Anti-macrophage migration inhibitory factor reduces transforming growth factor-ß1 expression in experimental IgA nephropathy
Joseph C. K. Leung,
Loretta Y. Y. Chan,
Anita W. L. Tsang,
Emily W. L. Liu,
Man Fai Lam,
Sydney C. W. Tang and
Kar Neng Lai
Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong
Correspondence and offprint requests to: Professor K. N. Lai, Department of Medicine, Room 411, Professorial Block, Queen Mary Hospital, The University of Hong Kong, Hong Kong. Email: knlai{at}hkucc.hku.hk
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Abstract
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Background. In human glomerulonephritis, including immunoglobulin-A nephropathy (IgAN), glomerular expression of macrophage migration inhibitory factor (MIF) is found to correlate with progressive renal injury. We have shown previously that polymeric IgA is capable of inducing MIF production in cultured human mesangial cells, suggesting a role in inducing inflammatory injury in IgAN. Herein, we examined whether IgA deposition and the subsequent renal injury can be ameliorated with anti-MIF treatment in an experimental murine model of IgAN.
Methods. Glomerular IgA deposition was induced in 4-week-old BALB/c mice by intravenous injection of immune complexes consisting of dinitrophenyl-conjugated bovine serum albumin (DNPBSA) and IgA MOPC-315 myeloma anti-DNP antibodies. To determine the therapeutic effect of anti-MIF, mice were given anti-MIF (5 mg/kg) or isotypic control antibody intravenously 2 h before the immune complexes administration. The mice were sacrificed 48 h after injection of DNPIgA. Proteinuria and haematuria were determined and the kidneys were removed for histopathology, immunostaining and immunoblotting. The effect of exogenous MIF on production of TGF-ß1 by cultured mesangial cells was also examined.
Results. IgA deposits were detected in glomeruli of all mice receiving the immune complexes while no glomerular deposit was detected in the control mice. Microscopic haematuria and mesangial hypercellularity were present in mice of the three experimental groups and were absent in the control group. Proteinuria was absent in all groups. Anti-MIF treatment also resulted in decreased renal expression of TGF-ß1. Moreover, the reduction in TGF-ß1 expression was confined mainly to glomerular mesangium. An in vitro culture experiment demonstrated that MIF increased TGF-ß1 production in a time- and dose-dependent fashion. MIF-induced TGF-ß1 synthesis was abolished by incubating cells with neutralizing antibody against MIF.
Conclusions. Our finding shows that anti-MIF treatment can ameliorate kidney injury and reduce glomerular TGF-ß1 expression in an experimental model of IgAN.
Keywords: animal model; IgA nephropathy; immunoglobulin A; macrophage migration inhibitory factor; mice; transforming growth factor-ß
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Introduction
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Immunoglobulin-A nephropathy (IgAN) is the commonest primary glomerulonephritis worldwide. Progressive glomerular injury is a striking feature of active IgAN [1]. The presence of polymeric IgA (pIgA) or IgA immune complexes in the circulation and in the glomerular mesangium strongly suggest a pathogenic role of IgA in IgAN. However, whether the pIgA is pathogenic and by what mechanism(s) pIgA affects the disease progression remain unclear. Macrophage migration inhibitory factor (MIF) is a cytokine that shares many activities with other pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumour necrosis factor-
(TNF-
) [2]. These cytokines play a pivotal role in mediating renal injury in experimental nephritis, including mesangial proliferative nephritis and anti-glomerular basement membrane antibodies-induced glomerulonephritis. In human glomerulonephritis (including IgAN), the up-regulation of MIF in glomerular mesangial cells and tubular epithelial cells correlates with progressive renal injury [3]. Recently, we have demonstrated that pIgA isolated from patients with IgAN is capable of inducing MIF production in cultured human mesangial cells [4]. Transforming growth factor-ß1 (TGF-ß1) belongs to a superfamily of multifunctional cytokines that participates in a wide range of biological events, including inflammation and wound repair [5]. TGF-ß1 induces synthesis and accumulation of extracellular matrix protein and has been implicated as the potent and key mediator of fibrogenesis. Increased renal TGF-ß1 expression occurs in human IgAN and experimental mesangial proliferative glomerulonephritis [6,7]. TGF-ß1 antagonist prevents the development of glomerular sclerosis in an experimental mesangial proliferative glomerulonephritis [8]. In the present study, we examined our hypothesis that mesangial deposition of pIgA induces glomerular injury through the local up-regulation of MIF and TGF-ß1. The potential therapeutic intervention by a neutralizing anti-MIF antibody was tested in an experimental model of IgAN.
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Subjects and methods
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Antibodies and reagents
The antibody III.D.9, a mouse anti-MIF monoclonal antibody (IgG1 subclass) that neutralizes the bioactivity of mouse MIF [3] and isotype-matched control antibody were gifts from Prof. Richard Bucala and Dr Christine Metz (Picower Institute for Medical Research, NY, USA). Rabbit anti-mouse TGF-ß1 antibody and blocking peptide to murine TGF-ß1 for immunoblotting and immunohistochemical staining were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-proliferating cell nuclear antigen (PCNA, clone PC-10) was obtained from Dako (Carpinteria, CA, USA). Anti-mouse pan macrophages (CD68) antibody was obtained from Serotec (Oxford, UK). All other chemicals were from Sigma (St Louis, MO, USA) unless stated otherwise.
Animals and treatment
All animal experiments were approved by the Committee on the Use of Live Animal in Teaching and Research of the University of Hong Kong and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Four-week-old BALB/c mice (1518 g) were used for establishing an animal model of IgAN. Glomerular deposition of IgA was induced by intravenous administration of dinitrophenyl-conjugated bovine serum albumin (DNPBSA) and IgA MOPC-315 myeloma anti-DNP antibodies (Calbiochem-Novabiochem, San Diego, CA, USA), as described previously [9]. Four groups of mice (eight in each group) were studied: three groups of mice received IgA anti-DNP (1 mg) and 0.5 mg BSADNP (0.5 mg) intravenously while the remaining group (the normal controls) received intravenous saline injection only. Two groups of mice receiving IgA anti-DNP and BSADNP (DNPIgA) complexes were given either anti-MIF (5 mg/kg) or control isotype antibody intravenously 2 h before the injection of DNPIgA complexes. The dose of anti-MIF used was determined by a pilot study, which showed maximal suppression of TGF-ß1 expression in kidney. All mice were sacrificed at 48 h after injection. Preliminary experiments were conducted showing mice sacrificed at 48 h after induction of IgA deposition was convenient and was associated with optimal synthesis of MIF or TGF-ß1. The kidneys were harvested, fixed and stored at 70°C before analysis.
Analysis of urine sample
Twenty-four hour urine samples were collected overnight in a metabolic cage before the mice were sacrificed. The urine samples were centrifuged at 600 g for 5 min. Erythrocyte number in 20 randomly selected high-power fields (magnification: x400) were counted. Microhaematuria was considered when the average count per field exceeded 10 red cells. The urinary protein concentration was measured by a Bio-Rad Protein Assay Kit using albumin as a standard (Bio-Rad Laboratories, Hercules, CA, USA).
Immunofluorescence detection of IgA deposit
Four-µm thick frozen kidney sections were air-dried and fixed in cold acetone for 10 min. After washing with phosphate-buffered saline (PBS), the sections were stained with fluorescein-conjugated rat anti-mouse IgA (BD Biosciences Pharmingen, Chicago, IL, USA). Immunofluorescence staining intensities of the kidney mesangium (15 glomeruli per coded section) were scored on a semi-quantitative scale from 0 to 4+ by two independent observers.
Histological and immunohistochemical examination
Four-micrometre thick paraffin-embedded kidney sections were deparaffinized with xylene and then rehydrated through a descending gradient of ethanol. The histological morphology was examined after staining with periodic acidSchiff (PAS). For determination of glomerular hypercellularity, 20 glomeruli free of artefact were selected and all nuclei within the glomerulus were counted. The result was expressed as the average number of nuclei per glomerulus. The expression of MIF-, TGF-ß1-, PCNA- and CD68-positive cells in the kidney was determined by immunohistochemical staining using specific antibodies. Briefly, the slides were incubated with 0.5% hydrogen peroxide for removal of endogenous peroxidase activity. Non-specific binding was blocked by incubation with blocking buffer (5% normal goat serum and 3% BSA in PBS) for 30 min. The sections were then incubated with anti-MIF (0.1 µg/ml) or anti-TGF-ß1 (0.5 µg/ml) overnight. The bound antibodies were visualized as brown colour using the Dako Envision Plus System (Dako). To ensure specificity of the staining, the following labelling controls were performed: (i) the primary antibodies were substituted with pre-immune rabbit immunoglobulin; (ii) staining was carried out without either the primary antibodies or the peroxidase-labelled polymer; and (iii) the primary antibodies were pre-incubated with 5 mg/ml blocking peptides. For double immunohistochemical staining of MIF and TGF-ß1 expression in the glomerulus, the section was first incubated with anti-TGF-ß1 and colour developed with 3,3-diaminobenzidine (brown colour), as described above. After colour development, the section was microwaved in 0.01 M sodium citrate buffer (pH 6) at 800 W for 5 min to denature bound antibodies and prevent antibody cross-reaction. The section was then stained with anti-MIF using a three-layer alkaline phosphataseanti-alkaline phosphatase method and developed with Fast Blue BB Base (blue colour).
Mesangial cell culture
Glomeruli were prepared from the cortex of 12-week-old mice by sieving and glomerular cells were grown in RPMI 1640 medium supplemented with glutamine (2 mmol/l), HEPES (10 mmol/l), penicillin (50 U/ml), streptomycin (50 µg/ml) and 20% fetal calf serum in an atmosphere of 5% CO2-95% air. Mesangial cells at fourth to tenth passages were used in our experiments.
RNA extraction and cDNA synthesis
Total RNA was extracted from cultured mesangial cells or a half kidney bisected transversely using the Totally RNA kit (Ambion Inc., Austin, TX, USA) as per the manufacturer's protocol. The RNA was dissolved in 50 µl DEPC-H2O and was stored at 70°C until assay. The quality of RNA was checked by formaldehyde agarose gel electrophoresis and quantified by absorbance at 260 nm. Five micrograms of total RNA was reverse transcribed to cDNA with the Superscript II reverse transcriptase (Life Technologies, Paisley, UK) in a 20 µl reaction mixture containing random 100 ng hexamer, 500 µM of each dNTP and 40 U RNase inhibitor for 10 min at 37°C, 60 min at 42°C and 5 min at 99°C. The cDNA was stored at 20°C until further use.
Gene expression of TGF-ß1 in kidney
Total cellular RNA was extracted and reverse transcribed to cDNA as described above. Specific primers for mouse MIF, TGF-ß1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed from known GenBank sequences (MIF: NM 010798; TGF-ß1: BC 013738; GAPDH: XM 132897). The sequences of the primers were as follows: (a) MIF sense primer 5'-CCAGAACCGCAACTACAG-3' and anti-sense primer 5'-CTCAAGCGAAGGTGGAAC-3'; (b) TGF-ß1 sense primer 5'-AACCGGCTGCTGACCCCCACTG-3' and anti-sense primer 5'-CGGGTTGTGTTGGTTGTAGAGG-3'; and (c) GAPDH sense primer 5'-ACCACAGTCCATGCCATCAC-3' and anti-sense primer 5'-TCCACCACCCTGTTGCTGTA-3'. The polymerase chain reaction (PCR) was carried out in the following profile: first cycle, 94°C for 3 min, 55°C for 1 min, 72°C for 1 min; second to 30th cycles, 95°C for 45 s, 55°C for 40 s, 72°C for 45 s. The final cycle was 94°C for 1 min and 72°C for 10 min. PCR amplicons were separated by 1.5% w/v agarose gels stained with ethidium bromide. The gel image was captured and analysed using the Gel Doc 1000 Gel Documentation System and Quantity One software (Bio-Rad Laboratories). The yield of MIF or TGF-ß1 mRNA was expressed as a ratio of MIF or TGF-ß1 amplicon to GAPDH amplicon. Precautions were taken to ensure the validity of the results as described previously [4].
Immunoblotting of TGF-ß1
Crude membrane extracts were prepared from the whole kidney using a solubilization buffer containing 10 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1% Triton X-100 and complete protease inhibitors. The extracts (5 µg) were electrophoresed through 15% SDSPAGE gel. For immunoblotting, the proteins were transferred to PVDF membranes. After blocking for 1 h at room temperature with the blocking buffer (1% gelatin in PBS with 0.05% Tween-20), the membrane was probed with anti-TGF-ß1 (1:500) or anti-actin (1:1000) in PBSTween for 16 h. The membrane was washed and incubated for 2 h at room temperature with a peroxidase-labelled goat anti-rabbit immunoglobulin and the antigenantibody reaction was detected with ECL plus chemiluminescence (Amersham Pharmacia Biotech, Arlington, IL, USA). The images were scanned on a flatbed scanner and the density of the bands was quantitated using ImageQuant software (Molecular Dynamic, Sunnyvale, CA, USA). Densitometry results were normalized to actin expression and reported as the ratio of average arbitrary integrated values (units) of TGF-ß1 to that of the actin value. To further confirm the specificity of the staining, control blots were probed with anti-TGF-ß1 pre-incubated with the blocking peptides of TGF-ß1.
Treatment of human mesangial cells with MIF
Murine mesangial cells (0.5 x 106 cells) were seeded onto six-well culture plates and were cultured for 24 h before growth was arrested for a further 24 h with plain culture medium (without fetal bovine serum). The cells were then cultured with 20 ng/ml MIF for various time intervals or cultured with increasing concentration of MIF (0.2580 ng/ml) for 48 h. In order to study whether TGF-ß1 synthesis by mesangial cells was induced by MIF, similar experiments were done in the presence of antibody against MIF (10 µg/ml). At the end of the experiments, cells were harvested for RNA extraction and the culture supernatant was collected for enzyme-linked immunosorbent assay (ELISA) of TGF-ß1. Biologically active TGF-ß1 in culture supernatants acidified by hydrogen chloride (HCl) was measured using a TGF-ß1 ELISA kit (Genzyme, Cambridge, MA, USA).
Statistical analysis
Results are expressed as means±SD. Statistical difference was assessed by a single factor variance (ANOVA) followed by an unpaired t-test, as appropriate. Non-parametric data were compared by the MannWhitney U-test. A P-value of <0.05 was considered significant.
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Results
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MIF and TGF-ß1 expression in mice after induction of IgA deposition
Figure 1 shows the mRNA and protein expression of MIF and TGF-ß1 in mouse kidney after IgAN induction. The mRNA expression peaked at 12 h for MIF and at 2 days for TGF-ß1 after induction of IgA deposition (Figure 1A). The protein expression peaked at days 2 for MIF and 4 for TGF after induction of IgA deposition (Figure 1B).

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Fig. 1. Time-course study of mRNA (A) and protein expression (B) of MIF (closed circles) and TGF-ß1 (open circles) in mouse kidney after IgAN induction.
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Glomerular IgA deposition
Kidney sections were examined for the presence of IgA deposits by immunofluorescence microscopy. Control animals had no detectable deposition of IgA (Figure 2A). Mesangial IgA deposition was demonstrated in all three groups of mice receiving intravenous DNPIgA immune complexes (Figure 2BD). The immunofluorescence was confined to the mesangial areas and was not observed in tubules or capillary endothelium. The patterns of diffuse and granular IgA deposits in the mesangium were similar in all positive animals. There was no difference in the intensity or the pattern of IgA deposition between mice receiving DNPIgA immune complexes alone (IgAN group) and mice receiving DNPIgA immune complexes followed by anti-MIF (anti-MIF group) or DNPIgA immune complexes followed by control antibody (control antibody group) (Table 1).

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Fig. 2. Representative immunofluorescence staining of IgA deposits in glomerular mesangium. (A) Normal control mice with no detectable IgA deposits, (B) mesangial IgA deposits in mice receiving intravenous DNPIgA immune complexes, (C) mesangial IgA deposits in mice receiving intravenous DNPIgA immune complexes followed by control antibody and (D) mesangial IgA deposits in mice receiving intravenous DNPIgA immune complexes followed by anti-MIF. There was no significant difference in the intensity and the pattern of IgA deposition between the IgAN group (B), the control antibody group (C) and the anti-MIF group (D). Magnification: x400. Results are from representative sections of eight different mice in each group.
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Urinalysis and glomerular histopathology
Microhaematuria was demonstrated in all three groups of mice receiving intravenous DNPIgA immune complexes. There was no significant difference in the degree of haematuria between these three groups of mice. Microhaematuria was not detected in the healthy control mice. Significant proteinuria was not detected in any group of mice. Histological examination revealed significant glomerular hypercellularity in both the IgAN group (Figure 3B) and the control antibody group of mice (Figure 3C) compared with the normal controls (Figure 3A) (P<0.005). There was no increased glomerular hypercellularity in the anti-MIF group when compared with normal controls (Figure 3D). The numbers of glomerular PCNA+ and CD68+ cells were significantly increased in all three groups of mice following intravenous administration of DNPIgA immune complexes when compared with the normal mice. Nevertheless, there was no increase in glomerular cellularity, glomerular PCNA+ or CD68+ cells in the anti-MIF group when compared with either IgAN group or the control antibody group (Table 1).

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Fig. 3. PAS staining of glomeruli. Glomerular hypercellularity was observed in mice from the IgAN group (B) and the control antibody group (C) compared with normal controls (A). For mice from the anti-MIF group (D), there was no increase in glomerular cellularity when compared with normal control mice (A). Magnification: x400. Results are from representative sections of eight different mice in each group.
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Gene expression of TGF-ß1 in the kidney
Semi-quantitative reverse transcriptionPCR with sequence-specific primers was performed using total RNA purified from whole kidneys. Figure 4 shows there was no difference in the gene expression of TGF-ß1 between the anti-MIF group (0.68±0.04) and the normal controls (0.62±0.05). However, the gene expression of TGF-ß1 in kidney from the IgAN (0.78±0.02) and control antibody (0.76±0.02) groups was significantly higher than that of normal controls (P<0.005 for both). With anti-MIF treatment, there was decreased gene expression of TGF-ß1 in IgAN mice induced by DNPIgA infusion (IgAN group 0.78±0.02 vs anti-MIF group 0.68±0.04; P<0.005) whereas a similar suppressive effect on gene expression of TGF-ß1 was not observed in IgAN mice treated with the control antibody.

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Fig. 4. Gene expression of TGF-ß1 in whole kidney. There was no difference in the gene expression of TGF-ß1 between the anti-MIF group and the normal control group. However, whole kidney from the IgAN group and the control antibody group exhibited higher gene expression of TGF-ß1 when compared with control mice (P<0.005). With anti-MIF treatment, there was decreased gene expression of TGF-ß1 in IgAN mice induced by DNPIgA infusion (anti-MIF group) (P<0.005) whereas a similar suppressive effect on gene expression of TGF-ß1 was not observed in IgAN mice treated with the control antibody. Data are means±SD of eight animals from each group.
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Tissue localization of TGF-ß1 proteins in the kidney
To localize the TGF-ß1 protein within the mouse kidney, 4-µm thick paraffin-embedded sections were stained with specific antibodies for TGF-ß1, as described earlier. The patterns of TGF-ß1 protein expression were similar in kidney sections from mice of the IgAN group, the anti-MIF group or the control antibody group. TGF-ß1 was found mainly in the glomerular mesangial area. Amongst all mice, mesangial expression of TGF-ß1 was strongest in the IgAN group and the control antibody group (Figure 5). There was a marked reduction in the expression of TGF-ß1 in the glomeruli from IgAN mice receiving anti-MIF treatment compared with those IgAN mice without anti-MIF treatment or treated with control antibody.

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Fig. 5. Representative immunostaining of TGF-ß1 expression in kidney. No staining of TGF-ß1 was detected in the control mice (A). Strong expression of TGF-ß1 in the glomerular mesangium of the IgAN group (B) and the control antibody group (C). There was a marked reduction in the glomerular TGF-ß1 expression in IgAN mice receiving anti-MIF treatment (D). Magnification: x400. Results are from representative sections of eight different mice in each group.
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Immunoblotting of TGF-ß1
The renal expression of TGF-ß1 was further assessed by immunoblotting (Figure 6). Densitometry analysis of the immunoblots revealed no difference between the protein synthesis of TGF-ß1 in kidneys from the IgAN group (108±13.11 U) and the control antibody group (101±13.56 U). However, the protein synthesis of TGF-ß1 in kidneys from the IgAN group, the anti-MIF group (90±9.70 U) and the control antibody group was significantly higher than that of the normal control group (67±14.67 U; P<0.005, P<0.05 and P<0.005, respectively). Anti-MIF treatment reduced the protein synthesis of TGF-ß1 in kidneys from IgAN mice induced by DNPIgA infusion (P<0.05) whereas a similar suppressive effect on protein synthesis of TGF-ß1 was not observed in IgAN mice treated with the control antibody.

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Fig. 6. Semi-quantitative immunoblotting of whole kidney extract. There was no difference in the TGF-ß1 expression between the IgAN group and the control antibody group. Mice from the IgAN group, the anti-MIF group and the control antibody group had higher TGF-ß1 expression when compared with normal controls. Anti-MIF treatment reduced the protein synthesis of TGF-ß1 in kidneys from IgAN mice induced by DNPIgA infusion (P<0.05) whereas a similar suppressive effect on protein synthesis of TGF-ß1 was not observed in IgAN mice treated with the control antibody. Data are means±SD of eight animals from each group.
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Double-antibody staining of MIF and TGF-ß1
The results of double-antibody staining of TGF-ß1 (brown) and MIF (blue) in mice glomerulus at 48 h after IgAN induction is shown in Figure 7. There is diffuse MIF staining in the glomerulus with strong MIF staining (arrow) in the area with intense TGF-ß1 signal (arrowhead).

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Fig. 7. Double-antibody staining of TGF-ß1 (brown) and MIF (blue) in mice glomerulus at 48 h after IgAN induction. Diffuse MIF staining in the glomerulus with strong MIF staining (arrows) in the area with intense TGF-ß1 signal (arrowheads). Magnification: x400.
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Effect of MIF on TGF-ß1 production in cultured mesangial cells
MIF increased synthesis of TGF-ß1 in a time- and dose-dependent manner (Figure 8). The synthesis of TGF-ß1 was abolished by incubating cultured mesangial cells with anti-MIF neutralizing antibodies.

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Fig. 8. Time- and dose-response curves of MIF-induced TGF-ß1 production in cultured mesangial cells. (A) Time-course of TGF-ß1 production when mesangial cells were cultured with 20 ng/ml MIF. (B) TGF-ß1 production at 48 h in mesangial cells cultured with different concentrations of MIF with (open circles) or without (closed circles) the presence of anti-MIF neutralizing antibodies (10 µg/ml). Results are means±SD of five individual experiments.
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Discussion
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The putative nephritogenic mediator of IgAN has been ascribed to the circulating IgA-containing immune complexes, since the prominent pathological feature of the disease is characterized by mesangial deposition of pIgA. This is further supported by the observation of early IgA deposition or recurrence of IgAN in renal allograft [10] and the spontaneous recovery of kidney from IgAN patient transplanted to non-IgAN recipient [11]. The finding of extra-renal IgA deposits also supports the notion that IgAN is mediated by circulating IgA-containing immune complexes [12]. Plasma concentration of pIgA is increased during acute exacerbation of IgAN [13]. Interestingly, IgA immune complexes and pIgA isolated from patients with IgAN have been shown to activate mesangial cells [14]. Activated mesangial cells release cytokines and chemokines, including IL-1, IL-6, TNF-
, monocyte chemotactic protein-1, TGF-ß and platelet-derived growth factor, that, in turn, stimulate proximal tubular epithelial cells and eventually lead to tubulointerstitial atrophy, interstitial infiltrate and fibrosis. We demonstrated previously that there was increased binding of pIgA from patients with IgAN to cultured human mesangial cells [15] and aberrant carbohydrate composition in the side chains of these pIgA favoured in vitro cell binding [16]. All of this evidence suggests that pIgA is pathogenic and plays an important role in the pathogenesis of IgAN.
In this study, we explored the role of MIF in the inflammatory cascade leading to renal injury in IgAN. The therapeutic potential of MIF blockade was examined in an experimental model of IgAN. IgAN was established in BALB/c mice based on the protocol by Rifai et al. [9]. Mesangial IgA deposits were detected after intravenous administration of immune complexes comprising DNPIgA and were accompanied by microhaematuria, mesangial hypercellularity and increased glomerular expression of TGF-ß1. The IgA deposition was not affected by treatment of IgAN mice with anti-MIF, but mesangial expression of TGF-ß1 was ameliorated by anti-MIF treatment. This MIF-induced TGF-ß1 was furthered supported by the results from in vitro culture experiments. The degrees of haematuria and mesangial hypercellularity were slightly decreased in IgAN mice with anti-MIF treatment, although the difference was not statistically significant. The finding that anti-MIF treatment had no effect on mesangial IgA deposition in experimental IgAN is not surprising, since mesangial IgA deposition in IgAN is determined by multiple factors, including size, electrical charge and carbohydrate composition of the IgA molecules and the nature of IgA receptors. There is no evidence that MIF alters the nature of the IgA molecule or that MIF modulates the expression of IgA receptors on mesangial cells.
The mechanism by which mesangial TGF-ß1 expression is up-regulated following IgA deposition is less well understood. Recent studies by others and us provide some clues to the mystery and MIF may contribute to the regulation of TGF-ß1 expression. MIF plays a key pathogenic role in the up-stream position for different inflammatory responses. Elevated serum levels of MIF and glomerular localization of MIF have been demonstrated in patients with various types of glomerulonephritis, including IgAN, especially during disease exacerbation [3]. MIF induces TNF-
and interferon-
production in macrophages via an amplifying pro-inflammatory loop. Our in vitro studies showed the gene expression and synthesis of MIF and TNF-
were up-regulated by pIgA from IgAN [4]. More intriguing is the down-regulation of pIgA-induced MIF synthesis by a neutralizing anti-TNF-
antibody. The fact that TNF-
is a potent inducer of TGF-ß1 expression in the kidney in vivo [17] raises the possibility that TNF-
may also contribute to the development of glomerulonephritis through the induction of renal TGF-ß1.
MIF also plays a pivotal role in the inflammatory response through activation of the T cells [18]. MIF mRNA and protein are constitutively expressed in many tissues. MIF protein is stored within the cytoplasm, being released when cells are stimulated by factors such as lipopolysaccharide and TNF-
[19]. In experimental mesangial proliferative glomerulonephritis (Thy-1 nephritis), marked up-regulation of MIF was found to associate with mesangial proliferation, macrophage accumulation and disease progression. Up-regulation of MIF expression is observed in IgAN and other glomerulonephritides [3]. The pro-inflammatory role of MIF is illustrated by its counter-regulatory effect on the suppression of cytokine production by glucocorticoid [2]. Indeed, MIF is the only molecule known to override the anti-inflammatory action of glucocorticoids. The importance of this mediator in the pathogenesis of glomerulonephritis is illustrated by the suppression of progressive renal injury in a rat model of crescentic glomerulonephritis by a neutralizing anti-MIF monoclonal antibody [20]. These observations implicate the crucial role of MIF in mediating inflammatory mechanisms of tissue damage and the therapeutic potential of antagonizing its effect. Our present findings demonstrate that anti-MIF can reduce TGF-ß1 synthesis in IgAN and this may have interesting bearing on the therapeutic approach to acute exacerbation of IgAN. Reducing the synthesis and release of MIF can alleviate the acute inflammatory injury. This may be achieved by pharmacological blockade of the pro-inflammatory loop involving MIF, TNF-
and TGF-ß1.
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Acknowledgments
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The authors thank Prof. Richard Bucala and Dr Christine Metz of the Picower Institute for Medical Research, NY, USA, for providing the anti-MIF antibodies. The study was supported by the Research Grant Committee (Hong Kong SAR) grant number 7329/00M.
Conflict of interest statement. None declared.
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Received for publication: 20. 4.03
Accepted in revised form: 28. 4.04