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Association of soluble CD89 levels with disease progression but not susceptibility in IgA nephropathy

2010; Elsevier BV; Volume: 78; Issue: 12 Linguagem: Inglês

10.1038/ki.2010.314

1523-1755

Mai Tuyet Vuong, Mirjana Hahn‐Zoric, Sigrid Lundberg, Iva Gunnarsson, Cees van Kooten, Lars Wramner, Maria Seddighzadeh, Anders Fernström, Lars Hanson, Lieu Thi, Stefan H. Jacobson, Leonid Padyukov,

Monoclonal and Polyclonal Antibodies Research

The Fc-α receptor (FcαR/CD89) is involved in IgA complex formation and may affect the development of IgA nephropathy (IgAN). In this study, we tested the genetic variations of the CD89 gene in relation to disease susceptibility in IgAN and the expression of soluble CD89 (sCD89) in sera of patients with IgAN and in controls. There was a significant difference between the levels of sCD89–IgA complexes, measured by sandwich enzyme-linked immunosorbent assay (ELISA), in 177 patients with IgAN with and without disease progression at the time of first diagnosis. No such difference was found in 42 patients with other renal diseases. The patients with IgAN without disease progression had stable but high levels of sCD89 over 5–15 years of follow-up in contrast to stable but low levels of sCD89 in the disease progression group. Moreover, levels of sCD89 complexes were correlated with one of the five CD89 genetic variants in 212 patients with IgAN and 477 healthy Caucasians; the single-nucleotide polymorphism (SNP) rs11084377 was significantly associated with a lower expression of sCD89. However, no association between CD89 gene polymorphisms and susceptibility to IgAN was detected. Thus, we found an association between the levels of sCD89–IgA complexes in serum and the severity of IgAN, and a possible genetic component in regulating the production or expression of sCD89. The Fc-α receptor (FcαR/CD89) is involved in IgA complex formation and may affect the development of IgA nephropathy (IgAN). In this study, we tested the genetic variations of the CD89 gene in relation to disease susceptibility in IgAN and the expression of soluble CD89 (sCD89) in sera of patients with IgAN and in controls. There was a significant difference between the levels of sCD89–IgA complexes, measured by sandwich enzyme-linked immunosorbent assay (ELISA), in 177 patients with IgAN with and without disease progression at the time of first diagnosis. No such difference was found in 42 patients with other renal diseases. The patients with IgAN without disease progression had stable but high levels of sCD89 over 5–15 years of follow-up in contrast to stable but low levels of sCD89 in the disease progression group. Moreover, levels of sCD89 complexes were correlated with one of the five CD89 genetic variants in 212 patients with IgAN and 477 healthy Caucasians; the single-nucleotide polymorphism (SNP) rs11084377 was significantly associated with a lower expression of sCD89. However, no association between CD89 gene polymorphisms and susceptibility to IgAN was detected. Thus, we found an association between the levels of sCD89–IgA complexes in serum and the severity of IgAN, and a possible genetic component in regulating the production or expression of sCD89. The Fc-α receptor (FcαRI/CD89) is a type I receptor glycoprotein expressed on myeloid cells, with known capability of high affinity binding of immunoglobulin A (IgA).1.Monteiro R.C. Van De Winkel J.G. IgA Fc receptors.Annu Rev Immunol. 2003; 21: 177-204Crossref PubMed Scopus (376) Google Scholar In addition, the existence of the soluble CD89 (sCD89) molecule has been detected both in vitro, in supernatants on activation of peripheral blood cells by enzyme linked immunosorbent assay (ELISA)2.van Zandbergen G. Westerhuis R. Mohamad N.K. et al.Crosslinking of the human Fc receptor for IgA (Fc-αRI/CD89) triggers FcR gamma-chain-dependent shedding of soluble CD89.J Immunol. 1999; 163: 5806-5812PubMed Google Scholar and in vivo, in human plasma, using western blotting.3.van der Boog P.J. van Zandbergen G. de Fijter J.W. et al.Fc-α RI/CD89 circulates in human serum covalently linked to IgA in a polymeric state.J Immunol. 2002; 168: 1252-1258Crossref PubMed Scopus (36) Google Scholar A 30 kDa sCD89 molecule has been demonstrated to be tightly bound to IgA and the CD89–IgA complex has been detected in relatively high concentrations (mean value 1900 ng/ml) in human plasma using semiquantitative dot blotting.3.van der Boog P.J. van Zandbergen G. de Fijter J.W. et al.Fc-α RI/CD89 circulates in human serum covalently linked to IgA in a polymeric state.J Immunol. 2002; 168: 1252-1258Crossref PubMed Scopus (36) Google Scholar Furthermore, as CD89–IgA complex is detectable in the circulation3.van der Boog P.J. van Zandbergen G. de Fijter J.W. et al.Fc-α RI/CD89 circulates in human serum covalently linked to IgA in a polymeric state.J Immunol. 2002; 168: 1252-1258Crossref PubMed Scopus (36) Google Scholar, it has been suggested that CD89 might contribute to the formation of polymeric serum IgA. However, no method for quantitation of sCD89 in human plasma or serum has previously been described. The FCAR gene encodes for the synthesis of FcαRI/CD89 and is located on human chromosome 19q13.4 (OMIM 147045) close to killer cell immunoglobulin-like receptor and leukocyte immunoglobulin-like receptor (also known as ILT and LIR) gene clusters. The FCAR gene has five exons and generates up to 10 different transcripts, several of which contain the transmembrane domain from exon 5. The function of its products or their expression might be influenced by genetic variances, such as single nucleotide polymorphisms (SNPs). There are two SNPs in the promoter of the FCAR gene (-311T/G and -142T/C) that are important for the promoter activity in a luciferase reporter gene assay.4.Shimokawa T. Ra C. C/EBP-α functionally and physically interacts with GABP to activate the human myeloid IgA Fc receptor (Fc-αR, CD89) gene promoter.Blood. 2005; 106: 2534-2542Crossref PubMed Scopus (13) Google Scholar These SNPs have been shown to be in association with chronic hepatitis C in a Japanese population,5.Watanabe A. Shimokawa T. Moriyama M. et al.Genetic variants of the IgA Fc receptor (Fc-αR, CD89) promoter in chronic hepatitis C patients.Immunogenetics. 2006; 58: 937-946Crossref PubMed Scopus (4) Google Scholar but were not reported to be high risk factors in allergic asthma.6.Jasek M. Obojski A. ntilde;czak M. et al.Are single nucleotide polymorphisms of the immunoglobulin A Fc receptor gene associated with allergic asthma?.Int Arch Allergy Immunol. 2004; 135: 325-331Crossref PubMed Scopus (8) Google Scholar According to the HapMap database there are two relatively small recombination blocks, one upstream of the gene within the promoter and the first two exons, and the other within the last exon. IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis, particularly in Western and Asian countries.7.Schena F.P. A retrospective analysis of the natural history of primary IgA nephropathy worldwide.Am J Med. 1990; 89: 209-215Abstract Full Text PDF PubMed Scopus (244) Google Scholar,8.D’Amico G. Natural history of idiopathic IgA nephropathy: role of clinical and histological prognostic factors.Am J Kidney Dis. 2000; 36: 227-237Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar Familial clustering studies suggest linkage of specific loci on chromosomes 6q22-q23, 4q26-31 and 17q12-22 with both susceptibility and progression of IgAN.9.Julian B.A. Quiggins P.A. Thompson J.S. et al.Familial IgA nephropathy. Evidence of an inherited mechanism of disease.N Engl J Med. 1985; 312: 202-208Crossref PubMed Scopus (161) Google Scholar, 10.Bisceglia L. Cerullo G. Forabosco P. et al.Genetic heterogeneity in Italian families with IgA nephropathy: suggestive linkage for two novel IgA nephropathy loci.Am J Hum Genet. 2006; 79: 1130-1134Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 11.Paterson A.D. Liu X.Q. Wang K. et al.Genome-wide linkage scan of a large family with IgA nephropathy localizes a novel susceptibility locus to chromosome 2q36.J Am Soc Nephrol. 2007; 18: 2408-2415Crossref PubMed Scopus (86) Google Scholar The polygenic complex nature of IgAN is broadly accepted, but very little is known about the specific genetic risk factors. Previously published data on the association of variations in the FCAR gene suggest the importance of FcαR expression in IgAN patients12.Tsuge T. Shimokawa T. Horikoshi S. et al.Polymorphism in promoter region of Fc-α receptor gene in patients with IgA nephropathy.Hum Genet. 2001; 108: 128-133Crossref PubMed Scopus (26) Google Scholar and on FcαR binding ability.13.Wu J. Ji C. Xie F. et al.Fc-αRI (CD89) alleles determine the proinflammatory potential of serum IgA.J Immunol. 2007; 178: 3973-3982Crossref PubMed Scopus (36) Google Scholar Interestingly, human FCAR transgenic mice develop spontaneous IgAN.14.Launay P. Grossetête B. Arcos-Fajardo M. et al.Fc-α receptor (CD89) mediates the development of immunoglobulin A (IgA) nephropathy (Berger's disease). Evidence for pathogenic soluble receptor-Iga complexes in patients and CD89 transgenic mice.J Exp Med. 2000; 191: 1999-2009Crossref PubMed Scopus (183) Google Scholar Our aim was to investigate the genetic influence of FCAR on the susceptibility and progression of IgAN with emphasis on the expression of sCD89 in plasma and its quantitation. To prove the presence of sCD89 in plasma of IgAN patients and healthy individuals western blotting was used. A 30 kDa protein band representing sCD89 was visualized in plasma samples of all tested individuals (Figure 1) using two different mouse monoclonal anti-CD89 antibodies (designated MIP8 and A3) and a polyclonal rabbit anti-CD89 antibody, all described in Materials and Methods. Both free sCD89 (MIP 8) and sCD89 bound to IgA (A3) can be detected in this way, although no free CD89 was found in the plasma samples and all reactivity was detected within the CD89–IgA complex (data not included). With the finding from western blotting as background, an ELISA method was developed using one monoclonal antibody (A3) specific for CD89 as capture antibody and a polyclonal anti-IgA antibody for detection of CD89–IgA complexes. Detectable levels of CD89–IgA complex in sera of IgAN patients and healthy controls were recorded in varying levels from 0.5 to 114% of a reference. The reference was a serum sample from one IgAN patient with a rather high level of sCD89, selected from western blot experiments. Distribution of sCD89 in the circulation of 115 healthy individuals has an approximate binomial shape with median (interquartile; 6.4 (2.5–27.2)). The majority of the individuals (n=78, 67.8%) had a sCD89 <20 relative units and 37 individuals (32.2%) exceeded 20 relative units. There was no significant difference between the sCD89 levels in the whole group of IgAN patients compared with the healthy control group. Furthermore, the serum levels of sCD89 were similar in the disease control group in comparison with the whole IgAN patient group. We found no significant correlations between the level of sCD89 and estimated glomerular filtration rate (GFR), P-value for the two-tailed Pearson's correlation coefficient=0.15, or between sCD89 levels and proteinuria, the Spearman's correlation coefficient=0.2. To investigate a possible association between sCD89 levels and disease severity in IgAN patients two groups were compared, IgAN progression (n=17) and non-progression group (n=92). Disease progression was defined as either doubling of serum creatinine during the follow-up period (at least 1 year of observation) or reaching stage 5 of chronic kidney disease (GFR 1%, but none demonstrated a significant association with IgAN (Table 2). No significant differences in genotype frequencies in either the codominant model or the dominant/recessive model were observed among the different severity groups with or without stratification for gender (data not included).Table 1Genotype frequencies of FCAR gene polymorphisms in IgAN patients in the dominant/recessive modelsSNPDominant/recessive modelχ2P-valueaNon-corrected.FCAR rs11084377TotalAAAG+GGGroup n=665Control46243 (9.3%)419 (90.7%)3.690.05Patient20310 (4.9%)193 (95.1%)FCAR rs4806605TotalCCCT+TT0.060.86Group n=679Control469306 (65.2%)163 (34.8%)Patient210135 (64.3%)75 (35.7%)FCAR rs10402324TotalAAAG+GG1.590.21Group n=676Control465207 (44.5%)258 (55.5%)Patient21183 (39.3%)128 (60.7%)FCAR rs1865097TotalAAAG+GG2.460.12Group n=677Control46647 (10.1%)419 (89.9%)Patient21130 (14.2%)181 (85.8%)FCAR rs16986050TotalAAAG+GG0.040.85Group n=670Control474318 (67.1%)156 (32.9%)Patient196133 (67.9%)63 (32.1%)Abbreviations: FCAR, Fc-α receptor; IgAN, immunoglobulin A nephropathy; SNP, single nucleotide polymorphism.a Non-corrected. Open table in a new tab Table 2Haplotype frequencies of FCAR gene polymorphisms in IgAN patients and healthy controlsBlockHaplotype frequencyControl/case ratio countsControl/case frequenciesχ2P-valueaNon-corrected.GCGAA0.265233.9:704.1, 124.9:293.10.249, 0.2993.6230.06GCAGA0.239235.8:702.2, 88.7:329.30.251, 0.2122.440NSACAGG0.168159.7:778.3, 67.6:350.40.170, 0.1620.153NSGTAGA0.16146.6:791.4, 70.0:348.00.156, 0.1670.266NSACAGA0.07473.2:864.8, 27.8:390.20.078, 0.0670.553NSACGAA0.04441.6:896.4, 18.6:399.40.044, 0.0440NSGTGAA0.02825.3:912.7, 12.1:405.90.027, 0.0290.042NSAbbreviations: IgAN, immunoglobulin A nephropathy; NS, not significant.a Non-corrected. Open table in a new tab Abbreviations: FCAR, Fc-α receptor; IgAN, immunoglobulin A nephropathy; SNP, single nucleotide polymorphism. Abbreviations: IgAN, immunoglobulin A nephropathy; NS, not significant. To address the possible influence of genetic variations on production of sCD89, sCD89–IgA complex levels were analyzed in individuals with different FCAR genotypes. We did not record any disease-specific effects in our study population. However, in the combined group, using a dominant/recessive model, it appeared that one of the SNP (rs11084377) was associated with lower production of sCD89 in the G dominant model (P=0.04, Mann–Whitney Test) (Figure 4). This marker is in close proximity to the alternative exon 2 in the FCAR gene structure, which is part of the common domain for Ig superfamily proteins. The most important finding in our study is the significant association between the levels of soluble Fc-α receptor (sCD89)–IgA complex in sera of IgAN patients and the progression of IgAN. In a disease control group of patients with other forms of glomerulonephritis, who had similar renal function and proteinuria as the IgAN group, no correlation to disease progression was observed. In addition, serum levels of sCD89 remained stable and low in IgAN patients with disease progression and continuously high in IgAN patients without progression, during a follow-up period of 5–15 years. We first developed an indirect ELISA method for measuring the levels of sCD89 in human sera through detection of IgA in the sCD89–IgA complex. The other important finding is the absence of an association of IgAN with variants of the FCAR gene, as well as the finding of a possible influence of FCAR gene polymorphism on production of sCD89. A considerable variation in levels of sCD89 in the circulation of patients with IgAN was also observed. In FCAR transgenic mice IgA immune complexes have been demonstrated to arise spontaneously in glomerular deposits. As this pathological condition is also seen in IgAN patients, it may be possible that sCD89 is also involved in human disease. We selected several gene variations for this analysis on the basis of our previous experience in genotyping of different gene loci. As even a small deviation from linkage disequilibrium may be important for detection of the association, we chose SNPs distributed all over the gene region and flanking regions. As is evident from our results, a recombination inside the FCAR gene is rare in the Caucasian population: out of 32 expected haplotypes, we recorded 8 that were common for the investigated population. This is evidence of strong linkage disequilibrium in this region, which in theory increases the chance for detection of an association by haplotype analysis (Figure 5). However, an association with susceptibility to IgAN was not detected for selected common variations in the FCAR gene. For the univariate allelic test there was only a 80% power to detect signals with odds ratio 1.9 or higher in our study population. As the genetic risk for a complex disease is expected to be low, our genetic association study may represent a type II error. On the other hand, the haplotype analysis suggests that there is a low, if any, probability of less common genetic variation, which may be important for the disease. In this study, we developed a new method for detection the of sCD89–IgA complex in human serum. Several previous attempts from other groups to quantify sCD89 directly in serum or plasma have failed, probably because of the fact that all sCD89 molecules are tightly bound to IgA within the CD89–IgA complexes and only a minor portion of the sCD89 molecule is exposed for antibody recognition. Alternatively, when these complexes were dissociated in vitro by strong detergents such as SDS, this reaction in solution was hindered because of antibody denaturation. Western blotting was effective, but is however not an accurate quantitative method. We resolved the problem by the detection of sCD89–IgA complexes instead of the free sCD89 molecules through indirect detection of this complex with antibodies against IgA, whereas the specificity of the reaction was ensured by capturing the sCD89 from the serum with a specific monoclonal anti-CD89 antibody. This method could be used for detection of circulating sCD89 in serum/plasma in different pathological conditions. One concern regarding the detection of this complex is related to its composition. We based our analysis on the assumption of its relative homogeneity (one IgA molecule bound to one sCD89 molecule). In fact, if there are differences in IgA/CD89 ratios in different individuals the measurements may be inaccurate. However, to our knowledge, there was no indication for such heterogeneity. Our data also suggest the influence of FCAR (CD89) gene polymorphisms in the regulation of production and/or expression of sCD89, which may relate to splicing mechanisms. This has to be proven in independent experiments because of the multiple testing in our study and relatively low effect value. We can only speculate regarding possible mechanisms related to sCD89–IgA complexes influence on progression of IgA. There are at least two possible explanations. One might speculate that these complexes are more ‘sticky’, precipitate at kidney, disturb its function and are less represented in the circulation in IgAN patients with disease progression. Another option is that circulating complexes are not pathogenic by itself, but glomerular IgA deposits occur as a reflection of low levels of sCD89, which cause an unusual reaction of IgA with other proteins in glomeruli and precipitation of IgA there. These issues might be addressed in future studies. Although we did not find a significant association with susceptibility of IgAN for the investigated SNPs, we here report an association between the serum levels of sCD89–IgA complex and the severity of IgAN as a novel marker for disease progression. A total of 212 unrelated patients (146 males and 66 females), mean age 39±14 (range 17–77 years) with biopsy-proven IgAN and 477 healthy Swedish Caucasians (321 males and 156 females), mean age 45±13 (range 18–80 years), matched by gender, were included in the study. The patients were recruited from the Department of Nephrology at the Karolinska University Hospital (n=117), Danderyd Hospital (n=31), Skövde Hospital (n=36) and Linköping Hospital (n=28), representing a population mainly resident in the central part of Sweden. Patients with Henoch-Schönlein purpura and patients with concomitant renal diagnosis were not included in the IgAN patient group. The sera from IgAN patients for comparison between the groups were collected within few days from the first diagnosis and biopsy. In all, 42 patients who had undergone renal biopsy and who had other forms of glomerular disease, namely lupus nephritis, minimal change disease or focal and segmental glomerulosclerosis were selected as the disease control group. They were matched by sex, gender, serum creatinine and proteinuria levels with IgAN patients. In seven IgAN patients (three without disease progression and four with progression) with serum samples collected longitudinally for 5–15 years of follow-up, sCD89 was analyzed at different time points in a total of 62 samples. All included individuals gave informed consent and the study was approved by the Ethics Committee of the Karolinska University Hospital, Stockholm, Sweden. A total of 109 IgAN patients from the Karolinska University Hospital who had been followed for up to 15 years since renal biopsy were investigated for the correlation between genotype and disease severity. GFR was estimated from yearly serum creatinine measurements using the Modification of Diet in Renal Disease equation.15.Levey A.S. Bosch J.P. Lewis J.B. et al.A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group.Ann Intern Med. 1999; 130: 461-470Crossref PubMed Scopus (12277) Google Scholar To investigate the correlation between genotype and disease severity the following criteria were used: IgAN progression (n=17) was defined as either doubling of serum creatinine during the time of follow-up (at least 1 year of observation) or reaching stage 5 of chronic kidney disease (GFR 99% reproducibility. Mann–Whitney or Kruskal–Wallis Tests were used to test for differences in the levels of sCD89 complex in patient and control plasma, the sCD89 complex levels in individuals with different genotypes and the severity of IgAN in different groups. To assess genotype, allele and haplotype frequencies Pearson χ2 and/or Fisher's Exact Tests were performed when appropriate, using SPSS 17.0 Software. Haplotype analysis was carried out using HaploView.17.Barrett J.C. Fry B. Maller J. et al.Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Crossref PubMed Scopus (11431) Google Scholar Power calculation was performed for two-tail or one-tail tests when appropriate for 5% threshold of significance. We thank Eva Jemseby, Neda Tahmasebifar, Per-Anton Westerberg, Susanne Schepull, Micael Gylling, Per Eriksson, and Arne Stahl for their helpful assistance and Robert Harris for linguistic advice. Jarl Ahlmén's involvement at the initial stage of this study is highly appreciated. We are grateful for the support for the project (7500730303 and 75007342) from SIDA's Secretariat for Research Cooperation for the bilateral cooperation between Vietnam and Sweden, Roche, and the Fund for Renal Research (Heart and Lung Foundation), Sweden.