Interleukin 12 induces crescentic glomerular lesions in a high IgA strain of ddY mice, independently of changes in IgA deposition

Fumiaki Nogaki1, Eri Muso1,, Ikei Kobayashi1, Hitoshi Kusano1, Kiichi Shirakawa1, Tadashi Kamata1, Atsushi Oyama1, Takahiko Ono1, Shigeki Miyawaki2, Haruyoshi Yoshida3 and Shigetake Sasayama1

1 Department of Cardiovascular Medicine, Kyoto University, Graduate School of Medicine, 2 Research Laboratories, Nippon Shinyaku Co. Ltd, Kyoto and 3 Division of Nephrology, Medical Research Institute, Kitano Hospital, Osaka, Japan



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Our recently established high immunoglobulin (Ig)A inbred strain (HIGA) of ddY mice showed constantly high serum IgA levels, progressive mesangial sclerosis accompanied by IgA deposits, and elevated renal expression of transforming growth factor (TGF)-ß, mimicking IgA nephropathy. In the present study, we assessed the role of the immune system, especially of T cells, in this strain.

Methods. The in vitro production of interferon (IFN)-{gamma}, interleukin (IL)-4 and TGF-ß1 by splenic CD4+ T cells was assessed in HIGA mice at 14 and 28 weeks of age by comparison with age-matched C57BL/6 and BALB/c mice, T-helper (Th) 1, and Th2 prone controls respectively. Moreover, recombinant murine IL-12 was administered intraperitoneally to HIGA mice and serum IgA and renal lesions were analysed.

Results. The production of IFN-{gamma} by splenic CD4+ T cells was markedly upregulated in HIGA mice at both ages as compared with age-matched C57BL/6 and BALB/c mice. Although splenic CD4+ T cells from HIGA mice produced less IL-4 than those from BALB/c mice at both ages, the former produced significantly more IL-4 with age, which contrasted with the age-associated decrease in the latter. Moreover, TGF-ß1 production of these cells in HIGA mice was equal to or greater than that in the two groups of control mice at both ages. Daily intraperitoneal administration of IL-12 for 1 week significantly enhanced crescent formation with glomerular macrophage accumulation and interstitial cell infiltration, whereas it reduced the serum IgA level.

Conclusions. In HIGA mice, Th1 is markedly upregulated from a young age and there is an age-associated Th2 increase with TGF-ß1 upregulation in helper T cells. The former may be related to the exacerbation of inflammatory renal lesions on IL-12 administration, while the latter may contribute to increased IgA production, leading to glomerular IgA deposition and progressive glomerulosclerosis in HIGA mice. The pathogenic role of T cell function and fluctuation of these subsets, especially the Th1/Th2 balance, is crucial to the immunopathological phenotype of the renal lesions in HIGA mice.

Keywords: crescent formation; high IgA strain of ddY mice; interleukin-12; interstitial cell infiltration; macrophages; T helper cells



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Human immunoglobulin (Ig)A nephropathy is the most common chronic glomerulonephritis, exhibiting mesangial IgA deposition with a wide variety of histological features [1]. Although this glomerulonephritis is relatively benign and the glomerular lesions, especially mesangial lesions, advance slowly, acute exacerbation associated with cellular crescent formation can occur and result in massive glomerular sclerosis, which leads to the rapid decline of renal function. As to the immunoregulatory background, specific T cell abnormalities that promote the frequently observed elevated serum IgA levels in these patients have been reported, as well as B cell abnormalities. For example, in a human in vitro study, T cells bearing receptors for the Fc portion of IgA have been reported to increase in number and to enhance the switch of IgM-positive B cells to those positive for IgA [2]. However, reports on the expression of the cytokines related to IgA production in this disease are somewhat controversial. Lai et al. [3] have reported that in IgA nephropathy patients, the expression of interleukin (IL)-2, interferon (IFN)-{gamma}, IL-4, IL-5 and transforming growth factor (TGF)-ß in CD4+ T cells was increased, whereas another group reported that the expression of IL-4, IL-5 and IL-6, but not IL-2 or IFN-{gamma} was enhanced in peripheral blood mononuclear cells (PBMC) [4]. For the analysis of the immunomodulatory mechanism that promotes the various clinicopathological features, an appropriate model of this chronically progressive disease has been needed. Recently, we established an inbred murine model of IgA nephropathy, a high IgA strain (HIGA) of ddY mice [5], by selective mating of pooled crude ddY mice which have been reported to spontaneously develop mesangioproliferative glomerulonephritis with glomerular IgA deposition [6]. HIGA mice showed constantly high serum levels of IgA from the age of 10–60 weeks with polymeric IgA dominant mesangial deposition and enhanced extracellular matrix (ECM) accumulation. Flow cytometric analyses of T and B cells among splenic mononuclear cells from these mice showed a significantly higher ratio of surface IgA positive (sIgA+) B cells and a higher expression of TGF-ß mRNA in B cell-depleted splenocytes [7].

IFN-{gamma} is the major cytokine secreted from T-helper (Th) 1 cells and promotes cellular immunity [8], whereas IL-4, which is secreted mainly from Th2 cells [9], is known to increase the population of B cells and facilitate IgA production [10], and to promote humoral immunity. TGF-ß1, a fibrogenic cytokine, has also been reported to be able to induce IgA production by the selective switch of sIgM+ to sIgA+ B cells [11], and helper T cells producing TGF-ß1 have been recently referred to as Th3 cells, which have been demonstrated to be important for immunoregulation [12]. Considering the significantly high serum IgA levels accompanying the marked expansion of matrix protein production with enhanced renal and splenic cell TGF-ß expression, a relatively important role for Th2 or Th3 cells in these lesions was hypothesized in this strain of mice.

Concerning the responsiveness of Th1 and Th2 cells, genetic determination has been demonstrated in murine strains, among which C57BL/6 and BALB/c mice have been known to develop Th1-and Th2-predominant immune responses respectively, on specific antigen stimulation [13]. In the present study, to disclose the relation between T-cell function and the renal lesions of HIGA mice, we assessed the productivity of IFN-{gamma}, IL-4, and TGF-ß1 from CD4+ splenic T cells of young and adult HIGA mice in comparison with age-matched C57BL/6 and BALB/c mice as Th1- and Th2-prone controls respectively. Moreover, to verify the possibility that the fluctuation of the Th1/Th2 balance affects the lesions of HIGA mice, the administration of IL-12, a cytokine that induces Th1-type responses and promotes cellular immunity [14], to adult mice was performed and its effects on pre-existing serological values and renal lesions were investigated.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Mice
HIGA mice were established by selective mating of ddY mice as described in detail previously [15]. Briefly, 52 female and male ddY mice of an outbred strain obtained at 2 months of age (Japan SLC, Inc., Shizuoka, Japan) were bred as the wild colony (generation 0, G0) and maintained at the Animal Laboratories of Nippon Shinyaku Co., Ltd (Kyoto, Japan). Six single-pairs with high serum IgA levels were selected for mating at an age of 4 months. The mice of the first offspring generation (G1) had higher serum IgA levels than those of G0. Similarly, an average of six single pairs from each generation were mated. At G15, mice were sterilized and sister-brother mating was continued until G20 with verification of the maintenance of high IgA. These mice were designated as the HIGA mice. C57BL/6 and BALB/c mice were obtained from Japan SLC as Th1- and Th2-prone control mice respectively.

Isolation of splenic CD4+ T cells
The analysis of immunopathological background was performed in groups of male HIGA mice at 14 and 28 weeks of age, which was before and after the expression of an apparent renal lesion respectively [5]. To assess the production of IFN-{gamma}, IL-4, and TGF-ß1 by splenic CD4+ T cells, spleens were removed from sacrificed male HIGA mice (14 weeks of age, n=10; 28 weeks of age, n=7), C57BL/6 mice as Th1-prone control (n=5, n=8 respectively) and BALB/c mice as Th2-prone control (n=5, n=6 respectively). Spleen cell suspensions were prepared by compression with the handle of a syringe in RPMI-1640 1% fetal calf serum (FCS) media followed by passage through a 100-µm nylon mesh. Red blood cells were lysed by ACK-lysing buffer (0.15 mol/l NH4Cl, 1.0 mmol/l KHCO3, 0.1 mmol/l Na2EDTA, pH 7.2) for 5 min at room temperature, and washed in RPMI-1640 1% FCS media. Then the isolation of CD4+ T cells was performed using magnetized beads coated with monoclonal antibody specific for mouse CD4 (Dynal, Oslo, Norway) following the manufacturer's protocol. Briefly, the magnetized beads were applied to splenic cell suspensions at 4°C for 20 min with gentle rocking and bead-bound CD4+ T cells were recalled using a magnetic separator (Dynal). Then a solution containing a polyclonal antibody that reacts with the Fab-fragments of the monoclonal antibody (Dynal) was added to the isolated cell suspensions at room temperature for 45 min and detached CD4+ T cells were obtained after removing the magnetized beads using the magnetic separator. The purity of CD4+ T cells was at least 90% as assessed by flow cytometry.

Measurement of cytokines produced by splenic CD4+ T cells in vitro
Splenic CD4+ T cells (1x106 cells/ml) were cultured in RPMI-1640 10% FCS media supplemented with 10 µg/ml phytohaemagglutinin (PHA) and 5 ng/ml phorbol myristate acetate (PMA) for 48 h at 37°C, 5% CO2 in 24-well tissue culture plates (Iwaki Glass, Chiba, Japan). IFN-{gamma} in culture supernatants was measured by a sandwich ELISA [16]. Briefly, microtitration plates (Nunc, Roskilde, Denmark) were coated with monoclonal rat anti-mouse IFN-{gamma} antibody (Pharmingen, San Diego, CA, USA) and were blocked with bovine serum albumin (BSA). After incubation with the samples, biotinylated monoclonal rat anti-mouse IFN-{gamma} antibody (Pharmingen) and then peroxidase–avidin (Zymed Laboratories, South San Francisco, CA, USA) was applied. The colours of the plates were developed, using 2,2'-azino-di-(3-ethylbenzthiazoline sulphonate (6)) (Boehringer Mannheim Biochemica, Mannheim, Germany) and absorbance was measured using an ELISA reader. IL-4 in culture supernatants was measured using a mouse IL-4 ELISA kit (Amersham, Buckinghamshire, UK). Biologically active TGF-ß1 in culture supernatants acidified by hydrogen chloride (HCl) was measured using a human TGF-ß1 ELISA kit (Genzyme, Cambridge, MA, USA) proven to cross-react with murine TGF-ß1.

Protocol of IL-12 administration
Female HIGA mice, 31–33 weeks old, received daily intraperitoneal injections of recombinant murine IL-12 (a gift from Dr Stan Wolf, Genetics Institute, Cambridge, MA, USA) at doses of 100 ng/mouse (n=6) or 300 ng/mouse (n=6), or the same volumes of PBS as controls (n=5) for 1 week (days 0–6) and were sacrificed at day 7. Blood samples were obtained just before the IL-12 treatment (day 0) and at day 7. Then the effects of IL-12 administration on serum Ig, splenic CD4+ T cells, and renal lesions were examined.

Measurement of serum Ig by ELISA
Total IgA and IgG in the sera were measured by a modification of the sandwich ELISA described previously [17]. Briefly, microtitration plates (Corning, Cambridge, MA, USA) were coated with goat anti-mouse IgA (Cappel Laboratories, Cochranville, PA, USA) or IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) antibodies and were blocked with BSA. Then, after incubation with the serum samples, alkaline-phosphatase (ALP)-conjugated goat anti-mouse IgA antibody (Zymed Laboratories) or peroxidase-conjugated goat anti-mouse IgG antibody (Cappel Laboratories) was applied. The plates were washed, and developed using p-nitrophenyl phosphate (Sigma, St Louis, MO, USA) as an ALP substrate or ABTS (Boehringer Mannheim Biochemica) as a peroxidase substrate. Absorbance was measured using an ELISA reader. Serum IgG1 and IgG2a were determined by a modification of the sandwich ELISA described previously [18]. Briefly, microtitration plates were coated with monoclonal rat anti-mouse IgG1 (BIOSYS, Compiegne, France) or IgG2a (BIOSYS) antibodies and were blocked with BSA. After incubation with the samples, biotinylated monoclonal rat anti-mouse Ig {kappa} antibody (Technopharm, Cedex, France) and then peroxidase-avidin (Zymed Laboratories) was applied. The colours of the plates were developed, using ABTS substrate (Boehringer Mannheim Biochemica) and absorbance was measured using an ELISA reader. The IgG1:IgG2a ratio was applied to the estimation of the change of Th1 and Th2 by IL-12 administration.

Renal function and urinary examination
Serum creatinine was measured by the Jaffe method [19]. Urinary occult blood and protein were determined semi-quantitatively using Multisticks (Bayer-Sankyo, Tokyo, Japan) when the mice were sacrificed.

Light microscopic study of renal tissues
Renal specimens were fixed in Doubosque Brazil solution and embedded in paraffin. Cross-sections (2 µm) of the central portion of the kidneys, containing about 100 glomeruli, were stained with periodic acid schiff (PAS). Glomerular hypercellularity was assessed by counting the total cell number in five selected glomerular sections containing vascular poles and no crescents from each mouse and expressed as the cell number per 1000 µm2 by measurement on NIH Image (NIH, Bethesda, MD, USA). The percentages of glomeruli involved in crescent formation were determined. Interstitial cell infiltration was also evaluated from (0) to (4+) in the entire cross-section (involving 100 glomeruli), as follows: (0) no lesion of cell infiltration, (1+) single focus of lesion, (2+) two isolated foci, (3+) more than two isolated foci, and (4+) diffuse cell infiltration.

Immunofluorescence study
Immunofluorescence studies of IgA and IgG on frozen renal sections (2 µm) were performed as described [6]. TGF-ß1 staining was performed using FITC-labelled polyclonal rabbit anti-human TGF-ß1 antibody (Santa Cruz Biochemistry, Santa Cruz, CA, USA) proven to react with murine TGF-ß1[5]. The grades of deposition were evaluated quantitatively by measuring the intensity of the fluorescence in glomerular areas with Photoshop 4.0 (Adobe, San Jose, CA, USA) and graded from 0 to 255.

Immunohistochemical staining of macrophages and CD4+ T cells
The three-layer immunoperoxidase technique [20] was performed for macrophage staining. Briefly, 2-µm frozen sections were soaked in 3% hydrogen peroxide (H2O2) to inactivate endogenous peroxides and then incubated sequentially with monoclonal rat anti-mouse macrophage antibody (M1/70.15; BIOSYS), peroxidase conjugated rabbit anti- rat IgG antibody (BIOSYS) and peroxidase conjugated goat anti-rabbit IgG antibody (Leinco Technologies, St Louis, MO, USA). The colour was developed with 3,3-diaminobenzidine and sections were counterstained with haematoxylin. CD4+ T cells were identified using the Vectastain® avidin–biotin complex (ABC) kit (Vector Laboratories, Burlingame, CA, USA) using monoclonal rat anti-mouse CD4 antibody (GK1.5; Southern Biotechnology, Birmingham, AL, USA) for the primary antibody according to the manufacturer's protocol. Glomerular positive cells were counted in the entire cross-section and expressed as the average number of cells per glomerulus. Extraglomerular positive cells were counted in an area of the cross-section not including the glomerulus, and expressed as the average number of cells per area (cells/mm2). Areas were calculated using Luzex 3U (Nikon Ltd., Tokyo, Japan).

Northern blot analysis
Renal cortices were shaved under the naked eye. Total RNA was purified by the acid guanidium thiocyanate–phenol–chloroform method [5]. Ten micrograms of total RNA was fractionated in 0.66 mol/l formaldehyde–1% agarose gel and transferred to a nylon membrane (Biodyne PALL, Glen Cove, NY, USA) by capillary action with 10xSSC overnight. After UV baking, the blot was prehybridized for 2 h at 65°C in prehybridization solution (4xSSC, 5xDenhardt's, 50 mmol/l Tris, 10 mmol/l EDTA and 0.5% SDS) and then was hybridized overnight at 65°C in hybridization solution (4xSSC, 5xDenhardt's, 50 mmol/l Tris, 10 mmol/l EDTA, 0.5% SDS, 100 mg/ml of salmon sperm DNA, 100 mg/ml of poly A (Boehringer Mannheim Biochemica) and 50% dextran sulphate containing [32P]cDNA probe labelled with a random primed DNA labelling kit (Amersham)). cDNA probes used were murine TGF-ß (a gift from Dr C. Shiota of Wakayama Medical College, Wakayama, Japan) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (ATCC, MD, USA). The blots was washed twice with 1xSSC, 0.5% SDS, for 15 min at 65°C and twice with 0.1xSSC, 0.5% SDS, for 15 min at 65°C. Each lane of the autoradiographs was analysed with BAS-2000 (Fujux, Tokyo, Japan). The signal densities of TGF-ß mRNA were corrected using that of GAPDH in each mouse.

Statistical analysis
The results are expressed as the mean±SEM. The Mann–Whitney U test was applied for comparisons with control mice or between younger and older mice in the immunological background study. In the study of IL-12 administration, the statistical significance of differences between groups was determined by Scheffe F test. Changes of serum Ig on IL-12 administration were analysed using Wilcoxon's signed rank test. P values <0.05 were taken as significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Immunoregulatory background of HIGA mice
The levels of serum IgA of HIGA mice were significantly higher than those of C57BL/6 and BALB/c mice at both 14 and 28 weeks of age, while all mice showed age-associated significant increases (Figure 1Go). As shown in Figure 2aGo, compared with the two control groups of mice, IFN-{gamma} production by splenic CD4+ T cells was greater in both young and adult HIGA mice. It should be noted that, especially at 14 weeks of age, HIGA mice produced significantly more IFN-{gamma} than C57BL/6 mice, which are known as Th1-prone mice possessing latently high levels of IFN-{gamma}. On the other hand, IL-4 production by CD4+ T cells of HIGA mice was lower than that of Th2-prone BALB/c mice at both ages; however, while an age-associated relative decrease was noted in BALB/c mice, a significant increase with age was observed in HIGA mice (Figure 2bGo). Referring to TGF-ß1 production by CD4+ T cells, HIGA mice tended to show an age-associated increase and at the age of 28 weeks, such production was higher than in the other two strains, significantly so compared with C57BL/6 mice (Figure 2cGo).



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Fig. 1. Serum IgA levels of C57BL/6, BALB/c and HIGA mice at 14 and 28 weeks of age. Symbols are: ({blacksquare}) C57BL/6; () BALB/c; () HIGA. The results are expressed as mean±SEM. *P<0.02 and **P<0.005. {dagger}P<0.05 compared to young mice.

 


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Fig. 2. Production of cytokines from splenic CD4+ T cells of C57BL/6, BALB/c and HIGA mice at 14 and 28 weeks of age. Isolated splenic CD4+ T cells were cultured in RPMI-1640 10% FCS media supplemented with 10 µg/ml PHA and 5 ng/ml PMA for 48 h and (a) IFN-{gamma}, (b) IL-4, and (c) TGF-ß1 in culture supernatants were measured by a sandwich ELISA. Symbols are: ({blacksquare}) C57BL/6; () BALB/c; () HIGA. The results are expressed as mean±SEM. *P<0.05, **P<0.01 and ***P<0.005. {dagger}P<0.05 and {dagger}{dagger}P<0.005 compared to young mice.

 

Effects of IL-12 administration on serum Ig levels and splenic CD4+ T cells
With a 1-week intraperitoneal administration of both a 100 and 300 ng/dose of IL-12 to adult HIGA mice, serum IgA levels decreased with statistical significance in the 300 ng/dose group (Figure 3Go). On the other hand, serum IgG levels in the group receiving the 300 ng/dose showed no significant change (639±105 mg/dl at day 0, 628±81 mg/dl at day 7). The IgG1:IgG2a ratio increased less in the groups receiving IL-12 than in the control group; however, the difference was not significant (Figure 4aGo). In vitro IFN-{gamma} and IL-4 production by splenic CD4+ T cells was downregulated by IL-12 administration in a dose-dependent manner (Figure 4bGo and cGo).



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Fig. 3. Effects of IL-12 administration on serum IgA levels in HIGA mice. IgA levels in the sera before (day 0) and after a 1-week administration of IL-12 (day 7) were measured. Symbols are: ({square}) 100 ng/dose; ({circ}) 300 ng/dose; (•) control. The results are expressed as the mean±SEM. *P<0.05 compared to the IgA level at day 0.

 


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Fig. 4. Effects of IL-12 administration on IgG1:IgG2a ratio and production of cytokines from splenic CD4+ T cells in HIGA mice. (a) IgG1:IgG2a ratio in the sera before (day 0) and after a 1-week administration of IL-12 (day 7) were calculated. Symbols are: ({square}) 100 ng/dose; ({circ}) 300 ng/dose; (•) control. Splenic CD4+ T cells isolated at day 7 were cultured in RPMI-1640 10% FCS media supplemented with 10 µg/ml PHA and 5 ng/ml PMA for 48 h, and (b) IFN-{gamma} and (c) IL-4 in culture supernatants were measured by a sandwich ELISA. The results are expressed as mean±SEM.

 

Renal function and urinary findings
IL-12 administration did not change the serum levels of creatinine (0.31±0.03 mg/dl in controls, 0.31±0.06 mg/dl with 100 ng of IL-12 and 0.33±0.02 mg/dl with 300 ng of IL-12). Before the IL-12 treatment, several mice already had mild proteinuria; however, there was no increase in either haematuria or proteinuria after 1 week of treatment (data not shown).

Renal histopathology
Microscopic findings showed prominent glomerular inflammatory lesions with cellular crescent formation in IL-12-treated mice (Figure 5aGo and bGo). Moderate glomerular hypercellularity and segmentally severe mesangial proliferation were seen in IL-12-treated mice (Figure 5bGo). Several severe crescents compressing the glomerular tuft were also observed (Figure 5aGo). In the interstitium, numerous foci of inflammatory cell infiltration appeared after IL-12 administration (Figure 5cGo). In mice treated with 300 ng of IL-12, the percentage of glomeruli involved in crescents significantly increased as compared with in the control mice (Figure 6aGo). Moreover, by quantitative analysis, glomerular cells, possibly including not only resident cells but also infiltrating leukocytes, significantly increased after administration of 300 ng of IL-12 (11.3±0.47 cells/1000 µm2 in controls, 12.1±0.42 cells/1000 µm2 with 100 ng of IL-12 and 13.5±0.40 cells/1000 µm2 with 300 ng of IL-12, P<0.05). Interstitial cell infiltration was significantly increased in IL-12-treated mice as determined by the semiquantitative analysis (Figure 6bGo).



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Fig. 5. Light microscopy of the inflammatory renal lesions of HIGA mice after IL-12 administration. (a) Crescent around Bowman's capsule, squashing the glomerular tuft (x400); (b) crescent accompanied by segmental mesangial proliferation (x400); (c) focus of interstitial cell infiltration (x200). PAS stain.

 


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Fig. 6. Effects of IL-12 administration on glomerular crescent formation and interstitial cell infiltration. (a) The percentages of glomeruli involved in crescent formation were counted in cross-sections (2 µm) of kidneys containing about 100 glomeruli; (b) interstitial cell infiltration was evaluated semi-quantitatively from (0) to (4+) in whole areas of cross-sections. The results are expressed as the mean±SEM. *P<0.05 and **P<0.005 compared to control.

 

Immunofluorescence findings
IL-12 administration tended to decrease glomerular IgA deposits, although a statistical significance was not obtained by quantitative analysis (17.0±3.7 in controls, 16.9±7.6 with 100 ng of IL-12 and 13.2±4.0 with 300 ng of IL-12). The changes of IgG deposition did not show any definite tendency after IL-12 administration (15.9±4.4, 21.8±1.1, 15.5±6.3 respectively). Apparent TGF-ß1 staining in mesangial areas did not show any significance of differences among groups (28.3±0.6, 28.6±0.1, 28.5±1.3 respectively).

Macrophage and CD4+ T cell infiltration in renal tissue
Using immunohistochemical staining, glomerular and interstitial infiltrating cells were identified. Glomerular macrophages increased in number dose dependently, with a significant increase in mice treated with 300 ng of IL-12 (Figures 7Go and 8aGo). Extraglomerular macrophages also tended to be increased by IL-12 administration (Figure 8bGo). Similarly, immunohistochemical studies revealed that glomerular CD4+T cells were increased dose dependently in IL-12 treated mice (Figure 8cGo). Although there was no significant difference between IL-12-treated and control mice with regard to extraglomerular CD4+ T cell infiltration (Figure 8dGo), foci of the accumulation of CD4+ T cells were scattered in the interstitium in two of 6 mice treated with 300 ng of IL-12.



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Fig. 7. Immunohistochemical staining of macrophages in renal tissue. The three-layer immunoperoxidase technique demonstrated infiltrating macrophages (arrows) in glomeruli (x400).

 


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Fig. 8. Effects of IL-12 administration on macrophage and CD4+ T cell infiltration in renal tissue. (a) Glomerular macrophages and (c) CD4+ T cells were counted in the whole cross-section and expressed as an average number of cells per single glomerular cross-section (cells/gcs). (b) Extraglomerular macrophages and (d) CD4+ T cells were counted in the area of the cross-section with the exception of glomerulus and were expressed as an average number of cells per area (cells/mm2). The results are expressed as the mean±SEM. *P<0.05 compared to controls.

 

Change of TGF-ß mRNA expression in renal cortices
Northern analysis revealed a dose-dependent increase of TGF-ß mRNA expression in renal cortices in IL-12 treated mice with a significant increase in the 300 ng-treated mice as compared with the control (Figure 9aGo and bGo).



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Fig. 9. Effect of IL-12 administration on TGF-ß mRNA levels in renal cortices. (a) Northern blot analysis for TGF-ß in renal cortices obtained from control and IL-12 treated mice was performed. Each lane shows a representative mouse. Corresponding GAPDH mRNA for each mouse is shown below the TGF-ß figure. (b) TGF-ß : GAPDH densitometric ratios were calculated for each group of mice. The results are expressed as mean±SEM. *P<0.005 compared to controls.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In the present study, we showed that in HIGA mice, a newly established inbred strain of ddY mice, Th1 is markedly upregulated from a young age and Th2 increases with age, accompanied by TGF-ß1 upregulation in helper T cells. The administration of IL-12 induced an active and crescentic inflammatory change of pre-existing renal lesions of HIGA mice. The modification of inflammatory renal lesions was accompanied by a marked infiltration of macrophages in association with a significant decrease of serum IgA levels. TGF-ß expression in renal tissue, which had been consistently upregulated in this mouse, showed a marked augmentation after IL-12 administration.

Although glomerular hypercellularity and crescent formation were enhanced by IL-12 administration, there was no apparent deterioration of urinary findings such as haematuria or proteinuria nor significant decrease of renal function possibly because of the relatively mild lesions with limited numbers of glomeruli involved in crescents (Figure 6aGo). This result may be due to the relatively short duration of IL-12 stimulation, which may not be sufficient to induce massive urinary lesions.

To analyse the immunological background of HIGA mice, ideally an inbred strain of ddY mice with a low IgA level and absence of renal lesions should be used as control mice. However, the establishment of such an inbred strain has not yet been achieved. The use of original ddY mice was thought to be inappropriate because these mice are an outbred strain with significant individual differences in genetic and immunological backgrounds. Instead, we used C57BL/6 and BALB/c as the control, because they are respectively typical Th1- and Th2-prone mice, without any renal lesions like HIGA mice.

Glomerular crescent formation has been reported to be associated with acceleration of cellular immunity in both human and murine experimental crescentic glomerulonephritis. In animal models, Th1 immune response and Th1-mediated delayed type hypersensitivity (DTH) have been demonstrated to be essential for crescent formation in experimental anti-GBM nephritis [21] and, moreover, treatment with IL-4 and IL-10, which are Th2 cytokines, attenuated crescentic glomerulonephritis accompanied by a decrease of glomerular infiltrating CD4+ T cells and macrophages in this model [22]. In contrast to C57BL/6 and BALB/c mice, in HIGA mice an age-associated significant increase of IL-4 production by splenic CD4+ T cells was noted in concordance with the significant, and also, age-associated elevation of serum IgA. As previously reported, in the process of selective mating of high IgA ddY mice it has been noted that the active inflammatory renal lesions with high cellularity in mesangium and a high incidence of cellular crescent formation of original mice become attenuated [5]. These results indicate that in HIGA mice an age-associated enhancement of Th2-cell activation may contribute to the modification of an active renal lesion to a relatively inactive lesion with the disappearance of crescent formation.

In HIGA mice, in addition to IL-4, TGF-ß1 derived from splenic CD4+ T cells was upregulated with age. This contrasts to the very little change or relative decrease in C57BL/6 and BALB/c mice. As IL-4 promotes the proliferation of B cells [10] and TGF-ß1 induces a selective class-switch to IgA-secreting B cells [11], the upregulation of these cytokines may contribute to the specific elevation of serum IgA, which is the essential serological characteristic of this murine model of IgA nephropathy. On the other hand, the present study demonstrated a significant decrease of serum IgA levels on intraperitoneal administration of recombinant IL-12 for a week in HIGA mice. In the synthesis of IgA, IL-12 has been reported to prevent the production of IgA antibodies in response to recombinant adenovirus [23] and to reduce antigen-specific faecal IgA in mice immunized orally with tetanus toxoid and cholera toxin [24]. In HIGA mice, age-associated clonal expansion of IgA-producing B cells was demonstrated [5,25]. The decrease of serum IgA levels on IL-12 treatment in HIGA mice may be attributed to the downregulation of this clonal expansion in addition to the reduced IL-4 and IL-5 production by Th2 cells due to the Th1 shift by IL-12.

In the present study, IL-12 administration to HIGA mice resulted in the development of crescent formation with a marked glomerular infiltration of macrophages, and to a lesser extent, CD4+ T cells. These results indicate that IL-12 administration accelerated the cellular immune reaction in glomeruli of HIGA mice due to the induction of Th1. However, changes in the IgG1:IgG2a ratio in sera and IFN-{gamma} and IL-4 production by splenic CD4+ T cells unexpectedly did not show the findings of Th1 shift upon IL-12 administration (Figure 4a–cGo). For the purpose of detecting a Th1/Th2 shift in experiments using cytokines such as IL-12 or IL-4, the measurement of antigen-specific IgG subclass antibodies or IFN-{gamma} and IL-4 from CD4+ T cells stimulated by a specific antigen in vitro might be better [24]. Although we could not detect an antigen-specific Th1/Th2 shift as the pathological antigen in HIGA mice is not known, it is possible to suggest that the local Th1 shift has occurred in the present study.

As a local factor to upregulate the macrophage infiltration in glomeruli, the specifically severe mesangial IgA deposition in HIGA mice should be considered. Duque et al. [26] reported that IgA molecule binding to human mesangial cells induces autocrine expression and synthesis of monocyte chemoattractant protein-1, IL-8, and IFN-inducible protein 10. However, in the present study, glomerular inflammatory cells increased significantly in spite of the relative decrease of IgA deposition in IL-12-treated HIGA mice. This result may indicate that not only IgA deposition but also other factors may contribute to the accumulation of inflammatory cells in these mice. Indeed, IL-12 treatment induced glomerular accumulation of T cells and macrophages in younger HIGA mice (10 weeks old) in which IgA deposition was not yet established [27]. On the other hand, the highly upregulated IFN-{gamma} productivity of splenic CD4+ T cells was demonstrated in the present immunoregulatory background study of young and old HIGA mice (Figure 2aGo). These results highly suggest that the role of peculiar T cells with high IFN-{gamma} productivity rather than the stimulation of IgA deposition is inevitable for the exacerbation of active inflammatory lesions of HIGA mice in response to IL-12.

IL-12 induced numerous foci of interstitial cell infiltration, which consisted of macrophages and T cells in HIGA mice. This finding may partly be the result of the deterioration of glomerular lesions on IL-12 administration; however, the possibility that IL-12 directly induced local cellular immunity, like that in glomeruli, could not be excluded. Considering that interstitial nephritis has occurred in patients with chronic HCV infection on administration of IFN-{alpha}, which is known to mimic IL-12 in inducing Th1 cells [28], that interstitial inflammatory lesions were induced by IL-12 without any exogenous antigen stimulation may indicate that this mouse is constantly under some priming condition of not only immunocytes but resident cells, including renal epithelial cells or interstitial fibroblasts.

Although the immunofluorescence study could not detect apparent upregulation of TGF-ß staining either in glomeruli or in interstitium, mRNA levels of TGF-ß in the cortices were significantly increased by IL-12 administration. This discrepancy may be caused in part by upregulated TGF-ß mRNA expression derived from infiltrating inflammatory cells. Taking into account the spontaneously enhanced renal expression of TGF-ß mRNA in HIGA mice from a young age [5], the contribution of genetically elevated inducibility of TGF-ß, the production of which has recently been reported to be positively regulated by TGF-ß itself [29], was also suggested to contribute to the highly expressed local TGF-ß mRNA in both glomeruli and interstitium after IL-12 treatment. These phenomena may explain the mechanism of the progressive renal matrix expansion after induction of inflammatory lesions as seen in human IgA nephropathy.

In conclusion, IL-12 administration promptly induced acute inflammatory renal lesions, including crescent formation and interstitial cell infiltration, in HIGA mice, a murine model of IgA nephropathy prone to develop high serum IgA and glomerulosclerosis. The pathogenic role of T-cell function and fluctuation of these subsets, especially the Th1:Th2 balance, is crucial to the immunopathological phenotype of the renal lesions in this mouse.



   Acknowledgments
 
This work was partly supported by a grant-in-aid for General Scientific Research from the Japanese Ministry of Education, Science and Culture. We are deeply indebted to Dr Stanley F. Wolf and Dr Meg O'Donnell, Genetics Institute, Cambridge, MA, USA, for the gift of recombinant murine IL-12, and for extremely helpful suggestions; to Dr C. Shiota, Wakayama Medical College, Wakayama, Japan, for providing cDNA probes and to Dr H. Yoshifusa, Research Laboratories, Nippon Shinyaku Co., Ltd., Kyoto, Japan, for assistance in the bleeding of mice.



   Notes
 
Correspondence and offprint requests to: Eri Muso MD, Department of Cardiovascular Medicine, Kyoto University, Graduate School of Medicine, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606–8397, Japan. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 25. 6.99
Revision received 16. 3.00.