Injection of recombinant Fc
RI/CD89 in mice does not induce mesangial IgA deposition
Paul J. M. van der Boog1,
Cees van Kooten1,
Ger van Zandbergen1,
Ngaisah Klar-Mohamad1,
Beatrijs Oortwijn1,
Nico A. Bos3,
Alexandra van Remoortere2,
Cornelis H. Hokke2,
Johan W. de Fijter1 and
Mohamed R. Daha1
1 Department of Nephrology and 2 Department of Parasitology, Leiden University Medical Center, Leiden and 3 Department of Cell Biology, University of Groningen, The Netherlands
Correspondence and offprint requests to: P. J. M. van der Boog, MD, Department of Nephrology, C3-P, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Email: p.j.m.van_der_boog{at}lumc.nl
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Abstract
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Background. Earlier studies have suggested that complexes of the human IgA receptor Fc
RI/CD89 with mouse IgA are pathogenic upon deposition in the renal mesangium. Transgenic mice expressing Fc
RI/CD89 on macrophages/monocytes developed massive mesangial IgA deposition and a clinical picture of IgA nephropathy (IgAN). Based on these findings, the purpose of this study was to design an experimental model of IgAN by injection of human CD89 in mice. The interaction of mouse IgA with CD89 was investigated further.
Methods. Recombinant human soluble CD89 and a chimeric CD89Fc protein were generated, produced, purified and injected in mice. Renal cryosections were stained for IgA and CD89. The interaction of mouse IgA with CD89 was analysed by fluorescence-activated cell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA) and plasmon resonance technology.
Results. Injection of recombinant human CD89 did not result in significant IgA or CD89 deposition in the renal mesangium. However, CD89 staining in the liver was found to be positive. CD89 was rapidly cleared from circulation without signs of complex formation with IgA. FACS analysis, ELISA and plasmon resonance techniques all revealed a dose-dependent binding of human IgA to recombinant CD89, while no detectable binding was seen of mouse IgA, either of serum IgA or of different monoclonal mouse IgA preparations.
Conclusions. An experimental model for IgAN in mice could not be obtained by injection of recombinant CD89. This is compatible with our in vitro biochemical data showing a lack of binding between recombinant human CD89 and mouse IgA.
Keywords: CD89; IgA; IgA nephropathy; mouse; receptor
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Introduction
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Primary IgA nephropathy (IgAN), the most common form of primary glomerulonephritis worldwide, is characterized by the deposition of predominantly macromolecular IgA of the IgA1 subclass in the glomerular mesangium [1]. The mechanisms leading to renal deposition remain elusive, but overproduction of IgA1 with increased levels of macromolecular IgA, secondary to a primary mucosal hypoimmune response [2] and abnormal glycosylation [3] have been suggested to play a role. Also, a reduced clearance of IgA-IC by the mononuclear phagocyte system, presumably due to saturation, reduction or altered function of IgA receptors, has been suggested [4].
Binding of IgA has been demonstrated in human mesangial cells [5]. The transferrin and Fc
/µ receptors have been suggested as possible IgA receptors [6,7]. The presence of the myeloid F
RI/CD89 receptor on these cells was excluded by several groups [5]. Recently, a pathophysiological role for the F
RI/CD89 receptor in IgAN has been proposed [8]. Two soluble forms of CD89 have been reported in human serum: a 30 kDa protein, which was not specific for IgAN [9] and a highly glycosylated 5070 kDa protein, which was found to be elevated in polyethylene glycol (PEG) serum precipitates from patients with IgAN [8]. In the latter study, transgenic mice, expressing human CD89 on monocytes/macrophages, spontaneously developed a clinical picture compatible with IgAN. It was deduced that the interaction between mouse polymeric IgA and circulating human CD89 in the transgenic mice resulted in generation of soluble CD89IgA complexes, subsequent deposition and renal inflammation.
In order to design an experimental model of IgAN, recombinant human CD89 and a soluble chimeric protein of CD89 [Fc(CD89)2] were generated and injected in mice. It was found that CD89 was cleared rapidly from the circulation, but kidney biopsies showed no CD89 and no significant mouse IgA deposition in the renal mesangium. This can be explained by the lack of interaction between human CD89 and mouse IgA, which we analysed using enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) and plasmon resonance techniques.
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Subjects and methods
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Production of recombinant CD89 and Fc(CD89)2
For the generation of a CD89Fc fusion protein, the complete extracellular region of CD89, as used for the production of monomeric sCD89 [10], was cloned into the pME-Fc vector [11], making use of the XhoI restriction site in the linker between the fusion product and the human IgG1 Fc tail (Figure 1A). Using EcoRI and NotI restriction enzymes, the whole coding sequence was cloned in the pME-Neo vector, introduced into Chinese hamster ovary (CHO) cells using electroporation and selected for G418 (500 µg/ml) resistance. Single clones with stable expression were obtained after limiting dilution, and those with the highest production, as determined by ELISA, were selected. Recombinant CD89 was purified using an IgA column, as described [10]. Purified proteins were analysed using SDSpolyacrylamide gel electrophoresis (PAGE).

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Fig. 1. CD89Fc fusion protein: schematic representation (A), SDSPAGE of three purified fusion protein samples under non-reducing and reducing conditions (B) and specific reactivity of fusion protein, measured by ELISA (C).
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In vivo studies
In order to determine whether in vivo injection of CD89 or Fc(CD89)2 was followed by renal deposition of mouse IgA, groups of three mice (Balb/c mice, 7 weeks old, weight 2025 g) were injected with 0.25 ml of phosphate-buffered saline (PBS) alone or with 0.25 ml of PBS containing 200 µg of human recombinant CD89 or 525 µg of Fc(CD89)2. After 3 h, all animals were sacrificed, and the kidneys removed and processed for immunohistochemistry. A similar experiment was performed in 15-week-old Balb/c and C57BL/6 mice, which were injected on three consecutive days with Fc(CD89)2 and sacrificed 24 h after the last injection. To investigate whether adequate levels of circulating CD89 were obtained, other groups of Balb/c and C57BL/6 were injected as described above and CD89 levels were monitored in blood, using a CD89-specific ELISA [10]. Liver biopsies were performed and processed for immunohistochemistry in order to analyse the clearance of the injected protein.
Immunohistochemistry
Mouse kidney and liver tissues were snap-frozen in liquid nitrogen and processed for immunofluorescence according to standard procedures. Between each step, tissue sections were washed three times for 5 min with PBS. In order to detect IgA depositions, tissue sections were incubated with goat anti-mouse IgA antibodies (1:1000, Nordic) followed by rabbit fluorescein isothiocyanate (FITC)-conjugated anti-goat antibodies (Dako, Glostrup, Denmark). The presence of CD89 in the tissue sections was analysed by incubation of the sections with rabbit anti-CD89/goat anti-rabbitFITC as described [5].
IgA preparations
Human IgA was purified from normal human serum as described earlier [12] by euglobulin precipitation, followed by zinc sulfate and ammonium sulfate precipitation. Subsequently, ion exchange chromatography was performed on a DEAE column. Dimeric IgA was obtained by gel filtration (S-300) and used in binding assays.
Monoclonal IgA anti-Thy-1 antibodies (ER4A) were used as mouse IgA. These antibodies were purified from ascites fluid by gel filtration column chromatography as described earlier [13]. Size analysis of ER4A anti-Thy-I antibodies showed that hybridoma ER4A predominantly secreted macromolecular IgA [13]. For binding studies with plasmon resonance analysis, culture supernatants of mouse IgA hybridomas, obtained after fusion of mesenteric lymph node cells with an Sp2/0 fusion partner, were also used [14]. Size fractionation of mouse serum was performed by HR200 gel filtration.
FACS analysis
The murine B-cell line IIA1.6 transfected with CD89 and murine FcR
-chain [15] was used to analyse the binding of IgA to CD89-positive cells. All binding experiments were performed at 4°C and, between all steps, cells were washed twice with cold PBS/0.5% bovine serum albumin (BSA) and/or 0.02% NaN3, and centrifuged at 1200 r.p.m. A total of 5 x 105 cells were incubated for 1 h at 4°C with serial sample dilu-tions of IgA or serum. Subsequently, cells were incubated with monoclonal mouse IgG anti-human IgA (4E8) or polyclonal rabbit IgG anti-mouse IgA. Thereafter, the cells were incubated with phycoerythrin (PE)-labelled goat anti-mouse IgG or FITC-labelled goat anti-rabbit IgG. Subsequently, cells were fixed with 1% paraformaldehyde in PBS and analysed on a FACScan. Data acquisition and analysis were done using Lysis II software (Becton Dickinson, San Jose, CA).
ELISA
The binding of mouse and human IgA to CD89 and Fc(CD89)2 was analysed by two ELISA methods. First, serial dilutions of human or mouse IgA (ER4A) were coated onto ELISA plates by overnight incubation at room temperature in coating buffer (0.1 M NaHCO3/Na2CO3, pH 9.6). Wells were washed three times using PBS, 0.02% Tween-20, and 100 µl of purified recombinant CD89 (14.7 µg/ml) or chimeric Fc(CD89)2 (16 µg/ml) was applied. All dilutions and subsequent antibody steps were performed in ELISA buffer [PBS, 0.02% Tween-20, 1% fetal calf serum (FCS)]. Following incubation for 1 h at 37°C, plates were washed as above and incubated with digoxigenin (DIG)-conjugated rabbit F(ab')2 anti-CD89 (1:1500), followed by horseradish peroxidase (HRP)-conjugated F(ab')2 anti-DIG (1:4000, Boehringer Mannheim) both for 1 h at 37°C, and washed in-between as above. The optical density at 415 nm was measured after addition of ABTS/H2O2 as substrate.
To ascertain that IgA was coated onto the wells properly, bound human IgA was detected by biotin-labelled monoclonal mouse anti-human IgA, followed by HRP-conjugated streptavidin and ABTS/H2O2. Bound mouse IgA was detected by DIG-conjugated affinity-purified rabbit anti-mouse IgA (1:1000), followed by HRP-conjugated F(ab')2 anti-DIG (1:4000) and ABTS/H2O2. Between each step, wells were washed three times with PBS, 0.02% Tween-20.
Secondly, IgACD89 interactions were analysed by coating ELISA wells with recombinant CD89 (1 µg/ml) or chimeric Fc(CD89)2 (1 µg/ml), followed by serial 2-fold dilutions of samples for 1 h at 37°C. Bound human IgA was detected by biotin-labelled monoclonal mouse anti-human IgA (4E8), followed by HRP-conjugated streptavidin and ABTS/H2O2. Bound mouse IgA was detected as described above with DIG-conjugated affinity-purified rabbit-anti-mouse IgA (0.5 µg/ml), followed by HRP-conjugated F(ab')2 anti-DIG (1:4000, 150 U/ml) and ABTS/H2O2. Between each step, wells were washed three times with PBS, 0.02% Tween-20.
Plasmon resonance analysis
Surface plasmon resonance spectroscopy was performed using a BIACORE 3000 instrument. Different channels of the same sensor chip CM5 (BIAcore AB, Uppsala, Sweden) were coupled with recombinant CD89 [6 µl of CD89 (1.4 mg/ml)], chimeric Fc(CD89)2 [10 µl of Fc(CD89)2 (1.6 mg/ml)] and BSA, following the manufacturer's instructions. Binding assays were performed at flow rates of 5 µl/min using HBS EP buffer [0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA and 0.005% (v/v) surfactant P20]. A 10 µl aliquot of analytes was injected and subsequently allowed to dissociate for 2 min. Subsequently, the surfaces were regenerated with 0.1 M glycineHCl, 0.3 M NaCl, pH 2.8, thereby regenerating the surface for a new binding cycle and returning to the baseline intensity.
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Results
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Construction of chimeric CD89
In order to investigate IgACD89 interactions, first a CD89Fc fusion protein was generated. The complete extracellular region of CD89 was fused with the human IgG1 Fc tail (Figure 1A). Recombinant soluble Fc(CD89)2 was produced in CHO cells and purified from culture supernatant, as described for sCD89 [10]. The purity of the preparations was checked by SDSPAGE and Coomassie brilliant blue staining, demonstrating a single band of 130 kDa under non-reducing and 65 kDa under reducing conditions (Figure 1B). Sandwich ELISAs directed against either the CD89 or Fc IgG part showed a specific reactivity, confirming both the identity and conformational integrity of the molecule (Figure 1C).
Injection of CD89 or Fc(CD89)2 in mice
In order to analyse the deposition of IgACD89 complexes upon intravenous (i.v.) administration, 7-week-old Balb/c and C57BL/6 mice were injected with purified recombinant CD89, chimeric Fc(CD89)2, or PBS as control. Injection of recombinant CD89 resulted in the rapid appearance of circulating CD89 (200 µg/ml) as measured by ELISA (Figure 2A). Within 3 h, nearly all injected protein was cleared, with the appearance of CD89 in the liver at 3 h, but not in the kidney (Figure 3). Injection of CD89 did not affect circulating IgA levels: all profiles of individual mice showed a similar 10% reduction after injection of CD89 or PBS at 3 min, which could be attributed to the dilution by i.v. injection (Figure 2B). Subsequently, three other groups of 7-week-old Balb/c-mice were injected in a similar way with CD89, Fc(CD89)2 or PBS, and sacrificed 3 h later. Kidneys were removed and stained for CD89 and IgA. No mesangial IgA or CD89 deposition could be detected in the renal tissue sections.

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Fig. 3. Staining of liver tissue sections for CD89 before (A) and 3 h after (B) injection of CD89. (C) A stained kidney tissue section 3 h after injection of CD89.
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In order to analyse age-, time- and mouse strain-dependent factors, the experiment was repeated with 15-week-old Balb/c and C57BL/6 mice. IgA levels in the Balb/c and C57BL/6 mice were 460±50 and 294±67 µg/ml, respectively. Moreover, Fc(CD89)2 was injected again on three subsequent days. Twenty-four hours after the last injection, kidneys were removed and stained for CD89 and IgA (Figure 4). Although no mesangial CD89 could be detected, there was a positive staining of mesangial IgA. However, this IgA deposition was not significantly different compared with control mice of this age, which were injected with PBS (Figure 4). The results of these experiments are summarized in Table 1. They indicate that no CD89 could be detected in the renal mesangium and that IgA staining was not different after injection of Fc(CD89)2 or PBS in both BALB/c and C57BL/6 mice.

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Fig. 4. Spontaneous mesangial IgA depostion in a 15-week-old BALB/c (B) and C57BL/6 mouse (C). The staining was specific for mouse IgA as when goat anti-mouse IgA antibodies were omitted, the staining was negative (A).
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Detection of IgACD89 interaction by FACS analysis
Since the results showed that injection of exogenous CD89 did not lead to detectable deposition of IgACD89 complexes in the renal mesangium, we further analysed the binding of mouse and human IgA to CD89-positive cells. In contrast to human serum IgA, which is mainly monomeric, mouse serum IgA is predominantly macromolecular. The murine B-cell line IIA1.6 transfected with cDNA encoding CD89 and murine Fc
-chain was used. In contrast to IIA1.6 cells, only cells transfected with CD89 bound human IgA [15]. IIA1.6-transfected cells were incubated with increasing concentrations of human IgA. In agreement with earlier results [15], human IgA bound to the CD89-positive cells. However, when cells were incubated with monoclonal dimeric mouse IgA (ER4A), no binding of mouse IgA to CD89 could be detected (Figure 5A and B). To mimic more physiological conditions, IIA1.6-transfected cells were also incubated with mouse and human serum in increasing dilutions. Figure 5C shows that under these conditions, only binding of human IgA to the CD89-positive cells was detected.
Detection of IgACD89 interaction by ELISA
To analyse the binding of mouse and human IgA to human CD89, different ELISAs were used. First, increasing concentrations of mouse and human IgA were coated onto ELISA plates. Fixed concentrations of purified CD89 or chimeric Fc(CD89)2 were added and the binding of CD89 or Fc(CD89)2 was detected with anti-CD89 antibodies. In both ELISAs, bound CD89 and Fc(CD89)2 was detected in wells coated with human IgA (data not shown). No binding of CD89 or Fc(CD89)2 to mouse IgA was found.
When ELISA plates were coated with Fc(CD89)2 and IgA was added, only human IgA was found to bind to coated Fc(CD89)2 (Figure 6A). Similarly, when total serum was added to coated Fc(CD89)2, only human IgA could be detected (Figure 6B). No binding of mouse IgA to Fc(CD89)2 was found.
Detection of IgACD89 interaction by plasmon resonance
Two channels of the same sensor chip were coupled with recombinant CD89 or chimeric Fc(CD89)2. A third channel was coupled with BSA as a control for unspecific binding. A representative sensorgram for the interaction with immobilized Fc(CD89)2 shows specific interaction with human but not mouse IgA (Figure 7A). A similar result was obtained with immobilized CD89. Even high concentrations of mouse IgA did not show interaction with CD89, whereas the interaction with human IgA was dose dependent (Figure 7B). Also size-fractionated mouse serum (Figure 7C) and 10 different monoclonal mouse IgA antibody samples showed no detectable binding to CD89 (Figure 7D). When heat-inactivated human and mouse serum were added, interaction was seen only in human serum (Figure 7D).

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Fig. 7. Detection of IgACD89 interaction by plasmon resonance, using purified IgA (A and B), fractionated mouse serum () (C) and 10 different mouse monoclonal IgA antibodies (D). Human IgA and serum were used as positive controls.
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Discussion
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Over the past decades, several investigators have tried to develop an experimental model of IgAN. A recent study showed that transgenic mice expressing Fc
RI/CD89 on macrophages/monocytes developed massive mesangial IgA deposition and a clinical picture of IgAN. In the present study, we tried to design an experimental model of IgAN by injection of the human IgA receptor Fc
RI/CD89 in mice. These injections did not result in deposition of IgA or IgACD89 complexes in the renal mesangium. In addition, although high circulating levels of CD89 were reached, followed by rapid clearance, no indication was found for a decrease in serum IgA levels. Using three different techniques (ELISA, FACS analysis and surface plasmon resonance spectroscopy), we could only demonstrate interaction of human CD89 with human but not with mouse IgA. Additional experiments have to be performed to clarify the pathogenesis of mesangial IgA deposition in CD89 transgenic mice.
IgACD89 complexes have been suggested to play a role in the pathogenesis of primary IgAN [8]. Transgenic mice, expressing human CD89 on macrophages/monocytes, spontaneously developed massive mesangial IgA deposition after 12 weeks. Transfer of serum from CD89 transgenic mice to controls induced heavy macroscopic haematuria for 24 h accompanied by mesangial IgA deposits and cellular infiltration in intra- and periglomerular regions [8]. It was found that dimeric mouse IgA could bind to CD89, and the authors hypothesized that interaction between mouse polymeric IgA and the CD89 transgenic product resulted in a release of pathogenic soluble CD89IgA complexes. Further IgACD89 complexes were found only in patients with IgAN. We recently confirmed the presence of IgACD89 complexes, but showed that these complexes were not specific for IgAN [9]. In the present study, we tried to develop an experimental model of IgAN by i.v. injection of CD89 or a chimeric construct of this receptor in 7-week-old Balb/c mice. High levels of CD89 and Fc(CD89)2 were obtained, but after 3 h, nearly all injected CD89 and Fc(CD89)2 was cleared, with appearance in the liver. Although the peak levels of CD89 and Fc(CD89)2 were 300 µg/ml (compared with IgA levels of
200 µg/ml), IgA levels did not change, suggesting that CD89 was not cleared as an IgACD89 complex. Three hours after administration, renal biopsies showed no IgA or CD89 depositions. To exclude age, time and mouse strain as confounding factors, Fc(CD89)2 was injected in a repetitive way in 15-week-old Balb/c and C57BL/6 mice. Although CD89 could be detected in the liver, in both strains no mesangial CD89 could be detected. However, we cannot exclude that the absence of CD89 staining was due to degradation of CD89 epitopes by mesangial proteases. Staining of mesangial IgA was present, but this was not significantly different from control mice of this age. Reviewing the literature, the presence of spontaneous IgA deposition in BALB/c mice was not found by various investigators [8,17,18], but positive reports have been published [19]. There are various explanations for this apparent discrepancy, ranging from different sensitivities of the assays used, to differences in strains and substrains, to the housing conditions of the animals and to differences in dietary proteins. There was a high inter-individual variability in spontaneous IgA deposition in the different mice. Therefore, it is difficult to exclude that some additional deposition might be caused by CD89 injection. This would require follow-up biopsies of the same kidney before and after injection. However, all biochemical data in this study and from the literature, and the absence of a reduced serum IgA level after injection, make it unlikely that there is a major contribution of newly formed complexes.
The data presented herein suggest that mouse IgA does not interact with human CD89. This seems to be in contrast to the reported interaction of dimeric mouse IgA with human CD89 [8]. In this study, IgA binding was inhibitable by a monoclonal antibody against CD89. In another study, an interaction between mouse IgA and human cells was demonstrated; however, in that study, the specificity of this binding was not explored in relation to human CD89 [21]. Therefore, we investigated the interaction of different forms of mouse IgA with CD89 systematically, using three different techniques. In FACS, ELISA and surface plasmon resonance analysis, we were unable to detect binding of mouse IgA to CD89. Also when monoclonal mouse IgA antibodies or total mouse serum were used, no binding to the human IgA receptor CD89 was found. The latter experiment with total mouse serum suggests that there are no additional serum factors which can promote IgACD89 interaction. Our data are in line with previous studies suggesting the absence of interaction between mouse IgA and human CD89 [20]. CD89 binds to the loop regions lying at the interface of the CH2 and CH3 domains of human IgA. Mouse IgA differs from human IgA1 at only two residues in these loops. In a recent study, a human IgA1 mutant was generated which mimicked mouse IgA in these interdomain loops, which were devoid of rosette formation with either neutrophils or CD89 transfectants, suggesting the absence of a binding site for human CD89 in mouse IgA [20]. Subtle differences may exist between the recombinant CD89 in our model as compared with the CD89 in transgenic mice. A difference in glycosylation of CD89 cannot be excluded as an explanation of the difference in CD89IgA binding in the two systems. In this regard, FACS results might be affected by the use of transgenic macrophages as compared with transfected B cells. Finally, use of different reagents, sensitivities of assays or differences in glycosylation of IgA may be additional explanations. For example, Th1/Th2 balance is different in the mouse strains used and may be one of the factors responsible for the glycosylation of mouse IgA [16]. However, in both the Balb/c and the C57BL/6 mice, which were employed as the transgenic background, similar results were obtained in our experimental model. Additional studies are required to clarify the nature of the interaction between human CD89 and mouse IgA.
In the transgenic mouse model, CD89 was permanently present on the surface of monocytes, whereas in our model only three daily injections of CD89 were performed. However, injection of serum of transgenic mice in RAG-2/ recipients and injection of purified patient IgA in SCID transgenic mice resulted in mesangial human IgA deposition within 48 h [8]. Therefore, we think that more injections of CD89 will not significantly change the results obtained. In addition, in our model, more prolonged administration will be hampered by a specific immune response against human CD89. Further experiments have to be performed to explain the pathophysiology of this interesting experimental model of IgA nephropathy.
In summary, in the present study, an experimental model of IgAN could not be established by injection of human IgA receptor F
RI/CD89 in mice. This is compatible with the observed absence of interaction between mouse IgA and recombinant human CD89. Additional experiments and detailed biochemical analysis of IgACD89 interactions in both mice and men will be required to understand its role in the pathogenesis of IgAN.
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Acknowledgments
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Part of this study was supported by a grant from the Dutch Kidney Foundation (C99-1822).
Conflict of interest statement. None declared.
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Received for publication: 24.12.03
Accepted in revised form: 15. 7.04