Institute of Anatomy, University of Zurich, Zurich, Switzerland
Correspondence and offprint requests to: Michel Le Hir, Institute of Anatomy, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Email: lehir{at}anatom.unizh.ch
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Abstract |
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Methods. BALB/c mice were immunized with rabbit IgG in incomplete Freund's adjuvant. Six days later, on day 0, they received rabbit anti-GBM serum intravenously. Proteinuria was assessed with dipsticks. Mice were killed on days 4, 8 or 14. Kidneys from days 4 and 8 were processed for immunofluorescence and histology. On day 14 mice were perfusion-fixed for electron microscopy.
Results. Proteinuria started on day 3. Autologous IgG and of C3 were found along the GBM. There was only slight infiltration with macrophages and no measurable infiltration by CD4 T cells, indicating the virtual absence of DTH. Besides infiltration with neutrophils there were little histological alterations on day 4. On day 8 many loops were hyalinized. On day 14, cellular crescents were found in 23% of glomeruli. Subendothelial spaces contained hyaline material, cells and fibrin. Podocytes displayed effacement of foot processes and apical microprotrusions. Podocyte bridges were common. These alterations were identical to those reported in the standard model that produces a DTH-like inflammation.
Conclusion. The qualitative pattern of histological damage in a murine model of anti-GBM glomerulonephritis does not depend on the underlying immunological process.
Keywords: crescentic; glomerulonephritis; histology; mouse
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Introduction |
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In humans anti-glomerular basement membrane (anti-GBM) glomerulonephritis is an autoimmune disease. The following findings suggest a major role of humoral mediators of immunity. Immunoglobulins and C3 are found in a linear pattern along the GBM [2,3]. Antibodies eluted from the kidneys of patients induce glomerulonephritis in squirrel monkeys [4]. Transfer of the disease by antibodies has also been achieved in a murine model [5]. However, infiltration of glomeruli with T helper cells and macrophages in human anti-GBM glomerulonephritis [6] suggest a role of cellular immunity and, more specifically, of delayed-type hypersensitivity (DTH). Furthermore, the severity of anti-GBM glomerulonephritis in rats and mice immunized against the alpha-3 chain of type IV collagen, a major antigen in the human disease [2,3], correlates better with the cellular immune response than with the humoral immune response [3,7]. It is thus likely that, depending on genetic factors and on environmental factors anti-GBM glomerulonephritis is driven by a cellular immune response, by a humoral immune response or by both.
The histopathology of glomerulonephritis driven by cellular immunity has been described previously in a murine model of anti-GBM glomerulonephritis [810]. In that model C57BL/6 mice receive an i.v. injection of anti-GBM serum a few days after immunization against the immunoglobulins of the donor species. Complete Freund's adjuvant (CFA) is used for immunization. That protocol yields a virtually pure DTH-type inflammation in the glomerulus [10,11]. Infiltration by macrophages and T-cells, the characteristic cellular mediators in DTH, is followed by intracapillary alterations and by the rapid development of cellular crescents [810].
The present study examined the pattern of histological lesions in anti-GBM glomerulonephritis under conditions where inflammation is initiated by humoral mediators of immunity. The aim was to compare the findings with those of the previous studies describing DTH-mediated anti-GBM glomerulonephritis in mice [810] and thus to discover glomerular alterations that were specific for cell-mediated inflammation or humorally mediated inflammation. BALB/c mice were used because in that strain, in contrast to the C57BL/6 strain, glomerulonephritis relies essentially on a humoral immune response when induced by the standard protocol outlined above [12]. In order to reduce further the influence of DTH in glomerular inflammation we immunized the mice with incomplete Freund's adjuvant (IFA) instead of CFA. As expected, with the modified protocol glomerulonephritis developed without measurable infiltration with CD4+ T cells and there was only slight infiltration with macrophages. Still, histopathology revealed qualitatively similar features as in the classical model of DTH-type glomerular inflammation in C57BL/6 mice immunized in the presence of CFA.
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Subjects and methods |
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Cutaneous test for DTH
Six female BALB/c mice were immunized with rabbit IgG as described above. Ten days after immunization 20 µl of 1 mg/ml rabbit IgG or of 1 mg/ml bovine serum albumin were injected in the right and left footpad, respectively. The difference of thickness between the two feet 24 h after injection of antigen was taken as measure of DTH.
Proteinuria
Micturition was evoked by holding the skin of the neck in a firm grip. Protein concentration in urine was assessed using dip sticks (Albustix, Miles Inc., USA). Values of 0 (traces), 0.3, 1, 3 or 20 mg/ml were attributed according to the colour scale provided by the producer.
Histology of immersion-fixed tissue
Mice were killed by inhalation of diethylether on days 4 or 8. The right kidney was immersion-fixed in 4% buffered paraformaldehyde (PFA) and processed for paraffin sectioning. Three slices from each kidney were embedded. Three micrometre thick sections were stained with the periodic acid-Schiff reagent (PAS), followed by haematoxilin. At least 50 glomeruli were evaluated per kidney for the presence of crescents.
Immunofluorescence
The left kidney was snap-frozen in isopentane cooled by liquid nitrogen. Six-micrometre thick sections were cut in a cryostat, air-dried and stored at 20°C. Fixation in acetone (10 min at 4°C) was performed just before the immunolabelling. After a rinse in Tris-buffered saline the sections were incubated for 16 h at 4°C with the primary antibodies diluted in TBS. The following rat anti-mouse monoclonal antibodies were used: anti-CD3 (clone KT3), anti-CD4 (clone YTS 191.1.2), anti-granulocytes (clone RB6-8C5) and anti-MHC class II (clone M5/114.15.2). The sections were then washed in TBS and incubated for 1 h at room temperature with a Cy3-labelled mouse anti-rat immunoglobulin antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA). After rinsing in TBS the sections were mounted in Immu-mount (Shandon, Pittsburgh, PA, USA). Immunoreactive cells and T helper cells were counted on cryosections in at least 50 glomerular cross-sections per mouse. Since the incidence of B cells identified with anti-B220 (clone RA36B2) was always <0.1 cell/glomerular cross-section, the majority of MHC class II-positive cells in glomeruli were probably macrophages.
Histology of perfusion-fixed tissue
On day 14, mice were anaesthetized with 17 mg/kg body weight xylazine hydrochloride and 50 mg/kg body weight ketamine hydrochloride, intraperitoneally. Kidneys were fixed by vascular perfusion via the abdominal aorta. The fixative consisted of 3% PFA, and 0.05 % picric acid. It was dissolved in a 3:2 mixture of 0.1 M cacodylate buffer (pH 7.4, adjusted to 300 mosm with sucrose) and 10% hydroxyethyl starch in saline (HAES; Fresenius AG, Germany). After 5 min of fixation in situ the kidneys were removed and cut into coronal slices. Some slices were embedded in paraffin. The remaining tissue was immersed for at least 24 h in the 3% PFA fixative solution, to which 0.5% glutardialdehyde was added. Thereafter, the tissue was post-fixed in 1% OsO4 and embedded in epoxy resin.
Light microscopical investigations were carried out on sections of 1 µm thickness, cut from epoxy resin-embedded tissue and stained with azur II-methylene blue.
For TEM, ultra-thin sections from epoxy resin-embedded tissue were contrasted with uranyl acetate and lead citrate.
Statistics
Unless otherwise specified each experimental group consisted of six mice. Mean values are given with the SEM. Variance analysis was performed using the ANOVA software, with a significance level of 5% in the Fischer PLSD test.
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Results |
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Mediators of inflammation
The aim of the next experiment was to examine the presence of inflammatory mediators in glomeruli. Twelve mice were immunized. Glomerulonephritis was induced in six of them by injection of anti-GBM serum whereas the other six were injected s.c. in the footpad with rabbit IgG for evaluation of the DTH response. The kidneys of the latter mice were used as controls in the evaluation of glomerulonephritis.
There was no measurable DTH response to rabbit IgG. Foot swelling was 0.04±0.02 mm. Using the same procedure we measured 1 mm swelling in C57BL/6 mice immunized with CFA [14].
In the anti-GBM-treated groups, proteinuria started on day 3 (Figure 1). The mice were killed on day 8 after injection of anti-GBM serum. Intraglomerular inflammatory cells were counted in immunolabelled cryosections (Table 1). There was a strong infiltration by neutrophile granulocytes and a small infiltration with macrophages (MHC class II-positive) on day 8. In glomerulonephritic mice the incidence of CD3+ cells was slightly higher than in controls but the incidence of CD4+ T cells was similar in both groups. Immunofluorescence revealed a linear deposition of mouse IgG and of C3 along the GBM (Figure 2).
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Histopathology
The kidneys of the mice used in the experiments described above were examined by histology on parafin sections (Figure 3). Endocapillary lesions on day 4 and on day 8 were evidenced mainly by increased amounts of PAS-positive material. This material was probably identical with the subendothelial hyaline proteinaceous material, which was seen in the electron microscope (see below). Capillary necrosis and hyalinization of loops was occasional on day 4 but frequent on day 8. There was no global glomerular hypercellularity, but on day 8 segmental hypercellularity was visible in some glomeruli. Elongated and segmented nuclei, which were found on day 4 and on day 8 in capillary loops represented probably neutrophile granulocytes. Crescents were found in 2.2±0.08% of glomeruli on day 8.
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Discussion |
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In order to evaluate the DTH response in glomeruli we counted T helper cells (CD4+) and macrophages (MHC II+). DTH reactions are characterized by their well-defined time course. The sensitization phase, which corresponds to the development of antigen-specific T helper cells, takes a few days after immunization. In the present study anti-GBM serum was injected 6 days after immunization against rabbit IgG. The specific effector phase, involving infiltration with T helper cells and macrophages, peaks between 1 and 3 days after antigen challenge and declines slowly thereafter. In the present study on days 4 and 8 after injection of anti-GBM serum there was no measurable infiltration with CD4 cells and there was little influx of macrophages in glomeruli. This contrasts with the large influx of these two cell populations in the standard model, in which C57BL/6 mice are immunized with CFA [10,16]. This is the first report of a model of anti-GBM glomerulonephritis in which there is no measurable infiltration with CD4 T cells. Humoral mediators of inflammation, autologous IgG and C3, were detected. Thus, the modified protocol yields a model for studying humorally mediated glomerulonephritis in the absence of a measurable DTH-like inflammatory response.
Surprisingly, all lesions observed in humorally mediated anti-GBM glomerulonephritis in the present study have been described previously in DTH-mediated anti-GBM glomerulonephritis in C57BL/6 mice immunized with CFA, and conversely. It has been proposed that a high incidence of cellular crescents might be an attribute of glomerulonephritis driven by DTH [10]. Although crescents developed more slowly in the present study than in various studies with C57BL/6 mice, 23.3% of glomeruli displayed crescents at day 14. The formation of cellular crescents following mesangiolysis in rats [21] also suggests that crescentic glomerulonephritis does not necessarily represent a DTH-type inflammation. Thus, crescents do not appear to be a safe criterion in order to discriminate between different types of inflammatory processes. This conclusion is in keeping with the large diversity of conditions that are associated with glomerular crescents in human pathology [1].
In animal models of glomerulonephritis quantitative differences have been found previously when comparing models in which the cellular immune response predominates and models in which the humoral immune response predominates. Whereas they contributed to the understanding of the pathophysiology of glomerulonephritis those observations are probably not very useful with respect to diagnostics in human pathology. Indeed, qualitative criteria are much more robust than quantitative criteria in histological diagnostics. In the present study we did not detect any specific histological feature, which might allow us discriminate between the two types of immune responses. This suggests that the glomerulus has only a very limited repertoire of structural adaptation to inflammatory insults. Similarly a variety of pathophysiological processes converge to the same pattern of histological lesions in focal segmental glomerulosclerosis [22].
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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