RI/CD89 are not specific for primary IgA nephropathy
RI/CD89 are not specific for primary IgA nephropathy.Primary IgA nephropathy, the most common form of glomerulonephritis worldwide1,2, is characterized by the deposition of predominantly polymeric IgA of the IgA1-subclass in the glomerular mesangium3,4. The mechanism leading to renal deposition remains largely unknown, but overproduction of IgA1 with increased levels of macromolecular IgA, secondary to a primary mucosal hypoimmune response5, and abnormal glycosylation6,7,8,9 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 suggested10. Until now, several IgA receptors or IgA-binding molecules have been identified: the hepatic asialoglycoprotein receptor (ASPGR), the myeloid Fc
RI/CD89-receptor, the polymeric Ig-receptor (pIgR) on epithelial cells in the mucosa, the Fc/
-receptor on the majority of B lymphocytes and macrophages, and the recently described transferrin receptor11,12,13,14,15. In addition, two poorly-characterized IgA receptors have been reported on various cell types16,17,18,19. The asialoglycoprotein receptor and the myeloid IgA-receptor FcRI/CD89 are thought to constitute the main receptors for the catabolism of circulating IgA. There is indirect evidence for impaired IgA clearance through the ASPGR-route20, and more recently, the FcRI/CD89-receptor has been reported to play a crucial role in the clearance of IgA-complexes20,21. CD89 has a higher affinity for large molecular weight IgA as compared to monomeric IgA22. In addition, it was found that monomeric and dimeric IgA from patients have a reduced capacity to bind recombinant CD8923.
The mechanism of IgA deposition in the renal mesangium remains unclear. Binding of IgA has been demonstrated to human mesangial cells16, but no specific receptor has been identified19,24,25,26. The presence of the FRI/CD89-receptor on mesangial cells was excluded by several groups16,19,24,27. In addition, no CD89 was detectable in deposits of IgA in the glomerular mesangium of patients with IgAN19 or in experimental models28. After binding of IgA, myeloid cells can release a soluble form of the FRI/CD89-receptor that retains its ability to bind IgA29. Recently it was suggested that complexes of IgA with CD89 deposit in mice kidneys and initiate an inflammatory reaction28. Further, soluble FRI/CD89-IgA complexes were found only in the circulation of patients with IgA nephropathy. In the same study, transgenic mice expressing human CD89 on monocytes/macrophages spontaneously developed a clinical picture compatible with IgA nephropathy28. However, in our very recent study, significant levels of IgA-CD89 complexes also were found in healthy individuals30. The majority of the circulating CD89 was associated with high molecular weight IgA and was resistant to dissociation upon denaturation with sodium dodecyl sulfate (SDS). To evaluate these conflicting results, we compared the levels and characteristics of IgA-CD89 complexes in patients with IgAN and matched healthy controls. The results indicate that equal amounts of IgA-CD89 complexes are present both in patients and controls.
METHODS
Human subjects
Thirty-five patients with biopsy confirmed IgAN were included, defined by mesangial deposits of IgA as the dominant isotype. None of the patients had clinical or laboratory evidence of Henoch-Schönlein purpura, systemic lupus erythematosus, liver disease or received immunosuppressive therapy. In 20 patients with limited disease (13 males; mean age 46-years-old, range 30 to 66 years) kidney function was normal or moderately impaired (serum creatinine 
175 mol/L) and proteinuria 1.5 g/24 h. In 15 patients with advanced disease (13 males; mean age 42-years-old, range 19 to 62 years) kidney function was impaired (serum creatinine>175 mol/L) and/or proteinuria>1.5 g/24 h was present. Clinical characteristics of both patient groups are summarized in Table 1. As controls, 30 age- and sex-matched healthy volunteers (20 males; mean age 44-years-old; range 20 to 63 years) were recruited. Informed consent was obtained from all subjects.
Detection of CD89, IgA and IgA-CD89-complexes by ELISA
CD89 in serum was detected by enzyme-linked immunosorbent assay (ELISA) as described previously30. In short, several monoclonal antibodies against the extracellular domain EC1 (2D11, 2H8) or EC2 (7D7, A77) of CD89 (2 g/mL in coating buffer)19,31 or polyclonal antibodies against CD89 (4 g/mL) from rabbit origin were used as the catching antibodies. Purified recombinant CD89 was used as a standard and serum samples were tested in serial dilutions. Subsequently, CD89 was detected with digoxygenin (DIG)-conjugated rabbit F(ab')2 anti-CD89 (1 g/mL), followed by horseradish peroxidase (HRP)-conjugated F(ab')2 anti-DIG. The detection limit of recombinant CD89 was 50 pg/mL both for the monoclonal antibody coating and polyclonal antibody coating.
For quantification of IgA in samples, ELISA wells were coated with polyclonal rabbit--huIgA antibody (concentration 1 g/mL; Dako, Glostrup, Denmark), followed by serial twofold dilutions of samples for one hour at 37°C. Bound IgA was subsequently detected by affinity purified biotin labeled goat F(ab')2 fragments directed against IgA heavy chains (concentration 0.5 mg/mL, dilution 1/1000; Tago, Burlingame, CA, USA). Concentrations in samples were calculated relative to a known standard of IgA that was included in each plate. The detection limit of IgA was 2 ng/mL.
For detection of IgA-CD89 complexes, a similar ELISA was used with monoclonal antibodies against the extracellular domain EC1 (2D11, 2H8) or EC2 (7D7, A77) of CD89 (concentration 2 g/mL) or polyclonal anti-CD89-antibodies (concentration 4 g/mL) as catching antibodies.
The presence of soluble CD89 and IgA-CD89 complexes was analyzed in both serum and in polyethyleneglycol (PEG) 6000 (Merck-Schuchardt, Hohenbrunn, Germany) precipitates of serum. For this purpose, 500 L of serum was precipitated with a final concentration of 6% (wt/vol) PEG 6000 and redissolved in 500 L phosphate-buffered saline (PBS).
Affinity purification of IgA and gel-filtration
IgA was purified by immunoabsorption as described previously30. In the fall through and eluate fractions protein content was assessed by Pierce BCA Protein Assay (Pierce, Munich, Germany) and IgA-concentrations were assessed by sandwich ELISA. Eluate fractions containing IgA were pooled and frozen in aliquots at -20°C until further processing.
Affinity purified IgA, or total serum as a comparison, was separated by size on a 26/60 HR200 Superdex column and fractions were assessed for protein, IgA and CD89 as described before30.
Western blot analysis
Recombinant soluble CD89, as well as serum, affinity purified IgA and fall through (non-binding proteins of an IgA-immunoabsorbent) obtained from healthy individuals and patients with IgAN, were separated on 6% SDS polyacrylamide gels under non-reducing conditions, and blotted onto polyvinylidine difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) as described before30. CD89 and IgA were detected with the monoclonal anti-CD89 antibody 7D7 (IgG1) at 10 g/mL and the monoclonal anti-IgA antibody 4E8 (IgG1) at 2 g/mL, respectively32. Mouse monoclonal antibodies were detected with HRP-conjugated goat-anti-mouse-immunoglobulins (Dako).
Dot-blot assays
CD89-concentrations were also quantified by a dot-blot assay as described before30. For this purpose 50 L samples of affinity purified IgA or recombinant CD89 were dotted in PBS onto PVDF-membranes using a special dot-blot device (Bio-Rad, Herts, UK), according to the manufacturer's instructions. Membranes were developed for CD89 reactivity with monoclonal mouse-anti-CD89 (7D7) as described for Western blotting. Dilutions of a standard recombinant CD89 solution were included in each blot. The detection limit of recCD89 was 1 ng/mL.
Statistical analysis
Statistical calculations were performed using the Student t test.
RESULTS
Detection of CD89 and IgA-CD89 complexes
To quantify the amount of CD89 in serum of patients with IgAN and healthy controls, we used different CD89-specific ELISAs, based on monoclonal antibodies directed against the EC1 or EC2 domain of CD89 or polyclonal antibodies against CD89 as coating and polyclonal reagents of rabbit origin as the detecting antibodies. RecCD89 could be detected in a dose dependent fashion with a detection limit of 50 pg/mL in ELISAs using either monoclonal or polyclonal anti-CD89 antibody coating Figure 1a. In both control sera and in sera of patients with IgAN, no significant levels of CD89 were detected. In order to exclude the possibility that our ELISA system was influenced by serum itself, a fixed concentration of 200 pg/mL recCD89 was added to dilutions of control or patients serum and subsequently assessed for CD89. This serum did not influence the detection of CD89 Figure 1b.
Figure 1.
Detection of circulating CD89 by ELISA. (A) Wells were coated with a monoclonal antibody against CD89 (7D7), followed by a dose-response of CD89 and detection of bound CD89 with polyclonal digoxygenin conjugated anti-CD89. (B) At an input of 200 pg/mL recombinant CD89 in buffer alone or in the presence of increasing concentrations of normal (
) and patient (
) serum CD89 was measured in the above mentioned ELISA. Further, dose-responses of sera of a healthy control (
) and a patient with IgAN (
) are shown. (C) Control and patient serum was precipitated with PEG 6,000 in a final concentration of 6%. Subsequently, CD89 was detected in the precipitates as described in panel B.
It has been suggested that CD89 can be found in PEG precipitates of sera of patients with IgAN in complexed form28. Therefore, CD89 concentrations were analyzed in PEG precipitates of normal and patient serum. No increased levels of CD89 were found in these precipitates, and the addition of CD89 to dilutions of PEG-precipitates did not influence its detection Figure 1c. Using an IgA-CD89-ELISA with monoclonal and polyclonal anti-CD89 as the catching antibodies, in sera and PEG-precipitates of both patients with IgAN and controls, no significant levels of IgA-CD89-complexes could be detected (data not shown).
Presence of IgA-CD89 complexes in patients with IgAN and controls
In normal human serum CD89 is predominantly complexed with IgA30. We analyzed for the presence of CD89 and IgA using Western blot analysis in serum and affinity purified IgA, both from patients and controls. Under non-reducing conditions two main bands of IgA were seen in both control and patient sera; representative examples are shown in Figure 2a (left blot). CD89 was detectable as a band of 180 kD and associated with the lower band of IgA (Figure 2a, right blot). As a positive control the previously described recombinant CD8929 was visualized as a 30 kD band. When IgA was purified from serum of a patient with IgAN by immunoabsorption, bands of IgA and CD89 of similar size as that of normals were seen in both serum and purified IgA Figure 2b. Both for patients and controls, similar results were obtained with six individual sera for each group.
Figure 2.
Circulating CD89 in complex with IgA. (A) In Western blots of patient serum (lane 1, 30 L of a dilution 1/300), control serum (lane 2, 30 L of a dilution 1/300) and recombinant CD89 (lane 3, 30 L of a concentration of 60 ng/mL) were detected by monoclonal antibodies against IgA (4E8) and CD89 (7D7). (B) Purified IgA was obtained from serum of a patient with IgAN by immunoabsorption. In Western blots of total serum (lane 1, 30 L of a dilution 1/300), purified IgA (lane 2, 30 L) and fall through of the anti-IgA affinity column (lane 3, 30 L) were detected by monoclonal antibodies against IgA (4E8) and CD89 (7D7). IgA and protein concentrations in respectively the purified IgA and fall through were the same as in the starting material.
Quantitative isolation of IgA
The results above suggest that both in healthy subjects and in patients with IgAN, CD89 is associated with IgA. Therefore, we purified IgA from serum by affinity chromatography using anti-IgA-Sepharose. The results of IgA isolation are illustrated by representative examples of a healthy subject and a patient with IgA nephropathy. Almost all IgA was bound to the immunoabsorbent and could be eluted with acidic buffer both from the sera of patients with IgAN and healthy controls Figure 3. IgA was isolated in a similar way from the sera of all patients with IgAN and healthy control subjects, followed by quantification of the IgA concentrations in the eluates and original sera. The recovery of IgA was comparable in patients with IgAN and controls (92 
5% and 94 5%, respectively). The average content of IgA in IgAN was higher than in controls as reflected in the total amount of IgA in the eluates (110 42 vs. 94 24 g IgA).
Figure 3.
Affinity purification of serum IgA. Fifty microliters of serum of a control subject (A) and a patient with IgA nephropathy (B) were diluted 20-fold and incubated with 1 mL packed Sepharose IgA-immunoabsorbent, left to rotate at 4°C overnight and subsequently poured in a small column. Bound IgA was eluted with acidic buffer and neutralized with Tris-HCl. In the fractions, protein () and IgA () were assessed.
Quantification of IgA-CD89 complexes
In order to quantify the amount of CD89 in the affinity purified IgA preparations (thus, IgA-CD89 complexes), we developed a detection method for CD89. Because we were only interested in IgA-CD89 complexes and wanted to prevent disturbance of the assay by other proteins, the IgA-CD89 complexes were quantified in affinity-purified IgA. Dot-blotting of a dose-response of recombinant CD89 and subsequent detection with monoclonal antibodies (7D7) revealed that CD89 could be detected up to a limit of 10 ng/mL Figure 4. IgA-eluates from patients and controls in the same dot-blot assay also revealed a dose dependent relationship in the assay Figure 4.
Figure 4.
Detection of recombinant CD89 by dot-blot. Increasing concentrations of recombinant CD89 (A) or dilutions of affinity purified IgA from a control or a patient with IgAN (B) were dotted onto PVDF membranes and developed for CD89-reactivity. The number of pixels was counted with densitometry. In all cases a linear dose-dependent response of detectable CD89 was found.
Full figure and legend (54K)IgA-CD89 complexes in patients with IgAN and healthy controls
Affinity purified IgA from patients with IgA nephropathy and controls was assessed for IgA and CD89. IgA concentrations were determined using ELISA and CD89 by dot-blot assay. In normals the CD89 concentrations were 161 56 ng/mL and there were no significant differences in the two patient groups (150 28 and 152 26 ng/mL). Using the recovery percentages of the IgA isolation procedure for each sample, as mentioned earlier in this article, the calculated serum levels of CD89 were 24.2 8.5 g/mL for normals and 22.6 4.2 g/mL and 22.9 4.0 g/mL for the two patient groups, respectively. Calculated concentrations of CD89 and CD89 per g IgA were not significantly different in the patient group and the healthy controls (P = 0.41 and 0.30, respectively; Figure 5). On a weight basis, the CD89/IgA ratio was 1.1% and 1.0% for the control and patient group, respectively. On a molar basis this was 6,6% and 6.0%, respectively, which indicates that about 6 to 7% of the circulating IgA contains CD89.
Figure 5.
CD89 concentrations in serum from controls and patients with IgAN. (A) Using the recovery percentages of the isolation procedure, levels of CD89 were calculated in serum from controls and two groups of patients with IgAN: patients with limited disease (creatinine 175 mol and proteinuria 1.5 g/24 h) and patients with advanced disease (creatinine>175 mol and/or proteinuria>1.5 g/24 h). (B) In affinity purified IgA of the same groups, concentrations of IgA were measured by ELISA and subsequently the CD89/IgA weight ratios were calculated for each individual.
Proteinuria and impaired renal function are risk factors for progressive disease in IgA nephropathy. Based on renal function and proteinuria, patients were divided in groups with limited and advanced disease Table 1. No significant differences of CD89 (P = 0.83) and CD89 per g IgA (P = 0.10) were found between the both groups of patients with IgAN Figure 5.
Isolation of IgA has no effect on size distribution
It is possible that the affinity isolation procedure may affect the size of the isolated IgA as compared to that in whole serum. Therefore, 2 mL serum from a healthy individual as well as affinity purified IgA were fractionated by size using gel filtration, and then assessed for IgA by ELISA and for protein by Pierce. The results illustrate for one representative subject that no significant shift in size distribution of IgA occurs in the affinity isolation procedure relative to that in untreated serum. Further, the IgA and protein profile in the affinity purified IgA exhibited an overlap, suggesting that IgA was the major protein present in the purified samples Figure 6. Similar results were obtained with the sera and isolated IgA from six patients with IgAN and six controls.
Figure 6.
Gel-filtration on Superdex HR200 of normal serum and affinity purified IgA. (A) Two milliliters of serum of a healthy control was fractionated by gel-filtration and assessed for protein () and IgA (). (B) IgA from 2 mL of the same control subject was purified by affinity chromatography and subsequently fractionated and analyzed for protein and IgA as described above. No significant difference between filtration profile of IgA in serum and purified IgA was found.
Size distribution of IgA-CD89 complexes in purified IgA
Recently, we have shown that covalently bound IgA-CD89 complexes in human serum circulate in a polymeric state30. In order to investigate the size distribution of CD89 in affinity purified IgA from patients with IgAN, purified IgA was fractionated by gel-filtration and assessed for IgA by ELISA and CD89 by dot-blot assay. The results indicate that CD89 both in patients with IgAN and controls is associated with high molecular weight IgA Figure 7. More than 80% of the IgA-associated CD89 was found in the fractions containing high molecular weight IgA.
Figure 7.
Size distribution of CD89 in IgA eluates from a control and patient with IgAN. Affinity purified IgA samples from a control (A) and patient with IgAN (B) were separated by size on Superdex HR200. Fractions were assessed for CD89 using dot-blot assay () and IgA using ELISA ().
DISCUSSION
The present study demonstrates that IgA-CD89 complexes are present in serum of patients with IgA nephropathy and healthy subjects. After affinity purification, the relative recovery of IgA from the sera of both groups was similar and the isolation procedure did not significantly affect the size distribution of IgA. CD89 was found associated with high molecular weight IgA and IgA-CD89 levels were comparable in both groups. These results do not support the hypothesis that circulating IgA-CD89 complexes are specific for patients with IgA nephropathy.
IgA-CD89 complexes have been suggested to play a role in the pathogenesis of primary IgA nephropathy. These complexes were found mainly in serum of patients with IgAN. In the same study transfer of serum from CD89 transgenic mice to controls led to rapid mesangial deposition of IgA28. However, recently we found that CD89 is present in the circulation of healthy human subjects30. To investigate the specificity of IgA-CD89 complexes for IgA nephropathy, we analyzed the presence and size characteristics of IgA-CD89 complexes in patients with primary IgAN and healthy controls.
Analysis of the levels of IgA-CD89 complexes with the dot-blot method revealed no significant differences between patients with IgAN and healthy controls. There was also no correlation between the presence and level of IgA-CD89 complexes and parameters associated with impaired renal prognosis. Patients with limited and patients with advanced disease had equal levels of IgA-CD89 complexes. Assuming that the monoclonal anti-CD89 antibody 7D7 has the same affinity for recombinant CD89 and CD89 complexed with IgA, concentrations of approximately 20 to 25 g CD89 per mL serum were found. This estimate is somewhat higher, but probably more accurate, than our initial dot-blot measurement using polyclonal antibodies and total serum30. At an average serum IgA-concentration of 2 mg/mL, on a weight basis the CD89/IgA-ratio was 1.1% and on a molar basis 6.6%. This indicates that about 6 to 7% of serum IgA might be complexed with CD89.
Experiments with fractionated purified IgA showed that the size distribution of IgA was not affected by the isolation procedure. In patients and control subjects CD89 was associated with high molecular weight IgA (300 to 600 kD). Under denaturing conditions of Western blotting these high molecular weight complexes appeared as smaller 180 kD IgA-CD89 complexes. Recently, we hypothesized that CD89 and monomeric IgA form a covalently linked complex, which seems to facilitate multimerization to polymeric serum IgA and circulates in high molecular mass fractions30. Most likely in an ELISA system these covalent IgA-CD89 complexes cannot be detected due to the tertiary structure of these complexes. In our dot-blot system the denaturing conditions might uncover these hidden antigenic determinants. This could explain the apparent discrepancy between the amount of CD89 detected in dot-blot (20 to 25 g/mL) and the absence of ELISA reactivity. In an earlier study, low levels of circulating IgA-CD89 complexes were found by ELISA exclusively in patients with IgAN28. Since under these conditions covalent complexes are most likely not detected, this might represent other forms of IgA-CD89 complexes.
In summary, our results indicate that the presence of IgA-CD89 complexes in the circulation alone is insufficient to explain mesangial deposition of IgA in patients with IgAN. Apparently other factors such as local cytokine production, altered glycosylation6,7,8,9 or expression of as yet undefined IgA receptors are required in addition to the presence of IgA- or IgA-CD89 complexes in the mesangial deposits.
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