1 Department of Structural Pathology, Institute of Nephrology and 4 Second Department of Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 2 Department of Pediatrics, Fukushima Medical University School of Medicine, Fukushima, 3 Department of Pediatrics, Niigata National Hospital, Kashiwazaki and 5 Department of Cell Pathology, Kumamoto University Faculty of Medical and Pharmaceutical Sciences, Kumamoto, Japan
Correspondence and offprint requests to: Hidehiko Fujinaka, MD, PhD, Department of Structural Pathology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, 757 Asahimachi-dori-1-bancho, Niigata 951-8510, Japan. E-mail: dexter{at}med.niigata-u.ac.jp
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Abstract |
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Methods. We examined correlations between the amount of urinary protein and the numbers of glomerular CD8+ cells or Mo/M in rats after administrating different doses of anti-GBM antibody (5.0, 7.5, 10.0 and 25.0 µl/100 g body weight). The roles of Mo/M
in induction of GN were examined in animals by depleting Mo/M
in the glomerulus. To do this, rats were injected intravenously with liposome-encapsulated dichloromethylene diphosphonate (liposome-MDP) from day 3 to day 7 after anti-GBM antibody injection and they were then sacrificed at day 8.
Results. Liposome-MDP treatment significantly reduced the number of ED-1+ Mo/M accumulated in glomeruli from 32.1±1.2 to 1.4±0.3/glomerular cross-section (mean±SD, P<0.01), and the amount of urinary protein from 103.8±19.8 to 31.8±15.9 mg/day (P<0.01), as well as the incidence of crescentic glomeruli from 91.3±2.7 to 23.3±7.6% (P<0.01) at day 8. This treatment also reduced the number of CD8+ cells accumulating in the glomeruli from 5.4± 0.7 to 0.5±0.1/glomerular cross-section (P<0.01). Upregulation of glomerular intercellular adhesion molecule 1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1) mRNA expression was suppressed by Mo/M
depletion.
Conclusion. These results indicate that Mo/M play an important role in the induction of crescentic anti-GBM GN and glomerular injury.
Keywords: anti-GBM glomerulonephritis; CD8; macrophage; monocyte; receptor; scavenger
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Introduction |
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In the present study, we examined correlations between Mo/M as well as CD8+ cell numbers and degree of glomerular injury to determine whether Mo/M
or CD8+ cells play a direct role in glomerular injury and lesions. Further, to test involvement of Mo/M
in anti-GBM GN more directly, we examined the effect of M
depletion on the development of the anti-GBM GN. Liposome-encapsulated dichloromethylene diphosphonate (liposome-MDP) was introduced specifically to deplete M
without affecting other leukocyte types, such as neutrophils, lymphocytes or Mo [48]. We treated WKY rats with liposome-MDP to deplete M
after induction of anti-GBM GN. We found that both the amount of protein excreted in the urine and the percentage of crescentic glomeruli were strongly correlated with glomerular ED-1+ Mo/M
numbers, but not with CD8+ cells. In addition, liposome-MDP treatment markedly suppressed lesion formation in this model, indicating a pivotal role for M
in glomerular injury and crescent formation.
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Subjects and methods |
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Preparation of MDP-entrapped liposome (liposome-MDP)
MDP was kindly provided by Boehringer Mannheim GmbH (Mannheim, Germany) and was encapsulated in liposome as described previously [4]. In brief, phosphatidylcholine (Nippon Fine Chemicals, Osaka, Japan), diacetyl phosphate (Nakarai Tesque, Ltd, Kyoto, Japan) and cholesterol (Wako Chemical Co., Tokyo, Japan) were dissolved in chloroform (Nakarai Tesque, Ltd) in a round-bottom flask. A thin film was formed on the interior of the flask under reduced pressure while using a rotary. Thereafter, MDP dissolved in distilled water was dispersed on the film. After gentle shaking, MDP was entrapped in liposomes and the product was washed with phosphate-buffered saline (PBS) by centrifugation, then suspended in PBS, and extruded through a polycarbonate membrane (pore size of 0.8 µm, Millipore, Tokyo, Japan) to remove large liposome particles. Finally, the liposome-MDP was suspended in PBS at an approximate concentration of 150 mM MDP. Control liposome without MDP entrapment was also prepared.
Depletion of M
Rats were divided into three groups (six rats each). Two of the groups received intravenous (i.v.) 25 µl/100 g body weight of rabbit anti-GBM antibody [1], and one of these was then given liposome-MDP while the other was given control liposome (150 µmol/rat, i.v.) daily from day 3 to day 7 after the anti-GBM antibody injection. The third group was given 25 µl/100 g of normal rabbit serum followed by daily treatment with liposome-MDP from day 3 to day 7. Urine specimens were collected on days 3 and 8 by housing the animals in metabolic cages for 24 h. The amounts of protein excreted in the urine were determined using a protein assay kit (Bio-Rad Laboratories, Tokyo, Japan). All animals were sacrificed at day 8 to obtain the kidneys. Peripheral blood was collected on days 3 and 8 from these rats, and the number of leukocytes in the circulation was counted.
Histology and immunohistochemistry
A portion of each kidney obtained on day 8 was fixed in methyl-Carnoy's solution and embedded in paraffin. The paraffin-embedded tissues were sectioned at 4 µm thicknesses and stained with haematoxylin and periodic acidSchiff reagent. The numbers of glomeruli with crescent formation were counted by observing >50 glomeruli in each section and calculating the frequency (%) of crescents. We defined a cellular crescent as a lesion consisting of proliferating epithelial and inflammatory cells filling part or all of Bowman's space and that consist of at least two layers of cellular proliferation (definition 1) [9]. We also defined it as a proliferative lesion in the Bowman's spaces of >25% of the circumference (definition 2).
For immunohistochemistry, the paraffin-embedded sections were dewaxed and incubated sequentially with normal goat serum (1:20 dilution) for 20 min, with monoclonal antibodies against rat Mo/M (ED-1; Dainippon Seiyaku Co., Tokyo, Japan, 1:500 dilution) or CD8+ cells (MRC-OX8; PHLS Centre for Applied Microbiology and Research, Wiltshire, UK, 1:200 dilution) for 1 h, and with horseradish peroxidase-conjugated goat anti-mouse immunoglobulins (EnVision, Dako Japan Co., Kyoto, Japan) for 1 h. The peroxidase reaction product was visualized with 0.5 mg/ml of 3'-diaminobenzidine tetrahydrochloride0.01% hydrogen peroxide as a substrate. The nuclei of cells stained with these antibodies were counted in >50 glomerular cross-sections in each kidney under a light microscope.
We previously reported that the amount of CD8+ cells that accumulated in the glomeruli of this model reached a peak at day 3 (10.1±5.2/glomerular cross-section), whereas the amount of ED-1+ Mo/M increased steadily from 19.5±7.6 at day 3 to 36.8±11.4/glomerular cross-section at day 6 [1]. To examine whether ED-1+ cells infiltrating the glomeruli were Mo or M
at days 1 and 3, the expression of class A scavenger receptor was examined as a M
marker by using a monoclonal antibody (MSR-A: CD204, SRA-E5, Funakoshi Co., Ltd, Tokyo, Japan) [10]. The paraffin-embedded sections were dewaxed and heated for 10 min by microwave in 0.01 M sodium citrate buffer (pH 2.0) to retrieve antigens. Then, they were incubated with the antibody (10 µg/ml) to identify the scavenger receptor by immunohistochemistry as described above. To determine whether the glomerular CD8+ cells bear ED-1, serial sections of the kidneys obtained at day 8 were immunostained.
Blood samples were collected on days 3 and 8 to determine total leukocyte counts. To examine percentages of Mo/M and CD8+ cells in blood, smear samples were immunostained with anti-rat Mo/M
(ED-1) and CD8 antibodies. The data were then presented as a percentage of ED-1- or OX-8- positive cell counts/total nucleated cell counts on each blood smear.
Serum samples were collected on days 3 and 8 to examine serum creatinine and blood urea nitrogen (BUN) concentrations (mg/dl) using a modified Jaffe and urease-indophenol methods (Wako Pure Chemical Industries, Ltd, Osaka, Japan).
Glomerular expression of ICAM-1 and MCP-1 mRNA
Glomeruli were isolated individually from renal cortex by a standard sieving method that yielded a 8994% purity, and total cellular RNA was extracted from the glomeruli by a modified acidphenolguanidium thiocyanate method (TRIzol, Gibco-BRL, Grand Island, NY). The RNase protection assay was employed to detect glomerular dehydrogenase (GAPDH) mRNA expression of intercellular adhesion molecule 1 (ICAM-1), MCP-1 and glyceraldehyde-3-phosphate as described previously [2,3]. For quantitation of the expression of each mRNA, the autoradiography films were scanned in an optical scanner (JX-330M, Sharp, Osaka, Japan) and the density of each protected band was quantified by image analysis software (NIH image, National Institutes of Health, Bethesda, MD). The data were represented as the ratio of specific mRNA/GAPDH mRNA band density to ensure a constant quantity of mRNA in each sample.
Statistical analysis
The data were expressed as means±SD. Statistical significance was analysed by the MannWhitney U-test. Differences with P<0.05 were considered statistically significant. Correlations between the amount of urinary protein (mg/day) or percentage of crescentic glomeruli (%) and the number of glomerular ED-1+ cells or CD8+ cells were analysed by the Pearson's correlation coefficient method.
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Results |
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As shown in Figure 1, the amount of urinary protein correlated significantly with both the numbers of glomerular ED-1+ Mo/M (r = 0.965, P<0.0001) and CD8+ cells (r = 0.639, P<0.05). The percentage of crescentic glomeruli also correlated with the numbers of both glomerular ED-1+ cells (r = 0.894, P<0.0001) and CD8+ cells (r = 0.628, P<0.05). However, the number of glomerular ED-1+ Mo/M
was more strongly correlated with proteinuria and crescent formation, suggesting that Mo/M
may be involved in induction of glomerular injury and lesion.
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The number of ED-1+ Mo/M infiltrating to glomeruli was 32.1±1.2 cells/glomerular cross-section in the anti-GBM antibody-injected rats at day 8 (Figure 3), which was also significantly reduced to 1.4±0.3 cells/glomerular cross-section by the liposome-MDP treatment (P<0.01). This number was still significantly larger than that of rats given normal rabbit serum and the liposome-MDP (0.6±0.1 cells/glomerular cross-section, P<0.01).
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The liposome-MDP treatment did not affect the total number of leukocytes or the differential fractions of Mo and CD8+ cells in the circulation (total peripheral leukocyte counts were 8620±340/mm3 and 8200±320/mm3 in rats before and after administration of normal rabbit serum and the liposome-MDP, respectively). The leukocyte count was not changed at day 8 in rats given the anti-GBM antibody and liposome-MDP (8160±360/mm3). The percentages of ED-1+ Mo and CD8+ cells in the peripheral blood were 16.7±5.5 and 20.5±4.0%, respectively, before the experiment and were unchanged after the liposome-MDP injection (Mo, 14.8±4.2%, P>0.05; CD8+ cells, 23.2±6.2%, P>0.05).
Immunohistochemistry using serial sections was used to examine co-expression of ED-1+ and CD8+ cells. The immunodetected ED-1+ cells were localized at sites different from where CD8+ cells were present in the sequential serial sections. No distinct ED-1+ cells were demonstrated to be positive for CD8 (Figure 5).
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Discussion |
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In a previous study, we found that suppression of glomerular Mo/M infiltration by neutralization of MCP-1 using a specific anti-MCP-1 antibody reduced urinary protein excretion and reduced the development of crescent formation, indicating that M
may cause direct glomerular injury [2]. Since M
have been identified as a component of the cell population in crescentic lesions, these cells have been thought to induce crescent formation [12,13]. The present study provides the first evidence for an important role for M
in glomerular injury and crescent formation during anti-GBM GN in WKY rats.
Administration of liposome-MDP was introduced as an efficient and selective method to kill M in vitro and in vivo without affecting other leukocyte populations [48]. Treatment efficiency was confirmed in the present study by suppression of the accumulation of M
in glomeruli. Liposome-MDP has been shown to exert its cytotoxic effect on M
after engulfment. We previously demonstrated that phagocytosed liposome-MDP caused apoptosis of M
[6]. Following treatment with liposome-MDP, the number of ED-1+ cells accumulating in glomeruli decreased whereas the number of these cells in the circulation was unaffected, indicating that most of the glomerular ED-1+ cells were M
and that circulating ED-1+ cells were Mo. This was confirmed by using immunostaining of anti-M
scavenger receptor class A as a M
marker, which showed that only a small population of ED-1+ cells was positive for the marker at day 1, whereas most ED-1+ cells bore the marker at day 3. Circulating Mo appear to be attracted to the glomeruli where they are differentiated to M
by various stimuli such as cytokines [18]. These M
are able to engulf liposome-MDP in the glomeruli and then cause apoptosis. The present study showed that depletion of M
in the glomeruli suppressed glomerular crescent formation and reduced the amount of urinary protein, which strongly suggests that M
cause direct injury to glomerular structure and function. A crucial role for M
in tissue injury has also been demonstrated in other disease models such as in experimental allergic encephalomyelitis [4] and arthritis [5]. In these models, liposome-MDP-induced depletion of M
caused inhibition of tissue injury.
Interestingly, the number of CD8+ cells in the glomeruli was also reduced to near zero levels by liposome-MDP treatment. This contrasts with previous findings, which showed that liposome-MDP administration had no effect on recruitment of CD8+ cells [7]. However, CD8+ cell involvement may have been different since the previous experiment used a completely different disease model. Nevertheless, if CD8+ cells represent a subset of M, as was reported recently [19], a reduction in CD8+ cells caused by liposome-MDP is possible. However, the immunostaining using serial sections in the present study showed that none of the CD8+ cells accumulating in the glomeruli at day 8 definitely bore ED-1. Since liposome-MDP treatment did not reduce CD8+ cells in the circulation, it is possible that the reductions in glomerular CD8+ cells were not caused by direct cytotoxicity of liposome-MDP on CD8+ cells but indirectly by a reduction in glomerular M
accumulation. Although the glomerular M
are thought to play a role in the accumulation of CD8+ cells in glomeruli, we demonstrated that CD8+ cells regulated accumulation of M
in the glomeruli [1]. Taken together, these findings indicate that both M
and CD8+ cells stimulate or cooperate with each other and play an important role in the induction and progression of anti-GBM nephritis in WKY rats.
Clinical trials using liposome-MDP have been already been initiated in patients having several disease types [20]. Inhibition of M activity may be a useful therapeutic strategy in human GN characterized by glomerular M
accumulation.
In summary, we demonstrated that the liposome-MDP treatment caused almost complete suppression of M accumulating in the glomeruli from WKY rats with anti-GBM nephritis, and that this treatment concomitantly reduced urinary protein excretion and frequency of glomerular crescent formation. These results suggest that glomerular M
play a pivotal role in the development of glomerular injury in this model, presumably through direct effects of M
causing glomerular structure injury and through indirect effects that stimulate recruitment of CD8+ cells in the glomerulus.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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