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
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Abstract |
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Methods. The in vitro production of interferon (IFN)-, 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- 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
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Introduction |
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IFN- 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-, 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.
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Subjects and methods |
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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-, 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- in culture supernatants was measured by a sandwich ELISA [16]. Briefly, microtitration plates (Nunc, Roskilde, Denmark) were coated with monoclonal rat anti-mouse IFN-
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-
antibody (Pharmingen) and then peroxidaseavidin (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, 3133 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 06) 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 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® avidinbiotin 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 thiocyanatephenolchloroform method [5]. Ten micrograms of total RNA was fractionated in 0.66 mol/l formaldehyde1% 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 MannWhitney 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.
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Results |
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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 3). 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 4a
). In vitro IFN-
and IL-4 production by splenic CD4+ T cells was downregulated by IL-12 administration in a dose-dependent manner (Figure 4b
and c
).
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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 5a and b
). Moderate glomerular hypercellularity and segmentally severe mesangial proliferation were seen in IL-12-treated mice (Figure 5b
). Several severe crescents compressing the glomerular tuft were also observed (Figure 5a
). In the interstitium, numerous foci of inflammatory cell infiltration appeared after IL-12 administration (Figure 5c
). 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 6a
). 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 6b
).
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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 7 and 8a
). Extraglomerular macrophages also tended to be increased by IL-12 administration (Figure 8b
). Similarly, immunohistochemical studies revealed that glomerular CD4+T cells were increased dose dependently in IL-12 treated mice (Figure 8c
). Although there was no significant difference between IL-12-treated and control mice with regard to extraglomerular CD4+ T cell infiltration (Figure 8d
), 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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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 9a and b
).
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Discussion |
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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 6a). 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- and IL-4 production by splenic CD4+ T cells unexpectedly did not show the findings of Th1 shift upon IL-12 administration (Figure 4ac
). 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-
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- productivity of splenic CD4+ T cells was demonstrated in the present immunoregulatory background study of young and old HIGA mice (Figure 2a
). These results highly suggest that the role of peculiar T cells with high IFN-
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-, 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.
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Acknowledgments |
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Notes |
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References |
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