1 Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan and 2 Department of Immunopathology, Bicht Medical School, INSERM E0225, Paris, France
Correspondence and offprint requests to: Yasuhiko Tomino, MD, Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. Email: yasu{at}med.juntendo.ac.jp
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Abstract |
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Methods. Japanese patients with IgAN (n = 124) and healthy controls (n = 100) were genotyped for FcR polymorphisms (Fc
RIIa-131H or R, Fc
RIIIa-176F or V and Fc
RIIIb-NA1 or -NA2). The genotyping of these polymorphisms was performed using allele-specific polymerase chain reaction (PCR) methods. Associations among Fc
R polymorphisms and susceptibility, age of onset, levels of serum immunoglobulins, intensity of glomerular IgG deposition and pathological severity were analysed.
Results. These three FcR polymorphisms showed no significant differences in genotype and allele frequencies between the IgAN patients and healthy controls. Each Fc
R polymorphism had no influence on age of onset, serum levels of IgG and glomerular IgG deposition in IgAN. However, Fc
RIIa-131R (R/R or H/R) or Fc
RIIIa-176V homozygous carriers (V/V) showed significantly more severe injury than Fc
RIIa-131H homozygous (H/H) (P<0.03) or Fc
RIIIa-176F carriers (F/F or F/V) (P<0.03), respectively.
Conclusion. The present study shows that polymorphisms of FcRIIa and Fc
RIIIa influence the severity of IgAN in Japanese patients but not the incidence, suggesting that IgG-IC may play important roles in the progression and prognosis of this disease via Fc
Rs.
Keywords: Fc receptor; IgA nephropathy; IgG; polymorphism
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Introduction |
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IgAN was originally described by Berger as Nephropathy with mesangial IgAIgG deposits [4]. Glomerular IgG deposits, mainly IgG1 or IgG3 [5,6] and circulating IgG/IgA-IC, have been observed in patients with IgAN [1,3,7]. In addition, it has been reported that glomerular IgG deposition in the presence of normal renal function is a risk factor for renal survival in patients with IgAN [8]. However, the role of IgG in the pathogenesis of this disease remains unclear.
Fc receptors for IgG (FcRs) have important functions in the regulation of immune responses, and they are also critical for IC clearance. Three functional polymorphisms of Fc
Rs (Fc
RIIa, Fc
RIIIa and Fc
RIIIb) have been described [9]. Each polymorphism is located in the extracellular domains, which are binding sites and affect binding affinity for each IgG subclass [9]. Indeed, many studies have reported skewed distributions of Fc
RIIa, IIIa and IIIb alleles in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [9].
FcRIIa is expressed on most myeloid cells, but not on glomerular mesangial cells [10,11]. The Fc
RIIa molecule exhibits both genetically determined structural and functional allelic forms, Fc
RIIa-131H and 131R. The Fc
RIIA-131H genotype has a greater ability to bind IgG1, IgG2 and IgG3. Interestingly, the Fc
RIIa-131R allele has almost no binding affinity for IgG2 [12,13].
On the other hand, FcRIIIa is expressed on macrophages, natural killer (NK) and glomerular mesangial cells [10,11]. The Fc
RIIIa molecule also exhibits both genetically determined structural and functional allelic forms, Fc
RIIIa-176V and 176F, and binds to IgG1, IgG3 and IgG4 subclasses which are more strongly bound by Fc
RIIIa-176V [14,15]. Fc
RIIIb bears a neutrophil antigen polymorphism caused by four amino acid substitutions. Fc
RIIIb-NA1 is more efficient in binding to IC containing IgG1 and IgG3 than Fc
RIIIb-NA2 [16,17].
Based on this background, the objective of the present study was to examine whether FcR polymorphisms influence the disease course of IgAN. The present findings may provide a clue to the pathological role of IgG in this disease.
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Materials and methods |
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FcRIIa allotyping (131R and 131H)
DNA fragments were amplified by polymerase chain reaction (PCR) using the following primers: sense, 5'-CTG AGA CTG AAA ACC CTT GGA ATC-3' and antisense, 5'-GCT TGT GGG ATG GAG AAG GTG GGA TCC ATA-3'. The amplification procedure consisted of initial denaturation for 5 min, 33 cycles of denaturation at 95°C for 20 s, annealing at 55°C for 20 s and extension at 72°C for 40 s, followed by a final extension at 72°C for 5 min. After the amplification, the PCR products were digested with restriction endonuclease, NdeI (Nippon Gene, Toyama, Japan). The restriction was performed with 4 µg of amplification products and 4 U of NdeI diluted in a specific buffer recommended by the manufacturer in a total volume of 20 µl reacted at 37°C overnight. The products including restriction fragments were electrophoresed in an 8% polyacrylamide gel, and visualized by staining with ethidium bromide. Restriction fragment length polymorphisms (RFLPs) of 131H and 131R were expressed as fragments of 200 and 231 bp in the presence of NdeI, respectively.
FcRIIIa allotyping (176V and 176F)
For this allotyping, nested PCR was performed as described previously [15]. DNA fragments were amplified by first PCR using the following two FcRIIIa-specific primers: Fc
RIIIa-sense; 5'-ATA TTT ACA GAA TGG CAC AGG-3' and Fc
RIIIa-antisense; 5'-GAC TTG GTA CCC AGG TTG AA-3'. The amplification procedure consisted of initial denaturation for 5 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 57°C for 1.5 min and extension at 72°C for 1.5 min, followed by a final extension at 72°C for 8 min. The fragment of
1.7 kbp containing the polymorphic site of Fc
RIIIa (176F and 176V) was amplified. A second PCR was performed with 1 µl of the first PCR product using the following primers: sense, 5'-ATC AGA TTC GAT CCT ACT TCT GCA GGG GGC AT-3' and antisense, 5'-ACG TGC TGA GCT TGA GTG ATG TTC AC-3'. The amplification procedure consisted of initial denaturation for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min.
After the amplification, the second PCR product was digested with NlaIII (New England Biolabs, Hitchin, UK). The restriction was performed with 4 µg of amplification products and 8 U of NlaIII diluted in a specific buffer recommended by the manufacturer in a total volume of 20 µl reacted at 37°C overnight. The products including the restriction fragment were electrophoresed in an 8% polyacrylamide gel, and visualized by staining with ethidium bromide. RFLPs of 176F and 176V were expressed as fragments of 94 and 61 bp in the presence of NlaIII, respectively.
FcRIIIb allotyping (NA1 and NA2)
Genotyping was performed as described previously [18]. DNA fragments were amplified by PCR using the following primers: FcRIIIb-Na1-specific primer, 5'-CAG TGG TTT CAC AAT GTG AA-3'; Fc
RIIIb-Na2-specific primer, 5'-CAA TGG TAC AGC GTG CTT-3'; and the reverse primer, 5'-ATG GAC TTC TAG CTG CAC-3'. The amplification procedure consisted of initial denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. The amplification products were electrophoresed in a 2% agarose gel and visualized by staining with ethidium bromide. The amplification products of Na1 and Na2 were expressed as 141 and 219 bp fragments, respectively.
In the above-mentioned allotyping, we preliminarily performed direct sequencing using nine DNA samples of controls. We compared these data with genotypes by several allotyping methods and finally chose each present method for this study.
Immunohistological analysis of glomerular IgG deposition in renal biopsy specimens
All renal biopsies used in this study were obtained from patients who had a glomerular filtration rate >70 ml/min. For the immunohistochemistry, the frozen sections (3 µm thick) from a part of the biopsy specimens were fixed in acetone for 10 min and rehydrated in phosphate-buffered saline (PBS). After blocking the sections with PBS containing 2% bovine serum albumin (BSA), 2% fetal calf serum (FCS) and 0.2% fish gelatin for 30 min, they were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-human IgG antibody (x100) (DAKO, CA, USA) diluted in blocking solution for 60 min, and washed three times with PBS.
For an evaluation of glomerular IgG, the intensity of IgG deposition in glomeruli was graded semi-quantitatively from 0 to 3+ (0 = none; 1+ = weak; 2+ = mild; 3+ = strong). This grading was determined by one pathologist and at least three nephrologists in a blind manner.
Classification of the severity of IgA-N
Formaldehyde-fixed renal biopsy specimens were embedded in paraffin and sectioned serially at 3 µm thick. All sections were stained with periodic acidSchiff (PAS). In this study, the severity of IgAN was classified into four groups by Clinical guidelines for the diagnosis and treatment of patients with immunoglobulin A (IgA) nephropathy in Japan [19]. The prognostic grading in this guideline using renal biopsy specimens was done by the Special Study Group on Progressive Glomerular Disease in the Ministry of Health and Welfare of Japan and the Japanese Society of Nephrology, with retrospective clinical data [19]. Each prognostic grade is based on light microscopic histological findings as summarized in Table 1. The renal prognosis by these criteria is divided into the following four groups; good prognosis group (group I) in which dialysis treatment will probably never be required; relatively good prognosis group (group II) in which the possibility of dialysis treatment is relatively low; relatively poor prognosis group (group III) in which dialysis treatment is likely to be required within 520 years; poor prognosis group (group IV) in which the possibility of dialysis treatment within 5 years is high [19]. This grading was determined by one pathologist and at least three nephrologists in a blind manner.
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Statistical analyses
To consider the linkages between each allele and genotype of FcR polymorphisms and clinical or pathological findings, the genotypes of each Fc
R were classified into two subgroups: each allele homozygous, and other allele carriers. Fc
RIIa genotypes were classified into two subgroups: Fc
RIIa-131H homozygous (H/H) and Fc
RIIa-131R carriers (R/R or H/R), or Fc
RIIa-131R homozygous (R/R) and Fc
RIIa-131H carriers (H/H or H/R). Fc
RIIIa genotypes were classified into two subgroups: Fc
RIIIa-176V homozygous (V/V) and Fc
RIIIa-176F carriers (F/F or F/V), or Fc
RIIIa-176F homozygous (F/F) and Fc
RIIIa-176V carriers (V/V or F/V). Fc
RIIIb genotypes were also classified into two subgroups: Fc
RIIIb-NA1 homozygous (NA1/NA1) and Fc
RIIIb-NA2 carriers (NA2/NA2 or NA1/NA2), or Fc
RIIIb-NA2 homozygous (NA2/NA2) and Fc
RIIIb-NA1 carriers (NA1/NA1 or NA1/NA2). In this study, this classification was used when we analysed the linkages between each allele of Fc
R polymorphisms and age of onset, intensity of IgG and histological severity.
Serum levels of IgG, IgA and IgM which were examined at the time of renal biopsy and the age of onset in this disease were expressed as mean±SE.
Statistical analyses for the association were performed using Stat View-J5.0 for Macintosh (Abacus Concept, Berkeley, CA). Serum levels of IgG, IgA and IgM and the age of onset and the duration from onset to renal biopsy in each pathological grade of IgA-N patients were compared using t-test analysis. Other data were analysed by the 2 test. When we found statistical significance in this test, we compensated these data with Yates correction. Statistical significance was defined as P<0.05.
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Results |
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Discussion |
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Disease susceptibility linked to FcR polymorphisms has been described for autoimmune diseases including SLE, RA, multiple sclerosis, GuillainBarre syndrome and anti-neutrophil cytoplasmic antibody (ANCA)-positive systemic vasculitis [9]. The crucial function of Fc
R is IC clearance. Fc
R can trigger the internalization of captured IC, which leads to degradation of antigenantibody complexes, as well as directing the antigenic peptides to the major histocompatibility complex (MHC) class I or class II antigen presentation pathway [9,20]. It is known that functional defects in IC clearance are linked to the initiation of autoimmune diseases such as SLE. Importantly, recent findings have indicated that antigen presentation is much more efficient if the IC is internalized by Fc
R rather than by non-specific uptake mechanisms such as fluid phase pinocytosis [9,20]. Other studies also demonstrated the functional differences in IC clearance between Fc
RIIa-131R and -131H [16], and between Fc
RIIIa-176V and -176F [15]. These findings support the idea that genetic polymorphisms of Fc
RIIa and Fc
RIIIa in SLE patients may influence disease susceptibility, because altered IgG-IC clearance may be critical for the incidence of SLE. However, our present results show that these Fc
R polymorphisms had no significant influence on the incidence, age of onset, levels of serum IgG and intensity of glomerular IgG deposition in IgAN. These findings suggested that IgG/IgG-IC may not be a triggering factor for IgAN, at least via Fc
R activation.
Nieuwhof et al. reported that the glomerular IgG deposition in IgAN patients with normal renal function was a risk factor for renal survival [8]. Moreover, the pathological role of circulating IgG-IC has been discussed in patients with IgAN [1,3]. Therefore, IgG/IgG-IC may contribute to the progression of IgAN. In previous studies, lower affinity alleles of FcRs (Fc
RIIIa-131R, Fc
RIIIa-176F and Fc
RIIIb-NA2 genotype) have been proposed as susceptibility factors for SLE and RA [9], possibly due to the inefficient clearance of circulating or tissue-deposited IC. The present data showed that one of the lower affinity alleles of Fc
Rs, Fc
RIIIa-131R, was linked to the severity of IgAN, suggesting that the impairment of IgG-IC clearance by this allele and subsequent glomerular deposition may also contribute to the glomerular lesions. However, our results also showed that the intensity of glomerular IgG deposition had no correlation with Fc
R polymorphisms. In the renal survival of IgAN, subclass restriction, mainly of IgG1 and IgG3, was observed in glomerular IgG deposition [5]. The Fc
RIIa-131R allele lacks binding affinity for IgG2. In this study, we only examined the correlation between the polymorphism and glomerular deposition of whole IgG including IgG2. Therefore, the correlation between their polymorphisms and each glomerular IgG subclass has to be examined carefully in a future study.
We also found that one of the higher affinity alleles of FcRs, Fc
RIIIa-176V, may influence the severity of the glomerular lesions in IgAN. Of note, Fc
RIIIa is expressed not only on circulating bone marrow-derived cells but also on glomerular mesangial cells [10,11]. Fc
RIIIa-176V binds more strongly to IgG1 and IgG3 than Fc
RIIIa-176F [14,15]. Therefore, it is speculated that greater activation of mesagial cells, presumably by IgG1/IgG3, may contribute to the progression of this disease.
It appears that polymorphisms of FcRIIa and Fc
RIIIa are associated with severity in patients with IgAN, suggesting the importance of Fc
RIIa and Fc
RIIIa in the prognosis of this disease. However, we found that the severity is linked to the low affinity allele of Fc
RIIa-131R and the high affinity allele of Fc
RIIIa-176V, suggesting that each polymorphism may influence the prognosis through different effector mechanisms. In this regard, in order to understand the contradictory results, it is interesting to examine the combination of these two genotypes. Although we have examined the combination in each patient, only two of eight Fc
RIIIa-176V/V patients in the progressive group (Table 7) had the Fc
RIIa-131R allele (data not shown). Since the total numbers of 176V/V were small, a future study with more patients will be required. In addition, a functional study with an appropriate model is needed to determine the role of Fc
R in the pathogenesis of IgAN.
Conflict of interest statement. None declared.
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References |
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