc
R bind to the Fc portion of IgG molecule in the form of immune complexes, leading to the activation of various Fc
R-expressing cells such as granulocytes, monocytes, macrophages, NK cells and subtypes of T cells (1115). These cells express each Fc
R subtype at different intensities and change the expression levels under inflammatory or pathological conditions (1621). The roles of FcR in various inflammatory diseases have been verified by using Fc
R-deficient mice. FcR
chain knockout mice, which exhibited no expression of Fc
R1, 2 and 3, failed to induce IgG-mediated endocytosis by macrophages. Fc
R were also shown to play crucial roles in antibody-mediated hypersensitivity and immune complex-mediated hypersensitivity (1619,22,23). Recent works demonstrated that Fc
R-deficient mice were resistant to nephrotoxic glomerulonephritis, indicating a critical role for Fc
R in the induction of murine glomerulonephritis (24,25). On the other hand, autoimmune glomerulonephritis occurred in Fc
R-deficient MRL/lpr mice as in wild-type MRL/lpr mice, indicating that the nephritis was Fc
R independent (26).
Since roles for Fc
R in the initiation of the crescentic glomerulonephritis of WKY rats had not been elucidated, we examined the involvement of Fc
R in this glomerulonephritis model and found a pivotal role for these receptors in the glomerular recruitment of macrophages, crescent formation and glomerular injury.
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Methods
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Animals
Animals used in this work were inbred male WKY rats (Charles River, Kanagawa, Japan), 200250 g body wt, aged 1216 weeks old.
Preparation of rabbit anti-GBM IgG and F(ab')2
Partially purified IgG was prepared from rabbit anti-GBM serum by 50% ammonium sulfate fractionation, followed by DEAEcellulose column chromatography. IgG was further purified by gel filtration on a Superdex 200 pg column using an AKTAexplorer 10S protein purification system (Amersham Pharmacia Biotech, Piscataway, NJ). F(ab')2 fragment was generated from the partially purified IgG fraction by incubation with immobilized pepsin [ImmunoPure F(ab')2 preparation kit; Pierce, Rockford, IL] as described in the manufacturers instructions. The F(ab')2 fragments were separated from undigested IgG and other proteolytic fragments by gel filtration on Superdex 200 pg. The purified IgG and F(ab')2 fragment showed apparent homogeneity on SDSPAGE gel stained with Coomassie brilliant blue R-250.
Induction of anti-GBM glomerulonephritis
Rats were injected with the intact IgG at different doses (3.0, 1.5 and 0.75 mg/kg body wt, five rats each). As we usually inject with 250 µl of anti-GBM antisera (containing 3.25 mg IgG)/kg body wt (4), the injected IgG doses in the present study were roughly equivalent to the amount of anti-GBM antisera used for the induction of anti-GBM glomerulonephritis in WKY rats. The ability to induce the glomerulonephritis was compared between intact IgG (1.5 mg/kg body wt, n = 5) and the F(ab')2 fragment (0.75 mg/kg; the same molar dose as 1.5 mg intact IgG/kg, n = 5). The animals were sacrificed at day 10 after the antibody injection. Urine specimens were collected on days 1, 4, 6 and 10 by housing animals in metabolic cages for 24 h. Urinary protein was estimated by using a protein assay kit (Nippon Bio-Rad, Tokyo, Japan).
Binding efficiency of intact IgG or F(ab')2 of the anti-GBM antibody to GBM was examined by immunofluorescence microscopy. Rats were sacrificed at 1 h, and days 4, 6 and 10 after intact IgG or F(ab')2 antibody injection. Kidney slices were snap-frozen in n-hexane at 70°C and sectioned in a cryostat. The cryostat sections (3 µm thick) were stained with fluorescein-conjugated affinity-purified anti-rabbit IgG F(ab')2 (Rockland Immunochemicals, Gilbertsville, PA).
Histological examination by light microscopy and immunohistochemistry
Kidney specimens were fixed with methyl Carnoys fixative, and embedded in paraffin for light microscopic examination and immunohistochemistry. The sections for light microscopy were stained with periodic acidSchiff. mAb against rat CD8 and ED1 (Dainippon Seiyaku, Tokyo, Japan) were used for staining of infiltrating CD8+ cells and macrophages respectively. To examine any involvement of platelets in the pathogenesis, they were immunolocalized in the kidneys using mouse monoclonal IgG antibody against rat platelet, PL-1 (kindly provided by Dr E. de Heer, Leiden University, The Netherlands). The numbers of immunostained cells were counted on >100 glomeruli in each kidney and expressed per glomerular cross-section. Rat Fc
R2-bearing cells were immunostained with a mouse anti-rat CD32 mAb (BD PharMingen, San Diego, CA) in cryostat sections of frozen kidney samples.
Detection of glomerular mRNA for Fc
R1, 2 and 3
Glomeruli were isolated from the renal cortex by a standard sieving method (27). Total cellular RNA was extracted from the isolated glomeruli homogenized using a modified guanidine thiocyanate method (TRIzol; Gibco/BRL, Grand Island, NY).
Ribonuclease protection assay was performed for the detection of Fc
R expression in the glomeruli. Fc
R1 (178 bp), 2 (249 bp) and 3 (289 bp) cDNA were amplified from the glomerular RNA obtained from rats 10 days after anti-GBM antibody injection by PCR with specific primers according to the sequences available in the GenBank database (AF143186, X73371 and M64369). Sense primers used for amplification of Fc
R1, 2 and 3 fragments were as follows: 5'-CGGGATCCCTTGCAGCCTCCATGGGTCAG-3', 5'-CGGGATCCCGAGTCTCATGCAGGTCTTCC-3' and 5'-GGAATTC-ATGTGTCCAAGCCTGTCACC-3' respectively; and antisense primers were 5'-CTGTCACCGCTTATGTCCACCCTTAAGG-3', 5'-GAGGTCTGGAGAGTTGACCACCTTAAGG-3' and 5'-CGAAGAAAAGTCGGTGTGGCCTAGGGC-3' respectively. As the sequence for rat Fc
R1 was not available, the primers were designed to the regions of mouse Fc
R1 sequence with the highest homology to human Fc
R1 sequence. The amplified PCR product was 87% homologous to the mouse Fc
R1 DNA sequence. The Fc
R1, 2 and 3 cDNA fragments were subcloned in pBluescript SK+ vectors and then linearized at the 5' ends to use as templates for preparation of 32P-labeled antisense cRNA probes. Ten micrograms of total glomerular RNA was hybridized with a mixture of 32P-labeled antisense cRNA probes (1 x 105 c.p.m.) for each Fc
R mRNA and GAPDH overnight at 45°C. Unhybridized cRNA probes were digested with ribonuclease T1 (120 U/ml; Gibco/BRL, Gaithersburg, MD) and ribonuclease A (4 µg/ml; Boehringer Mannheim, Tokyo, Japan) for 1 h at 30°C. The ribonucleases were then digested with proteinase K (500 µg/ml; Promega, Madison, WI) for 30 min at 37°C. After phenol:chloroform extraction and sodium acetate:ethanol precipitation the hybridized RNA probes were denatured at 95°C for 5 min and electrophoresed on a 6% polyacrylamide gel. Detection and analysis of bands were performed by phosphor-imaging techniques using the Molecular Imager FX (Bio-Rad, Hercules, CA). The data was represented as a ratio of specific mRNA:GAPDH mRNA to verify the constant quantity of mRNA in each sample.
Blocking of Fc
R by heat-aggregated IgG (HAIgG)
HAIgG was prepared as described previously (28,29). Briefly, rat IgG was extracted from normal rat serum by 33% ammonium sulfate precipitation and heated at 63°C for 30 min. Resulting aggregates were ultracentrifuged at 10,000 g for 90 min to obtain the soluble fraction of HAIgG. Then, the soluble HAIgG was diluted in PBS and used in the experiment. Rats were injected with HAIgG (n = 5) and unaggregated rat IgG (n = 5) at a dose of 50 mg/100g body wt i.v. 30 min before the injection with anti-GBM antibody. Boost injections of HAIgG and unaggregated rat IgG were made on days 2 and 4 after the administration of anti-GBM antibody, and animals were sacrificed on day 5.
Depletion of CD8+ cells
CD8+ cells were depleted from the circulation by administrating mAb against rat CD8 (MRC-OX8, 28 mg of
-globulin/kg body wt i.p. and 6 mg/kg i.v.) at two different time points of this glomerulonephritis model. One group of rats (pre-treated group, n = 20) was given the anti-CD8 antibody 2 days before the administration of 25 µl/100 g body wt of anti-GBM antibody as described in our previous studies (1,2), where we demonstrated that CD8+ cells in the circulation, spleen, cervical lymph node and thymus were completely depleted by this MRC-OX8 administration protocol, and the depletion was maintained for nearly 10 days. The other group of rats (post-treated group, n = 10) was given the antibody 3 days after anti-GBM antibody administration. As a control, rats were injected with irrelevant mAb and anti-GBM antibody or untreated. The animals from the pre-treated and control group were sacrificed at days 1, 3, 7 and 14, and the post-treated group animals were sacrificed at days 7 and 14 (five rats at each time point) for histological examination and RNA isolation from the glomeruli.
Urine specimens were collected on days 1, 3, 7 and 14 by housing animals in metabolic cages for 24 h. Urinary protein was estimated by using a protein assay kit (Nippon Bio-Rad).
Flow cytometry analysis
Flow cytometry was employed to examine Fc
R2-bearing leukocytes obtained from the glomeruli and spleens of rats with anti-GBM glomerulonephritis. Glomeruli isolated from the renal cortices were disintegrated using a stainless mesh (30). Then, leukocytes were isolated by centrifugation on 45% Percoll solution and stained with different combinations of antibodies: FITC-conjugated mouse anti-rat CD32 (clone D34-485) mAb, R-phycoerythrin (PE)-conjugated mouse anti-rat CD8 mAb (clone OX-8), PE-conjugated mouse anti-rat CD11b/c mAb (clone OX-42) and PE-conjugated mouse anti-rat CD4 mAb (clone OX-35). All antibodies were obtained from BD PharMingen (San Diego, CA). Stained cells were analyzed by two-color flow cytometry using a FACScan (Becton Dickinson Immunocytometry System, San Jose, CA).
Statistical analysis
Statistical evaluations were performed using the MannWhitney test (data meet the requirements for using the test). Analysis was performed with the use of Prism 3.0 for Windows (GraphPad, San Diego, CA). Data were considered statistically significant at P < 0.05. All statistical tests were two-sided.
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Results
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Binding of anti-GBM antibody to the GBM
Both intact IgG (1.5 mg/kg) and the F(ab')2 fraction (0.75 mg/kg) of rabbit anti-GBM antibody were localized by immunofluorescence microscopy along the GBM in a linear pattern at 1 h after injection (Fig. 1A and B). No significant difference in the intensity and distribution of rabbit IgG staining was observed between these groups throughout the experiment up to day 10 (Fig. 2A and B). No deposition of rat IgG was found in the glomeruli until day 5, and a faint but significant signal was observed at day 7 and later after the anti-GBM antibody injection (Fig. 2C and D). The intensity of immunofluorescence for rat IgG was much weaker than that for rabbit IgG.

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Fig. 1. Glomerular binding of intact IgG (A) or F(ab')2 (B) of anti-GBM antibody at 1 h after injection. Light microscopic findings in glomeruli of rats injected with anti-GBM antibody IgG (C) or F(ab')2 (D). Glomerular accumulation of CD8+ T cells (E) and macrophages (G) was observed in the intact anti-GBM antibody IgG-injected rats, but no recruitment of CD8+ T cells (F) and macrophages (H) was detected in the glomeruli of rats injected with F(ab')2 fragment of the anti-GBM antibody.
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Fig. 2. Immunofluorescence microscopy shows the deposition of rabbit IgG (A and B) and rat IgG (C and D) in the glomeruli of WKY rats at day 1 (A and C) and day 7 (B and D) after the administration of anti-GBM antibody. The immunofluorescence intensity for rabbit IgG along the GBM at day 5 (E) is not affected by HAIgG administration (F).
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Induction of anti-GBM glomerulonephritis by intact IgG and F(ab')2 of anti-GBM antibody
Although the binding of both IgG and F(ab')2 fractions of antibody to GBM was nearly identical, anti-GBM glomerulonephritis was induced only in animals injected with intact IgG at day 10, and was characterized by hypercellular glomeruli with proteinaceous material deposition and crescent formation (Fig. 1C). Immunohistochemical analysis revealed a significant number of CD8+ T cells (5.0 ± 0.8 cells/glomerular cross-section, mean ± SD, P < 0.01) and ED1+ macrophages (27.4 ± 3.4 cells/glomerular cross-section, P < 0.01) in the glomeruli of intact IgG-injected rats (Fig. 1E and G). High levels of proteinuria also confirmed the induction of anti-GBM glomerulonephritis (Fig. 3). In contrast, no apparent glomerular histological changes, glomerular infiltration of CD8+ T cells and ED1+ macrophages or proteinuria were observed in the F(ab')2-injected animals (Fig. 1D, F and H). Considerable numbers of platelets were detected in the glomerular capillaries, but not in the crescentic lesions. In addition, the number and density of platelets in the glomeruli were almost the same as those detected in the interstitial capillaries (data not shown).

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Fig. 3. Urinary protein excretion in WKY rats injected with intact IgG (filled circles) or F(ab')2 (open circles) anti-GBM antibody. Proteinuria was completely absent in the group injected with F(ab')2 fragment of the anti-GBM antibody.
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Expression of mRNA for Fc
R in the glomeruli of rats from two experimental groups
Expression of Fc
R1, 2 and 3 mRNA was detected in the glomeruli of rats injected with intact anti-GBM IgG, but not in the glomeruli of F(ab')2-injected animals (Fig. 4). Fc
R1, 2 and 3 mRNA was intense in the glomeruli of rats injected with 3.0 mg of anti-GBM IgG/kg; the mRNA:GAPDH ratio was 0.142 ± 0.002 (Fc
R1), 0.352 ± 0.003 (Fc
R2) and 0.434 ± 0.003 (Fc
R3) respectively; or in 1.5 mg/kg-injected rats, 0.121 ± 0.001 (Fc
R1), 0.302 ± 0.002 (Fc
R2) and 0.473 ± 0.003 (Fc
R3). The expression was a little less in animals given a smaller dose of the antibody; 0.103 ± 0.001 (Fc
R1), 0.161 ± 0.001 (Fc
R2) and 0.189 ± 0.002 (Fc
R3) respectively in 0.75 mg/kg-injected rats. Rats injected with 1.5 or 3.0 mg of anti-GBM antibody IgG/kg and 250 µl of anti-GBM antisera/kg body wt expressed a similar intensity of mRNA for all three types of Fc
R in the glomeruli. The mRNA:GAPDH ratios were 0.113 ± 0.001 (Fc
R1), 0.282 ± 0.002 (Fc
R2) and 0.394 ± 0.00 (Fc
R3) respectively in the group injected with the anti-GBM sera. The histological glomerular changes and the amounts of protein excreted in the urine were also almost identical in rats given anti-GBM antibody IgG at 1.5 or 3.0 mg/kg and in rats given anti-GBM antisera of 250 µl/kg (data not shown).

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Fig. 4. Expression of three types of Fc R in glomeruli of rats 10 days after injection with anti-GBM IgG or F(ab')2. (1) Anti-GBM IgG 0.75 mg/kg; (2) anti-GBM IgG 1.5 mg/kg; (3) anti-GBM IgG 3.0 mg/kg; (4) anti-GBM antiserum 0.25 ml/kg; (5) anti-GBM F(ab')2 0.75 mg/kg.
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Blocking of Fc
R by HAIgG
Control animals given unaggregated rat IgG + anti-GBM antibody exhibited high levels of proteinuria at day 5. In contrast, animals given HAIgG + anti-GBM antibody demonstrated a significant reduction in protein excretion in the urine (Fig. 5).

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Fig. 5. Effect of HAIgG treatment on proteinuria in WKY rats injected with anti-GBM antibody. Levels of excreted protein are significantly lower in the treated group (open circles) in comparison with the positive control group (filled circles).
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Rabbit anti-GBM antibody was localized by immunofluorescence microscopy along the GBM in a linear pattern in both groups at day 5 after injection. No significant difference in the intensity of staining was observed between the two groups (Fig. 2E and F). The marked endocapillary hypercellularity and crescent formation induced in the control group rats were reduced in rats treated with HAIgG. The frequency of glomeruli with crescent formation was decreased from 76.8 ± 3.5% in the control rats to 30 ± 9.7% (P < 0.01) in the HAIgG-treated rats. Accumulation of CD8+ T cells and ED1+ macrophages was observed in the glomeruli of the control rats (Fig. 6A and B). The numbers of macrophages detected in the glomeruli were significantly decreased by the HAIgG treatment from 26.4 ± 3.4 to 15.8 ± 3.0 cells/glomerular cross-section (P < 0.01), while that of CD8+ T cells was unaffected (4.7 ± 1.1 versus 3.9 ± 0.8 cells/glomerular cross-section, P = 0.24) (Fig. 6C and D).

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Fig. 6. CD8+ T cell and ED1+ macrophage accumulation in glomeruli of the control group (A and B) and the group treated with HAIgG (C and D) at day 5 after the injection of anti-GBM antibody.
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The expression of mRNA for all three types of Fc
R in the glomeruli was significantly decreased by the HAIgG treatment: Fc
R1:GAPDH mRNA ratio from 0.163 ± 0.002 to 0.081 ± 0.001 (P < 0.01), Fc
R2:GAPDH mRNA ratio from 0.604 ± 0.004 to 0.261 ± 0.002 (P < 0.01) and Fc
R3:GAPDH mRNA ratio from 0.402 ± 0.003 to 0.214 ± 0.002 (P < 0.01). The decrease was also demonstrated in the cortices: Fc
R1:GAPDH mRNA ratio from 0.065 ± 0.006 to 0.046 ± 0.002 (P < 0.01), Fc
R2:GAPDH mRNA ratio from 0.085 ± 0.003 to 0.064 ± 0.005 (P < 0.01) and Fc
R3:GAPDH mRNA ratio from 0.089 ± 0.009 to 0.054 ± 0.007 (P < 0.01) (Fig. 7).

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Fig. 7. The mRNA expression of three types of Fc R in the renal cortex and glomeruli of rats isolated from the control group (unaggregated rat IgG + anti-GBM antibody, lanes 26 and 12), and the group treated with HAIgG (lanes 711 and 13) and normal glomeruli (lane 1).
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Effect of CD8+ cell depletion on Fc
R expression
Glomerular hypercellularity caused by the accumulation of CD8+ T cells (4.5 ± 1.7 cells/glomerular cross-section) and ED1+ macrophages (25.3 ± 6.0 cells/glomerular cross- section) was found in the rats 7 days after anti-GBM antibody administration (Fig. 8A and B). In contrast, glomerular accumulation of CD8+ T cells was negligible (0.0 ± 0.0 cells/glomerular cross-section, P < 0.01) and that of macrophages (14.4 ± 4.2 cells/glomerular cross-section, P < 0.01) was significantly reduced by anti-CD8 antibody administration before anti-GBM antibody injection (Fig. 8C and D). Through treatment with anti-CD8 antibody after anti-GBM antibody injection, the numbers of CD8+ T cells and ED1+ macrophages were also significantly reduced in the glomeruli (0.1 ± 0.0, P << 0.01 and 10.6 ± 3.0, P < 0.01 respectively) (Fig. 8E and F).

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Fig. 8. CD8+ T cell (A, C and E) and ED1+ macrophage (B, D and F) accumulation at day 7 after injection of anti-GBM antibody in glomeruli of control anti-GBM glomerulonephritis rats (A and B), anti-rat CD8 mAb pre-treated rats (C and D) and post-treated rats (E and F).
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Excretion of urinary protein was almost completely suppressed in animals treated with anti-CD8 antibody before anti-GBM antibody injection (Fig. 9). In contrast, the amount of urinary protein was unaffected by day 7 in animals given anti-CD8 antibody after anti-GBM antibody injection. However, urinary protein excretion was significantly decreased at day 14 in these rats (208.5 ± 50.2 versus 109.5 ± 13.1 mg/day, P = 0.02) (Fig. 9).

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Fig. 9. Urinary protein changes in WKY rats with anti-GBM glomerulonephritis treated with anti-CD8 mAb. Proteinuria is significantly decreased in rats treated with anti-CD8 antibody before (open circles) and after (open squares) anti-GBM antibody injection in comparison with the positive control group (filled circles).
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Glomerular expression of mRNA for all three types of Fc
R gradually increased during the time course of anti-GBM glomerulonephritis, reached a plateau at day 3 and noticeably decreased at day 14 (Fig. 10). By administration of anti-CD8 antibody before anti-GBM antibody injection, expression of mRNA for Fc
R1 and Fc
R3 became faint, and Fc
R2 expression was undetectable in the glomeruli until day 7. However, expression of all Fc
R greatly increased in the glomeruli at day 14, which corresponds with the influx of CD8+ T cells and ED1+ macrophages into the glomeruli at this time point as detected by immunohistochemical staining. While intense mRNA expression for all Fc
R was demonstrated in the glomeruli at day 7 in animals treated with anti-CD8 antibody after anti-GBM antibody injection, expression of Fc
R1 and 3 was obviously decreased, and that of Fc
R2 was undetectable at day 14 (Fig. 10).

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Fig. 10. Fc R mRNA expression in glomeruli of rats from the normal control group (1); untreated anti-GBM group: day 1 (2), 3 (3), 7 (4) and 14 (5); anti-CD8 antibody pre-treated group: day 1 (6), 3 (7), 7 (8) and 14 (9); and anti-CD8 antibody post-treated group: day 7 (10) and 14 (11).
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Flow cytometric analysis of leukocytes accumulating in the glomeruli
Immunostaining of frozen kidney cross-sections revealed that the number and distribution of Fc
R2+ cells was comparable to those of ED1+ macrophages. In addition, Fc
R2 staining was clearly observed in giant cells, which were apparently transformed from macrophages since they were ED1+. These results bring us to the conclusion that Fc
R2 is expressed by infiltrating macrophages (Fig. 11A and B).
To identify the exact type of Fc
R2-bearing immune cells in the glomeruli, leukocytes isolated from glomeruli were stained with different combinations of antibodies and examined by flow cytometry. The results showed that nearly equal proportions (
10%) of glomerular infiltrates were CD4+ T cells and CD8+ T cells, and that no expression of Fc
R2 was detected on both CD4+ T cells and CD8+ T cells. In contrast, macrophages detected as CD 11b/c-bearing cells expressed Fc
R2 (Fig. 11CE).
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Discussion
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Anti-GBM glomerulonephritis in WKY rats, initiated by a single injection with a small dose of anti-GBM antibody, is characterized by the predominant infiltration of monocytes/macrophages and considerable accumulation of CD8+ T cells, and by the high frequency of glomerular crescent formation. The respective involvement of cell-mediated and humoral immunity in the development of this model is still indistinct. The present study was designed to examine the role of Fc
R for the anti-GBM IgG antibody in the development of the crescentic glomerulonephritis model, which has not been clearly established in previous studies on this model.
In the current study we demonstrated that crescentic glomerulonephritis was induced only in animals injected with intact IgG of anti-GBM antibody, but not in animals injected with F(ab')2 fractions of anti-GBM antibody, in spite of the equal binding of them to GBM. The gradual increase of expression of Fc
R1, 2 and 3 mRNA during the time course of the disease was parallel to the progress of the disease. The observation suggested the significance of these molecules in this model. To corroborate these results we performed in vivo blocking of Fc
R by administering HAIgG. In a previous study, macrophage accumulation and subsequent glomerular injury in an experimental anti-GBM glomerulonephritis model in rabbits were demonstrated to be dependent on the Fc portion of the disease-initiating IgG molecule (31). As HAIgG is a high-affinity ligand for Fc
R (28,32), its injections were expected to block all Fc
R types of leukocytes in experimental animals and to elucidate the roles of these receptors in anti-GBM glomerulonephritis. The study showed a significant amelioration of this model by Fc
R blocking, suggesting that pathological changes and urinary protein excretion are dependent on the immune adherence of infiltrating cells to heterologous antibody planted along the GBM through Fc
R. The immune adherence may be mediated by binding between the complement receptors of leukocytes and the complement components activated by the immune complex in this model. However, we recently demonstrated no effects of decomplementation by cobra venom administration to rats on the development of this model (33), indicating that complement receptor-mediated immune adherence was not essential.
However, these data leave undecided the following questions: which type of Fc
R is important and which type of infiltrating cells express the Fc
R? Previously, the macrophage was shown to be a major cell type accumulating in the glomeruli and crescentic lesions, and one of the most important mediators of glomerular injury and crescent formation (34,35). Furthermore, the crucial role of CD8+ T cells in the induction of the glomerulonephritis was demonstrated by almost complete suppression of the glomerular changes and injury in CD8+ T cell-depleted rats (1,2,36). The severity of glomerulonephritis, infiltration by T cells and macrophages, and, particularly, the formation of crescents was greatly reduced by the treatment of experimental animals with anti-CD8 mAb (1,2,36). We assumed a linkage between CD8+ T cells and Fc
R expression, leading to the progression of glomerulonephritis. To clarify this linkage, we examined Fc
R expression in the glomeruli of anti-GBM glomerulonephritis rats in a CD8+ T cell-depleted condition. This experiment demonstrated that expression of mRNA for Fc
R1 and 3 was significantly decreased, but had not become null in the glomeruli, where no CD8+ T cells and a substantial number of macrophages were detected. The decrease of Fc
R expression was associated with the decrease of macrophages accumulating to the glomeruli. These facts may indicate that these glomerular Fc
R were expressed on the macrophages. On the other hand, the observation that Fc
R2 expression was undetectable in the glomeruli of CD8+ T cell-depleted rats may indicate that Fc
R2 are also expressed on the CD8+ T cells as well as macrophages. However, flow cytometry analysis of leukocytes isolated from glomeruli of anti-GBM glomerulonephritis rats showed that cells expressing Fc
R2 were restricted to macrophages, but not to CD8+ T cells. Immuno histochemistry also showed Fc
R2 immunoreactivity in a close association with ED1+ staining. In addition, glomerular accumulation of CD8+ T cells was not affected by HAIgG treatment in the present study. It has been reported that the cell-surface expression of Fc
R is up-regulated by various agents, including such cytokines as IFN-
and IL-10 (3740). CD8+ T cells could be the source of the cytokines which up-regulate the expression of Fc
R on the surface of infiltrating macrophages. Thus, our results support the data that macrophages are the major population of Fc
R-bearing cells in the glomeruli of this model and CD8+ T cells stimulate expression of Fc
R, especially Fc
R2 in macrophages in the glomeruli. The role of other Fc
R-bearing cells, such as mast cells or platelets, in this model is considered to be minor since no glomerular accumulation of mast cells has been shown in this model. Although considerable numbers of platelets were detected by immunostaining in the glomerular capillaries, similar numbers of platelets were also observed in the glomeruli of normal rat kidneys or interstitial capillaries. Therefore, both mast cells and platelets may not essentially contribute to the induction of this model, although we could not neglect the possibility of their minor participation.
Taken together, the present study proposes the following initiation mechanism of anti-GBM glomerulonephritis in WKY rats. At the beginning the heterologous anti-GBM antibody binds to GBM and attracts macrophages through the interaction between the IgG Fc portion of the antibody and Fc
R on their surface. Macrophages begin to accumulate in glomeruli of this model from day 1 after the injection of anti-GBM antibody and their number increases during the time course of the disease (1). Rat antibody against rabbit IgG produced at the late phase binds to the rabbit anti-GBM antibody on GBM at day 5 after anti-GBM antibody injection, which was demonstrated by immunofluorescence microscopy. The rat anti-rabbit IgG antibody should also contribute to the macrophage accumulation at the late phase. The distribution of macrophages in glomeruli at the late phase better corresponded with the deposition of rat IgG rather than that of rabbit IgG, which also supports this suggestion.
The recognition of the Fc portion of anti-GBM antibody by Fc
R should stimulate the macrophages to produce and release pro-inflammatory cytokines and chemokines, such as tumor necrosis factor, IL-1 and MCP-1 as described previously (1,2,4). Our previous study also showed that this model was CD8+ T cell dependent, and glomerular accumulation of CD8+ T cells was mediated through interaction between ICAM-1 and LFA-1 (1,3). Cytokines released from macrophages might stimulate induction of ICAM-1 expression on endothelial cells and activate LFA-1 on CD8+ T cells in the glomeruli. However, it is unclear how CD8+ T cells play a key role in this model. Up-regulation of IFN-
in the glomeruli of this model was demonstrated in our previous study (2), suggesting that CD8+ T cells and CD4+ T cells might be stimulated by another cytokine, such as IL-12, released from macrophages in the glomeruli. IL-12 is a cytokine known to stimulate CD4+ T cells to differentiate to Th1, and also stimulates CD8+ T cells and CD4+ T cells to produce IFN-
(2,17). Then, IFN-
could further activate macrophages to express Fc
R and to promote macrophage-mediated glomerular injury as delayed-type hypersensitivity.
In conclusion, this is the first study providing direct evidence of the crucial participation and role of Fc
R in the initiation of anti-GBM antibody-induced crescentic glomerulonephritis in WKY rats and the recruitment of macrophages to the glomeruli. Although the type of Fc
R mostly contributing to the progression of this glomerulonephritis has not yet been identified, the important role of the Fc
R-mediated immune adherence was clarified in the macrophage accumulation in this model.
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Acknowledgements
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This work was presented in parts at the 34th and 35th Annual Meetings of the American Society of Nephrology, San Francisco, CA, October 2001 and Philadelphia, PA, OctoberNovember 2002. We thank Mr Kan Yoshida and Ms Sachiko Morita for technical assistance. P. K. is the recipient of a Japanese Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho) scholarship.
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Abbreviations
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FcRFc receptor
GBMglomerular basement membrane
HAIgGheat-aggregated IgG
PEphycoerythrin
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