Interactions of human mesangial cells with IgA and IgA-containing immune complexes

Kidney International, Vol 62 (2002), pp 465–475 Interactions of human mesangial cells with IgA and IgA containing immune complexes1 JAN NOVAK, HUONG L VU, LEA NOVAK, BRUCE A JULIAN, JIRI MESTECKY, and[.] Kidney International, Vol 62 (2002), pp 465–475 Interactions of human mesangial cells with IgA and IgA-containing immune complexes1 JAN NOVAK, HUONG L VU, LEA NOVAK, BRUCE A JULIAN, JIRI MESTECKY, and MILAN TOMANA Departments of Microbiology, Pathology, and Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA Interactions of human mesangial cells with IgA and IgA-containing circulating immune complexes Background IgA nephropathy (IgAN) is characterized by IgA1-containing immune complexes in mesangial deposits and in the circulation The circulating immune complexes (CIC) are composed of galactose- (Gal) deficient IgA1 and IgG or IgA1 antibodies specific for the Gal-deficient IgA1; interactions of these CIC with mesangial cells (MC) were studied Methods Binding, internalization, and catabolic degradation of myeloma IgA1 protein as a standard control and the isolated CIC were studied using human MC, hepatoma cell line HepG2 expressing the asialoglycoprotein receptor (ASGP-R), and monocyte-like cell line U937 expressing the Fc␣-R (CD89) Biochemical and molecular approaches were used to assess expression of CD89 and ASGP-R by MC Results At 4⬚C, radiolabeled IgA1 bound to MC and HepG2 cells in a dose-dependent and saturable manner The binding was inhibited by IgA-containing CIC or excess IgA1 or its Fc fragment but not by the Fab fragment of IgA1 At 37⬚C, the cell-bound IgA1 was internalized and catabolized In addition to IgA1, HepG2 cells also bound (in a Ca2⫹-dependent manner), internalized, and catabolized asialoorosomucoid (ASOR), other asialo-(AS)-glycoproteins, and secretory component (SC) The binding by MC appeared to be restricted to IgA1 or ASIgA1 and was not Ca2⫹-dependent Furthermore, MC and HepG2 cells internalized and catabolized IgA1-containing CIC Using RT-PCR with ASGP-R- or CD89-specific primers, mRNAs of the two respective genes were not detected in MC Conclusions The data showed that the ability of MC to bind IgA1 and IgA1-containing CIC in vitro was mediated by an IgA receptor that was different from CD89 or ASGP-R and had a higher affinity for IgA-CIC than for uncomplexed IgA vated levels of IgA1 and IgA1-containing circulating immune complexes (CIC) are found in sera of most IgAN patients [4–7] IgA1-containing immune deposits are observed in the renal mesangium (usually with C3 complement component, and often with IgG, IgM, or both) [8] The progression of the disease is characterized by proliferation of mesangial cells and expansion of extracellular matrix (ECM), leading ultimately to glomerular sclerosis [9] About 30 to 40% of IgAN patients develop kidney failure within 20 years of clinical onset; these patients require either dialysis or kidney transplantation [10] IgAN recurs in approximately 50 to 60% of patients after kidney transplantation [11] On the other hand, a kidney transplanted from a donor with subclinical IgAN to a patient without IgAN clears the immune deposits after several weeks [12] It is therefore apparent that IgAN arises from formation and deposition of immune complexes rather than from a defect inherent in the kidney [2, 4–6, 8, 13–16] IgA immune deposits are likely derived from CIC, however, the nature of the antigens in the CIC is unknown [4, 5, 14] Based on our experimental data, we postulated that aberrant glycosylation of IgA1 immunoglobulins in the circulation of IgAN patients leads to formation of CIC [6] Incomplete galactosylation of O-linked glycans in the IgA1 hinge region results in the exposure of underlying neo-antigenic N-acetylgalactosamine (GalNAc) [6, 16] that is recognized by naturally occurring antibodies (IgG or IgA1 specific for GalNAc) [6] We further postulated that these CIC deposit on mesangial cells (MC), resulting in MC proliferation and overexpression of extracellular matrix [15, 17] Recently, highly undergalactosylated IgA1 was detected in the mesangial deposits in IgAN patients, and this further supports our hypothesis [18, 19] In this regard, understanding the mechanisms by which CIC bind to MC is of utmost importance [10] Several studies evaluating the nature of an IgA receptor expressed on MC have provided conflicting results Some Idiopathic IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world [1–3] Ele1 See Editorial by Go´mez-Guerrero, Suzuki, and Egido, p 715 Key words: renal mesangial cells, circulating immune complexes, IgA nephropathy, glomerulonephritis Received for publication March 26, 2001 and in revised form March 7, 2002 Accepted for publication March 22, 2002  2002 by the International Society of Nephrology 465 466 Novak et al: Mesangial cells and IgA-containing complexes found Fc␣R (CD89) or the asialoglycoprotein receptor (ASGP-R) expressed by cultured MC or in some renal biopsy samples from IgAN patients [20–23] Other studies showed binding of IgA1 to MC but did not confirm expression of CD89 by MC [24–27] Most of these studies were conducted using uncomplexed [monomeric (m), polymeric (p)] or heat-aggregated IgA1 However, in light of recent findings on the role of CIC containing aberrantly glycosylated IgA1 in the pathogenesis of IgAN [6, 10, 17–19], it is important to examine interactions of MC with not only free IgA1 and de-galactosylated IgA1, but also with galactose-deficient IgA1 bound in CIC Our study reports that human MC in primary culture bind, internalize, and catabolically degrade IgA1 and IgA1-containing CIC HepG2 cells expressing ASGP-R [28, 29] and U937 expressing Fc␣R (CD89) served as a positive controls ASGP-R is a hetero-oligomer of two subunits, H1 and H2, encoded by separate genes [30, 31] ASGP-R on hepatocytes binds glycoproteins with terminal Gal or GalNAc residues, and the bound glycoproteins are subsequently internalized and catabolized [28, 29, 32] A standard probe to detect ASGP-R is asialoorosomucoid (ASOR) [28, 29] that is rapidly internalized and catabolized by hepatocytes upon binding [6, 29] ASGP-R also binds secretory component (SC) [28], a highly glycosylated fragment of the pIg receptor (pIgR) that remains bound to secretory IgA or IgM [33–35] Eventual catabolic degradation of SC or ASOR by MC thus would confirm the previously postulated presence of ASGP-R on MC [21] Fc␣R (CD89) is a glycoprotein expressed on myeloid cells that binds both IgA subclasses [36] Binding of IgA to the Fc␣R also may lead to internalization and catabolic degradation [28] Using these control cells, we have demonstrated differences in properties and binding specificities of IgA1 receptors expressed by human MC, HepG2 and U937 cells Our observations indicated that the ability of MC to bind IgA1 and IgA1-containing CIC in vitro is mediated by a new type of IgA receptor with higher affinity for IgACIC than for uncomplexed IgA METHODS Cells Human MC were isolated from the normal portions of kidney cortexes of tumor nephrectomy specimens (2 cell cultures) as described before [37, 38] or purchased as primary cells (2 cell cultures) from a commercial source (Clonetics, San Diego, CA, USA) Cells from passages to were used in our experiments MC were maintained in RPMI 1640 supplemented with 20% fetal calf serum (FCS), l-glutamine (2 mmol/L), penicillin G (100 U/mL), streptomycin (0.1 mg/mL) in humidified 5% CO2 atmosphere at 37⬚C Purity and identification of MC was based on cell morphology and immunohistochemical features: (a) positive staining for vimentin, and (b) negative staining for factor VIII-related antigen and cytokeratin (to exclude contamination with endothelial and epithelial cells, respectively) Human hepatocarcinoma cell line HepG2 and human monocytic cell line U937 (both obtained from ATCC, Rockville, MD, USA) were maintained in RPMI 1640, 10% FCS, and antibiotics [28] MC for binding experiments were serum starved (medium contained only 0.5% FCS) 24 hours before the experiment Isolation of IgA1 proteins, their Fab and Fc fragments, and IgA1-containing CIC IgA1 and IgA2 myeloma proteins were isolated from plasma of several different patients with multiple myeloma by precipitation with ammonium sulfate, starchblock electrophoresis, and size-exclusion and ion-exchange chromatography [6, 33] The polymeric forms of IgA1 (Mce) and IgA2 (Fel) were used [6] and their polymeric character was demonstrated by size-exclusion chromatography and sodium dodecyl sulfate (SDS)-gel electrophoresis under non-reducing conditions The Fab fragment of IgA1 myeloma protein (Ste) was prepared by cleavage with IgA1 protease from Haemophilus influenzae (HK50); Fab and Fc fragments of IgA1 (Mce) were generated using IgA1 protease from Neisseria gonorrhoeae [39, 40] The resulting Fab and Fc fragments were purified by size-exclusion chromatography [33] and their purity was verified by SDS electrophoresis Fractions rich in Gal-deficient IgA1-containing CIC were prepared from sera of IgAN patients by size-exclusion chromatography on a calibrated Superose column [6]; high-molecular-mass fractions reactive with Helix aspersa (HAA) lectin (which binds terminal GalNAc of Gal-deficient O-glycans of IgA1; Sigma, Chemical Company, St Louis, MO, USA) and containing IgA and IgG were pooled [6] Asialo- (AS) IgA1, asialo-agalacto IgA1 and ASOR were prepared from native proteins by incubation with neuraminidase (from Vibrio cholerae) and ␤-galactosidase (from bovine testes that preferentially cleaves ␤1,3 linkages) [6, 16, 28, 41] SC was isolated from human milk [33] IgG was purified from normal human serum by ammonium sulfate precipitation, and subsequent ionexchange (DEAE-cellulose) and affinity chromatography on a Protein-G column [42] To prepare in vitro a complex of IgG bound to Galdeficient pIgA1, we incubated 125I-labeled degalactosylated IgA1 (Mce) [6] with purified human IgG with antiGalNAc activity [6] Free IgA1 was separated from the IgG-IgA1 complexes by size-exclusion chromatography on a Superose column The fractions containing IgG bound to the radiolabeled IgA1 were identified by using Protein-G-coated Immulon Removawell strips (Dynatech Laboratories, Alexandria, VA, USA) and a Packard Novak et al: Mesangial cells and IgA-containing complexes model 5110 gamma spectrometer (Packard Instrument Company, Downers Grove, IL, USA) Radioiodination and tetramethylrhodamine isothiocyanate (TRITC)-labeling Proteins were radiolabeled with carrier-free Na125I by the lactoperoxidase method [43] The excess free Na125I was separated from the protein by size-exclusion chromatography on a column of Sephadex G-25 [28] IgA1 (Mce) was TRITC-labeled using 0.02 mg TRITC per mg protein in 2% bicarbonate buffer, pH 8.2 After overnight incubation, free TRITC was removed on a column of Sephadex G-50 and the TRITC-IgA1 conjugate was isolated using ion-exchange chromatography on a column of DEAE-cellulose The TRITC-labeled protein was aliquoted and stored at ⫺70⬚C Binding experiments Mesangial cells (or HepG2 cells) grown in 24-well plates (60-80% confluent) were washed twice with 20 mmol/L HEPES buffer, pH 7.3, containing 140 mmol/L NaCl, 0.8 mmol/L MgCl2, 0.34 mmol/L K2HPO4, 0.34 mmol/L KH2PO4, (buffer A), and incubated with a radiolabeled protein in 0.2 mL buffer B [buffer A supplemented with 2.7 mmol/L CaCl2 and 1% bovine serum albumin (BSA)] on ice for one hour [28] Buffer B without CaCl2 supplement also was used in some experiments, as described in the text In the inhibition experiments, MC were preincubated for one hour with inhibitors on ice, before radiolabeled IgA1 was added and incubated for another one hour Following extensive washing with buffer B, cells were lysed with 0.4 mL 0.3 N NaOH and the radioactivity of the lysate was determined in a ␥ spectrometer Cell count was determined with cells released by trypsin/ ethylenediaminetetraacetic acid (EDTA) treatment using a hemocytometer Catabolic degradation of internalized proteins Mesangial cells and HepG2 in tissue culture flasks were incubated with radiolabeled proteins (⬃1 ␮g of ASOR, AS-IgA1, or immune complexes) in mL buffer B at 37⬚C for four hours Radioactivity was determined in (a) incubation medium, (b) cells washed and released by trypsinization, and (c) released cells after additional trypsinization to remove protein bound on cell surface Catabolic degradation was expressed as percentage of total radioactivity not precipitable by 10% trichloroacetic acid (TCA; from incubation medium or cell lysate, as specified in the text) [28] Protein in the cell lysate was determined spectrophotometrically using a BioRad assay (BioRad, Hercules, CA, USA) with BSA as the standard To determine molecular masses of proteins catabolically degraded, MC were incubated in mL HEPES buffer, pH 7.3 containing 1% BSA with 10 ␮g 125I-labeled IgA1 at 37⬚C for 16 hours The cells were washed with 467 HEPES buffer, lysed with a polyacrylamide gel electrophoresis loading buffer containing 2% SDS, and analyzed by SDS-polyacrylamide gel electrophoresis (SDSPAGE), using 1.5 mm thick vertical slab gels (20 ⫻ 20 cm) with to 20% polyacrylamide gradient [28] The gels were fixed, frozen with dry ice, and sliced with a gel slicer The radioactivity in 1-mm sections was measured by a gamma spectrometer Confocal microscopy Mesangial cells grown on a microscope slide were incubated with TRITC-labeled IgA1 (Mce; 10 ␮g) overnight at 4⬚C, extensively washed with RPMI medium containing 0.5% FCS and then incubated at 37⬚C for four hours, followed by incubation at room temperature for two hours Imaging was performed on a Leica DMIRBE inverted epifluorescence/Nomarski microscope outfitted with Leica TCS NT Confocal optics The system is equipped with UV, argon ion, krypton ion, and helium/neon lasers for imaging in a wide range of blue, red, and far-red fluorescence The laser was set to optimal TRITC excitation wavelength to observe the internalized TRITCIgA1 Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA isolated from the cells (MC, HepG2, and U937) with RNA-Stat-60 reagent (Tel-Test, Friendswood, TX, USA) was used for RT-PCR HepG2 and U937 cells served as controls for ASGP-R and CD89 expression, respectively First-strand cDNA synthesis was performed at 42⬚C for 15 minutes, followed by 37⬚C for 45 minutes using murine leukemia virus reverse transcriptase and oligo(dT)16 as the primer The cDNA was amplified using the following primers that we designated based on the sequences of the correspond ing genes submitted to GeneBank [30, 31, 44] for Fc␣ receptor (CD89): CD89F 5⬘-AGCACGATGGACCCCAAACA GA-3⬘; CD89R 5⬘-CTGCCTTCACCTCCAGGTGTT-3⬘, and the following primers for H1 and H2 genes encoding the two ASGP-R subunits: H1F 5⬘-CTGGACAAT GAGGAGAGTGAC-3⬘; H1R 5⬘-TTGAAGCCCGTC TCGTAGTC-3⬘; H2F CCTGCTGCTGGTGGTCATC TG-3⬘; H2R 5⬘-CCCATTTCCAAGAGCCATCAC-3⬘ A pair of primers specific to ␤-actin (F 5⬘-TTCCAGCC TTCCTTCCTGG-3⬘; R 5⬘-TTGCGCTCAGGAGGAG CAA-3⬘) was used as a control PCR was performed in a DNA thermal cycler using 94⬚C melting, 60⬚C annealing, and 72⬚C extension temperatures for 35 cycles PCR amplicons were analyzed on 2% NuSieve 3:1 agarose gels RESULTS Binding of IgA1 and IgA1-containing CIC to MC To determine binding characteristics of IgA1, MC were incubated for one hour on ice with various concentra- 468 Novak et al: Mesangial cells and IgA-containing complexes Fig Binding of 125I-labeled IgA1 (Mce) myeloma protein (䊏) and 125 I-labeled IgA1 (Mce) with partially degalactosylated and desialylated O-linked glycans ( ) to mesangial cells (MC) MC were incubated for one hour on ice with ␮g radiolabeled proteins Cells were washed and the bound radioactivity determined as described in the Methods section tions of radiolabeled pIgA1 (Mce) myeloma protein Consistent with earlier reports, the binding of the pIgA1 to MC was dose-dependent and saturable The binding of radiolabeled pIgA1 was inhibited by 68% using a 150fold excess of unlabeled pIgA1 Analyses of IgA1 binding to MC using a Scatchard plot (not shown) suggested a single population of receptors with approximately ⫻ 105 binding sites per cell and Ka 4.79 ⫻ 106 mol/L⫺1 The calculations were based on the assumption that pIgA1 (Mce) was predominantly polymeric, as judged from its elution profile on a calibrated size-exclusion high pressure liquid chromatography (HPLC) column (TSK 5000) Furthermore, we assumed that each receptor bound only one IgA1 molecule Because the mesangial immune deposits in IgAN patients contain aberrantly glycosylated (Gal-deficient O-linked glycans) IgA1 [18, 19], we examined MC binding of Gal-deficient IgA1 myeloma protein The binding of IgA1 that was modified by treatment with neuraminidase and ␤-galactosidase (that removes preferentially ␤1,3 bound Gal) was more than twofold greater compared with the unmodified control (Fig 1) In sera of IgAN patients, however, the aberrantly glycosylated IgA1 [16, 45–48] is not free, but rather is complexed with IgG (or IgA1) in CIC [5, 6, 16] To prepare IgA1containing CIC, serum from an IgAN patient was fractionated using size-exclusion chromatography Fractions with Gal-deficient IgA1 were identified with GalNAcspecific lectin (HAA) in ELISA (Fig 2) The HAAreactive fractions (CIC of molecular mass 700 to 900 kD), designated as CIC containing Gal-deficient IgA1, were pooled and used in further experiments Binding of radiolabeled Gal-deficient pIgA1 was inhibited by unlabeled Gal-deficient pIgA1 (Fig 3A), but Fig Isolation of Gal-deficient IgA1 containing CIC from serum of an IgAN patient Serum (0.5 mL) was fractionated on a Superose column (0.9 ⫻ 60 cm), 0.25 mL fractions were collected and analyzed by ELISA for IgA (䉭), IgG (䊐), and reactivity with HAA lectin (䊉; specific for terminal GalNAc, thus reacting with Gal-deficient IgA1) Fractions containing CIC with aberrantly glycosylated IgA1 (molecular mass about 700 to 900 kD) were pooled and used in the experiments not by the Fab fragment of IgA1 (that contained a portion of the hinge region) Surprisingly, CIC containing aberrantly glycosylated IgA1 appeared to be better inhibitors than uncomplexed monomeric or polymeric IgA1 (Fig 3A) The same amount of uncomplexed IgA1 (Galdeficient or normally glycosylated) had no significant inhibitory effect (data not shown), suggesting that properties such as spatial organization of IgA1 in CIC may play a role in MC binding By comparing the inhibitory activity of Fab and Fc portions of IgA1, respectively, we concluded that IgA1 bound to a MC IgA receptor by its Fc portion (Fig 3B) This is consistent with our finding that both IgA subclasses (IgA1 and IgA2) bound to MC (data not shown) The results in the prior section suggested better binding of IgA1 in CIC compared with free IgA1 To verify this finding, sera from three IgAN patients were fractionated using size-exclusion chromatography on a calibrated Superose column and analyzed for IgA, and for reactivity with HAA The fractions corresponding to mIgA and to IgA complexed in CIC were incubated with MC grown on microscope slides After three hours of incubation at 4⬚C, MC were washed with phosphate-buffered saline (PBS) and stained with TRITC-conjugated F(ab⬘)2 fragment of anti-human IgA antibody and examined with a fluorescence microscope MC incubated with CIC showed strong IgA binding, while MC incubated with uncomplexed IgA exhibited only background-level fluorescence Novak et al: Mesangial cells and IgA-containing complexes 469 Fig Inhibition of 125I-labeled Gal-deficient pIgA1 (Mce) binding to MC MC grown to 80% confluence in a 24-well plate were pre-incubated with inhibitors for one hour on ice and then about ␮g radiolabeled protein was added Cells were washed and the bound radioactivity was determined as described in Methods (A) Control is no inhibitor, and inhibitors are Gal-deficient pIgA1 Mce (100 ␮g), mIgA from serum of an IgAN patient (⬃50 ␮g), and Gal-deficient-IgA1-containing CIC from serum of an IgAN patient (⬃1 ␮g) (B) Control is no inhibitor, and inhibitors include Gal-deficient pIgA1 Mce (100 ␮g), Fab fragment of IgA1 (100 ␮g), and Fc fragment of IgA1 Mce (100 ␮g) These results indicated that IgA1 bound to MC through the Fc part of its molecule because IgA1 or its Fc fragment, but not its Fab fragment, inhibited IgA binding Furthermore, abnormalities of O-linked glycans of IgA1 and spatial organization of IgA1 molecules clustered in CIC influenced binding to MC, favoring IgA in CIC over uncomplexed IgA Catabolism and internalization of IgA1 by MC To detect potential degradation products of cell-associated and internalized IgA1, the radiolabeled protein was added to MC and incubated for 16 hours; the cells were then washed, lysed, and the lysate was analyzed by SDS gel electrophoresis under non-reducing conditions (Fig 4) Generation of protein fragments smaller than 30 kD was observed after incubation with MC, indicating catabolic degradation of the 125I-labeled IgA1 This finding is consistent with decreased TCA-precipitable radioactivity To visualize internalization of IgA1 by MC, we incubated MC grown on a microscope slide with TRITClabeled IgA1 The cells were then fixed and observed with a confocal laser scanning microscope Many MC showed intracellular fluorescent vesicles, which indicated internalization of IgA1 (Fig 5) Internalization and catabolism of AS-IgA1, ASOR, and SC by HepG2 and MC Asialoglycoprotein receptor has been reported as one of the receptors responsible for binding of IgA1 to MC [21] To verify this report, we incubated 125I-labeled ASIgA1 with MC and HepG2 cells HepG2 cells express Fig Catabolism of 125I-labeled IgA1 (Mce) by MC About 10 ␮g 125 I-labeled protein was added to the cells in tissue culture flask and incubated in mL HEPES buffer with 1% BSA at 37⬚C for 16 hours The cells were then washed, and lysed using 2% SDS buffer and the lysate was analyzed by SDS gel electrophoresis under non-reducing conditions (solid line) The distribution of radioactivity in the gel was determined by assaying the radioactivity of 1-mm sections of the gel in a gamma counter Control 125I-labeled IgA1 (dotted line) was electrophoresed in parallel Migration of standards is shown by arrows ASGP-R [28, 29] and served as a positive control The cell cultures were then washed, treated with trypsin to release cells from the flasks and surface-bound radiolabeled protein from cells After further washing, the cells were lysed with NaOH The internalized protein was de- 470 Novak et al: Mesangial cells and IgA-containing complexes Fig Confocal laser scanning photomicrograph of IgA1 internalized by a MC MC grown on a microscope slide were incubated with TRITC-labeled IgA1 (Mce; 10 ␮g) overnight at 4⬚C, extensively washed with RPMI 1640 medium containing 0.5% FCS and incubated at 37⬚C for four hours, followed by incubation at room temperature for two hours A single MC is shown with fluorescent vesicles in the cytoplasm Bar depicts 20 ␮m tected as radioactivity in the trypsin-treated and washed cells AS-IgA1 was internalized by both cell cultures (Fig 6) Under the same conditions, HepG2 internalized sixfold more AS-IgA1 per mg cell protein than did MC To estimate the kinetics of catabolic degradation of internalized proteins, percentage of the radiolabeled protein not precipitable with TCA was determined HepG2 cells catabolized 47% of the internalized AS-IgA1 compared to 39% for MC Unlike with HepG2, the binding and internalization of AS-IgA1 by MC was not Ca2⫹-dependent (data not shown) Earlier studies demonstrated that ASGP-R on hepatocytes or HepG2 cells is responsible for rapid internalization and catabolic degradation of ASOR [28] We investigated whether ASOR also is internalized and catabolized by MC 125I-labeled ASOR incubated with HepG2 and MC was internalized 144-fold more effectively by HepG2 than MC (Fig 6) Likewise, the catabolic degradation was more effective in HepG2 cells Furthermore, it was also reported that HepG2 internalize and catabolize SC [28] Therefore, we compared catabolism of SC and IgA1 in MC (Table 1) More than 99% of original 125I-labeled SC incubated with MC remained intact, while the original 125I-labeled IgA1 was partially (about 13%) catabolically degraded during the four-hour incubation These data demonstrated that SC, but not IgA1, escaped catabolic degradation by MC Therefore, ASGP-R is missing or nonfunctional on MC Binding and internalization of IgA1-containing CIC by HepG2 and MC Studies described above indicated that IgA1-containing CIC bound to MC more effectively than free IgA1 Novak et al: Mesangial cells and IgA-containing complexes Fig Internalization of 125I-labeled ASOR and 125I-labeled asialo(AS) IgA1 (Mce) by HepG2 and MC About ␮g of each radiolabeled protein was added to the cells in tissue culture flask and incubated in mL buffer B at 37⬚C for four hours Cells were washed and radioactivity measured after trypsinization to release surface-bound molecules Results represent an average from experiments conducted in triplicates 471 Fig HepG2 and MC cell-associated (bound and internalized) 125 I-labeled Gal-deficient IgA1-containing CIC isolated from serum of an IgAN patient (䊏) and CIC from a healthy control ( ) About ␮g aliquots of the radiolabeled proteins were added to the cells in tissue culture flasks and incubated in mL buffer B at 37⬚C for four hours Cells were washed before the cell-associated radioactivity was measured Results are averages from experiments conducted in triplicates Table Catabolism of radioiodinated secretory component (SC) and IgA1 (Mce) by human mesangial cells TCA precipitable protein % Before incubation After incubation SC IgA1 (Mce) 93.9 93.0 95.6 82.8 One-microgram aliquots of the SC or IgA1 proteins were added to MC in cultivation flasks and incubated for hours at 37⬚C Then, the supernatant was collected, precipitated with TCA, and the intact protein (TCA-precipitable radioactivity measured using gamma counter) was expressed as % of TCA-precipitable radioactivity The experiment was conducted in triplicate To examine the possible role of liver cells and ASGP-R in binding and processing of these CIC, we compared the binding and catabolism of these CIC by MC and HepG2 Gal-deficient IgA1-containing CIC were purified from serum of an IgAN patient and control CIC were isolated from serum of a healthy volunteer using size-exclusion chromatography [6] These CIC (molecular mass about 700 to 900 kD) were radioiodinated, and incubated with MC and HepG2 MC bound and internalized more protein from the IgAN-CIC than from control CIC On the other hand, HepG2 cells bound less protein from the IgAN-CIC (Fig 7) We hypothesized that the lower degree of internalization of IgAN-CIC by hepatoma cells may be due to the presence of GalNAc-specific IgG [6] that bound to IgA1 and thus prevented IgA1 hinge region O-glycans [32] or Fc glycans [49, 50] from binding to ASGP-R on HepG2 cells To test this hypothesis, we prepared in vitro 125I-labeled Gal-deficient IgA1 in a free form and bound to GalNAc-specific IgG and used HepG2 cells to assess the effect on internalization of the radiolabeled IgA1 Complexing IgG with the IgA1 reduced the binding and internalization by HepG2 cells (Fig 8) Fig Internalization of a complex of IgG-Gal-deficient pIgA1 (䊏) and free Gal-deficient pIgA1 ( ) by human hepatoma cell line HepG2 125 I-labeled degalactosylated IgA1 was incubated with purified human IgG with anti-GalNAc activity Free IgA1 was separated from the IgGIgA1 complexes by size-exclusion chromatography on Superose column The fractions containing IgG bound to the radiolabeled IgA1 were detected by capture radioimmunoassay using Protein-G-coated Removawell strips and gamma-counter detection The proteins were incubated with HepG2 cells at 37⬚C for three hours, then the cells were washed, treated with trypsin, and the radioactivity was measured with a gamma counter and expressed per mg of cell protein In summary, these experiments indicated that MC bound and internalized IgA1-containing CIC via a receptor different from ASGP-R MC bound more effectively the CIC from an IgAN patient than that from a healthy control Furthermore, CIC from an IgAN patient were less efficiently internalized by hepatoma cells (HepG2) than control CIC The IgG bound to Gal-deficient IgA1 apparently masks the binding sites on IgA1 glycans from ASGP-R or interferes with an efficient internalization 472 Novak et al: Mesangial cells and IgA-containing complexes Fig RT-PCR of Fc␣R (CD89) transcripts in MC and U937 cells Total RNA was reversetranscribed and the cDNA was PCR-amplified with CD89-specific primers The amplicons were separated on 2% agarose gel and photographed under UV light Lane 1, molecular size standards; lanes 2-5, ␤-actin RT-PCR in MC; lane 6, ␤-actin RT-PCR in U937; lane 7, RT-PCR of Fc␣R transcripts in U937 with three signals detected that correspond to the a.1, a.2, and a.3 splicing variants; lanes 8-11, RT-PCR of mRNA from MC grown under various conditions failed to reveal any CD89specific signal (lanes 10, 11, MC were supplemented with insulin-like growth factor; lanes 9, 10, 5% glucose was added to the storage medium) CD89 and ASGP-R mRNA expression in MC To determine a possible involvement of CD89 in binding IgA to MC, RT-PCR was used to examine whether the CD89 gene is transcribed in MC Total RNA isolated from MC grown with, or without, insulin-like growth factor and from U937 cells served as templates for RT followed by PCR amplification with CD89-specific primers The results did not indicate the presence of CD89 mRNA in MC, although all three major splicing products, a.1, a.2, and a.3, (observed as PCR amplicons of about 0.9 kb, 0.85 kb, and 0.62 kb, respectively) of CD89 mRNA were detected in samples from CD89-positive U937 cells (Fig 9) Adding insulin-like growth factor (known to alter gene expression in MC [51] and used in experiments by others [20, 52, 53]) to the growth medium or glucose [54] to the storage medium did not induce expression of CD89 (Fig 9) Furthermore, we determined whether MC express ASGP-R cDNA prepared from total RNA from MC and HepG2 cell cultures served as templates for RTPCR with two sets of primers specific for H1 and H2 subunits of the ASGP-R MC did not yield any specific signals, while samples from HepG2 contained RNA for both ASGP-R subunits, detected on an agarose gel as bands of about 0.6 kb and 0.5 kb, respectively (data not shown) In summary, MC expressed a receptor that bound IgA1 and IgA1-containing CIC, but this receptor did not exhibit properties of Fc␣R (CD89) or ASGP-R Greater binding affinity of this receptor for Gal-deficient IgA1containing CIC compared with uncomplexed IgA may explain mesangial deposition of these CIC in IgAN DISCUSSION IgA deposits in the glomerular mesangium in IgAN are apparently derived from CIC [2, 4, 5, 8, 10, 15, 17]; however, the nature of antigens and ensuing CIC is unknown The evidence suggesting that the mesangial im- mune deposits originate from CIC includes: (a) IgA1, but not IgA2, is present in CIC in the circulation of most IgAN patients [4, 5] and in their mesangial deposits [55]; (b) shared idiotypic determinants are expressed on CIC and in mesangial deposits [56], however, without a disease-specific idiotype [57]; (c) Gal-deficient IgA1 is present in CIC [6, 16, 45–48] and mesangial deposits [18, 19] in IgAN; and (d) Gal-deficient IgA1 is also found in the circulation of Henoch-Schoănlein purpura patients, but only in those with clinical nephritis [58] We have postulated that aberrant glycosylation of the hinge region of some IgA1 molecules of IgAN patients exposes antigenic determinant(s) comprised of GalNAc linked to Ser or Thr of the polypeptide chain [16] The Gal-deficient IgA1 is, in turn, recognized by naturally occurring antibodies (IgG or IgA1 specific for GalNAc) that form CIC [6, 16], some of which deposit in the mesangium Indeed, two groups have detected highly undergalactosylated IgA1 in the kidney mesangial cells of IgAN patients [18, 19] While binding of human IgA1 to human and rat MC has been well documented [20, 24, 26, 27, 53], there had been no such study with CIC containing undergalactosylated IgA1 isolated from sera of IgAN patients This study compared uncomplexed Gal-deficient IgA1 and CIC containing Gal-deficient IgA1 for the binding, internalization, and catabolism by human MC Intact IgA1 or the Fc portion but not its Fab fragment inhibited binding of IgA1 to MC This finding indicates that IgA1 bound to MC through the Fc portion of the molecule MC bound asialo-agalacto-IgA1 better than normally glycosylated IgA1 [59, 60] Results of inhibition experiments indicated that CIC from IgAN patients bound to MC more efficiently than complexes from healthy controls, or than normally glycosylated IgA1 or asialo-agalacto-IgA1 Interestingly, binding of CIC to MC was partially inhibited by normally glycosylated IgA1 or asialo-agalacto-IgA1 but only marginally by IgG These Novak et al: Mesangial cells and IgA-containing complexes findings underscore the importance of IgA receptor(s) for binding of IgA1-CIC and are consistent with observations that IgG receptors are significantly expressed only after MC activation or stimulation [61] Our study showed for the first time that: (a) the IgAN CIC containing Gal-deficient IgA1 bound to MC more efficiently than uncomplexed IgA; (b) a greater amount of CIC from an IgAN patient bound to MC than CIC from a healthy control; and (c) a novel IgA Fc receptor was important for CIC binding to MC These findings suggest a direct role for aberrant IgA1 glycosylation in the formation of CIC and their binding to MC Preliminary experiments suggested reduced binding of CIC from an IgAN patient to HepG2, implying that these CIC may more easily escape hepatic catabolism This characteristic may be one of the factors responsible for increased circulating IgA1 levels in IgAN patients While MC in vitro bind IgA1 in a saturable manner and the binding is inhibited by an excess of unlabeled IgA1 [20, 21, 24, 26, 27, 52], the nature of the receptor(s) has remained controversial Several IgA receptors have been identified on human cells: ASGP-R on hepatocytes [28, 32]; pIgR on epithelial cells [62]; Fc␣R (CD89) on monocytes, neutrophils, and eosinophils [29, 44, 63, 64]; CD71 (transferrin receptor) [65]; and Fc␣/␮ receptor [66, 67] The pIgR [26] and surface-bound galactosyltransferase can be excluded as possible candidates because the bound proteins are not catabolically degraded [68–75] and pIgR binds polymeric but not monomeric IgA Asialoglycoprotein receptor is a hetero-oligomer of two homologous subunits, H1 and H2, encoded by separate genes [30, 31] ASGP-R on hepatocytes binds and internalizes some glycans or glycoproteins with terminal Gal and GalNAc residues The internalized proteins are then catabolically degraded [28] ASOR and IgA1 are excellent probes for ASGP-R as they are efficiently bound, internalized, and catabolized by human hepatocytes and the hepatoma cell line HepG2 [28, 29, 76] To examine the postulated presence of ASGP-R on human MC [21], we compared binding, internalization, and catabolism of radiolabeled AS-IgA1 and ASOR by MC in primary culture and by a HepG2 cell line Our experiments showed that only HepG2 bound, internalized, and catabolized both AS-IgA1 and ASOR, while MC bound and degraded AS-IgA1, but not ASOR This finding was consistent with our observation that only HepG2 cells exhibited mRNAs encoding the two ASGP-R subunits Therefore, it was unlikely that MC, under the conditions of our experiments, expressed ASGP-R Other investigators recently reached the same conclusion [26] Fc␣R (CD89) is a glycoprotein expressed on myeloid cells that binds both IgA subclasses [36] Some investigators have postulated that this receptor accounts for IgA1 binding to MC [20, 22, 23] In contrast to earlier reports 473 [20, 22, 23], we did not detect its mRNA in MC from normal kidney tissue or commercial sources Concordant with our results, other recent studies also failed to detect CD89 on human MC [24–27] Insulin-like growth factor was used by others as a supplement in the culture medium [20] and, therefore, we also tested whether this growth factor would induce CD89 expression However, no induction was detected Fc␣R (CD89) has several isoforms that originate from alternative splicing [77] Because some reports have shown conflicting data about the expression of CD89 on MC

Kidney International, Vol 62 (2002), pp 465–475 Interactions of human mesangial cells with IgA and IgA-containing immune complexes1 JAN NOVAK, HUONG L VU, LEA NOVAK, BRUCE A JULIAN, JIRI MESTECKY, and MILAN TOMANA Departments of Microbiology, Pathology, and Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA Interactions of human mesangial cells with IgA and IgA-containing circulating immune complexes Background IgA nephropathy (IgAN) is characterized by IgA1-containing immune complexes in mesangial deposits and in the circulation The circulating immune complexes (CIC) are composed of galactose- (Gal) deficient IgA1 and IgG or IgA1 antibodies specific for the Gal-deficient IgA1; interactions of these CIC with mesangial cells (MC) were studied Methods Binding, internalization, and catabolic degradation of myeloma IgA1 protein as a standard control and the isolated CIC were studied using human MC, hepatoma cell line HepG2 expressing the asialoglycoprotein receptor (ASGP-R), and monocyte-like cell line U937 expressing the Fc␣-R (CD89) Biochemical and molecular approaches were used to assess expression of CD89 and ASGP-R by MC Results At 4⬚C, radiolabeled IgA1 bound to MC and HepG2 cells in a dose-dependent and saturable manner The binding was inhibited by IgA-containing CIC or excess IgA1 or its Fc fragment but not by the Fab fragment of IgA1 At 37⬚C, the cell-bound IgA1 was internalized and catabolized In addition to IgA1, HepG2 cells also bound (in a Ca2⫹-dependent manner), internalized, and catabolized asialoorosomucoid (ASOR), other asialo-(AS)-glycoproteins, and secretory component (SC) The binding by MC appeared to be restricted to IgA1 or ASIgA1 and was not Ca2⫹-dependent Furthermore, MC and HepG2 cells internalized and catabolized IgA1-containing CIC Using RT-PCR with ASGP-R- or CD89-specific primers, mRNAs of the two respective genes were not detected in MC Conclusions The data showed that the ability of MC to bind IgA1 and IgA1-containing CIC in vitro was mediated by an IgA receptor that was different from CD89 or ASGP-R and had a higher affinity for IgA-CIC than for uncomplexed IgA vated levels of IgA1 and IgA1-containing circulating immune complexes (CIC) are found in sera of most IgAN patients [4–7] IgA1-containing immune deposits are observed in the renal mesangium (usually with C3 complement component, and often with IgG, IgM, or both) [8] The progression of the disease is characterized by proliferation of mesangial cells and expansion of extracellular matrix (ECM), leading ultimately to glomerular sclerosis [9] About 30 to 40% of IgAN patients develop kidney failure within 20 years of clinical onset; these patients require either dialysis or kidney transplantation [10] IgAN recurs in approximately 50 to 60% of patients after kidney transplantation [11] On the other hand, a kidney transplanted from a donor with subclinical IgAN to a patient without IgAN clears the immune deposits after several weeks [12] It is therefore apparent that IgAN arises from formation and deposition of immune complexes rather than from a defect inherent in the kidney [2, 4–6, 8, 13–16] IgA immune deposits are likely derived from CIC, however, the nature of the antigens in the CIC is unknown [4, 5, 14] Based on our experimental data, we postulated that aberrant glycosylation of IgA1 immunoglobulins in the circulation of IgAN patients leads to formation of CIC [6] Incomplete galactosylation of O-linked glycans in the IgA1 hinge region results in the exposure of underlying neo-antigenic N-acetylgalactosamine (GalNAc) [6, 16] that is recognized by naturally occurring antibodies (IgG or IgA1 specific for GalNAc) [6] We further postulated that these CIC deposit on mesangial cells (MC), resulting in MC proliferation and overexpression of extracellular matrix [15, 17] Recently, highly undergalactosylated IgA1 was detected in the mesangial deposits in IgAN patients, and this further supports our hypothesis [18, 19] In this regard, understanding the mechanisms by which CIC bind to MC is of utmost importance [10] Several studies evaluating the nature of an IgA receptor expressed on MC have provided conflicting results Some Idiopathic IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world [1–3] Ele1 See Editorial by Go´mez-Guerrero, Suzuki, and Egido, p 715 Key words: renal mesangial cells, circulating immune complexes, IgA nephropathy, glomerulonephritis Received for publication March 26, 2001 and in revised form March 7, 2002 Accepted for publication March 22, 2002  2002 by the International Society of Nephrology 465 466 Novak et al: Mesangial cells and IgA-containing complexes found Fc␣R (CD89) or the asialoglycoprotein receptor (ASGP-R) expressed by cultured MC or in some renal biopsy samples from IgAN patients [20–23] Other studies showed binding of IgA1 to MC but did not confirm expression of CD89 by MC [24–27] Most of these studies were conducted using uncomplexed [monomeric (m), polymeric (p)] or heat-aggregated IgA1 However, in light of recent findings on the role of CIC containing aberrantly glycosylated IgA1 in the pathogenesis of IgAN [6, 10, 17–19], it is important to examine interactions of MC with not only free IgA1 and de-galactosylated IgA1, but also with galactose-deficient IgA1 bound in CIC Our study reports that human MC in primary culture bind, internalize, and catabolically degrade IgA1 and IgA1-containing CIC HepG2 cells expressing ASGP-R [28, 29] and U937 expressing Fc␣R (CD89) served as a positive controls ASGP-R is a hetero-oligomer of two subunits, H1 and H2, encoded by separate genes [30, 31] ASGP-R on hepatocytes binds glycoproteins with terminal Gal or GalNAc residues, and the bound glycoproteins are subsequently internalized and catabolized [28, 29, 32] A standard probe to detect ASGP-R is asialoorosomucoid (ASOR) [28, 29] that is rapidly internalized and catabolized by hepatocytes upon binding [6, 29] ASGP-R also binds secretory component (SC) [28], a highly glycosylated fragment of the pIg receptor (pIgR) that remains bound to secretory IgA or IgM [33–35] Eventual catabolic degradation of SC or ASOR by MC thus would confirm the previously postulated presence of ASGP-R on MC [21] Fc␣R (CD89) is a glycoprotein expressed on myeloid cells that binds both IgA subclasses [36] Binding of IgA to the Fc␣R also may lead to internalization and catabolic degradation [28] Using these control cells, we have demonstrated differences in properties and binding specificities of IgA1 receptors expressed by human MC, HepG2 and U937 cells Our observations indicated that the ability of MC to bind IgA1 and IgA1-containing CIC in vitro is mediated by a new type of IgA receptor with higher affinity for IgACIC than for uncomplexed IgA METHODS Cells Human MC were isolated from the normal portions of kidney cortexes of tumor nephrectomy specimens (2 cell cultures) as described before [37, 38] or purchased as primary cells (2 cell cultures) from a commercial source (Clonetics, San Diego, CA, USA) Cells from passages to were used in our experiments MC were maintained in RPMI 1640 supplemented with 20% fetal calf serum (FCS), l-glutamine (2 mmol/L), penicillin G (100 U/mL), streptomycin (0.1 mg/mL) in humidified 5% CO2 atmosphere at 37⬚C Purity and identification of MC was based on cell morphology and immunohistochemical features: (a) positive staining for vimentin, and (b) negative staining for factor VIII-related antigen and cytokeratin (to exclude contamination with endothelial and epithelial cells, respectively) Human hepatocarcinoma cell line HepG2 and human monocytic cell line U937 (both obtained from ATCC, Rockville, MD, USA) were maintained in RPMI 1640, 10% FCS, and antibiotics [28] MC for binding experiments were serum starved (medium contained only 0.5% FCS) 24 hours before the experiment Isolation of IgA1 proteins, their Fab and Fc fragments, and IgA1-containing CIC IgA1 and IgA2 myeloma proteins were isolated from plasma of several different patients with multiple myeloma by precipitation with ammonium sulfate, starchblock electrophoresis, and size-exclusion and ion-exchange chromatography [6, 33] The polymeric forms of IgA1 (Mce) and IgA2 (Fel) were used [6] and their polymeric character was demonstrated by size-exclusion chromatography and sodium dodecyl sulfate (SDS)-gel electrophoresis under non-reducing conditions The Fab fragment of IgA1 myeloma protein (Ste) was prepared by cleavage with IgA1 protease from Haemophilus influenzae (HK50); Fab and Fc fragments of IgA1 (Mce) were generated using IgA1 protease from Neisseria gonorrhoeae [39, 40] The resulting Fab and Fc fragments were purified by size-exclusion chromatography [33] and their purity was verified by SDS electrophoresis Fractions rich in Gal-deficient IgA1-containing CIC were prepared from sera of IgAN patients by size-exclusion chromatography on a calibrated Superose column [6]; high-molecular-mass fractions reactive with Helix aspersa (HAA) lectin (which binds terminal GalNAc of Gal-deficient O-glycans of IgA1; Sigma, Chemical Company, St Louis, MO, USA) and containing IgA and IgG were pooled [6] Asialo- (AS) IgA1, asialo-agalacto IgA1 and ASOR were prepared from native proteins by incubation with neuraminidase (from Vibrio cholerae) and ␤-galactosidase (from bovine testes that preferentially cleaves ␤1,3 linkages) [6, 16, 28, 41] SC was isolated from human milk [33] IgG was purified from normal human serum by ammonium sulfate precipitation, and subsequent ionexchange (DEAE-cellulose) and affinity chromatography on a Protein-G column [42] To prepare in vitro a complex of IgG bound to Galdeficient pIgA1, we incubated 125I-labeled degalactosylated IgA1 (Mce) [6] with purified human IgG with antiGalNAc activity [6] Free IgA1 was separated from the IgG-IgA1 complexes by size-exclusion chromatography on a Superose column The fractions containing IgG bound to the radiolabeled IgA1 were identified by using Protein-G-coated Immulon Removawell strips (Dynatech Laboratories, Alexandria, VA, USA) and a Packard Novak et al: Mesangial cells and IgA-containing complexes model 5110 gamma spectrometer (Packard Instrument Company, Downers Grove, IL, USA) Radioiodination and tetramethylrhodamine isothiocyanate (TRITC)-labeling Proteins were radiolabeled with carrier-free Na125I by the lactoperoxidase method [43] The excess free Na125I was separated from the protein by size-exclusion chromatography on a column of Sephadex G-25 [28] IgA1 (Mce) was TRITC-labeled using 0.02 mg TRITC per mg protein in 2% bicarbonate buffer, pH 8.2 After overnight incubation, free TRITC was removed on a column of Sephadex G-50 and the TRITC-IgA1 conjugate was isolated using ion-exchange chromatography on a column of DEAE-cellulose The TRITC-labeled protein was aliquoted and stored at ⫺70⬚C Binding experiments Mesangial cells (or HepG2 cells) grown in 24-well plates (60-80% confluent) were washed twice with 20 mmol/L HEPES buffer, pH 7.3, containing 140 mmol/L NaCl, 0.8 mmol/L MgCl2, 0.34 mmol/L K2HPO4, 0.34 mmol/L KH2PO4, (buffer A), and incubated with a radiolabeled protein in 0.2 mL buffer B [buffer A supplemented with 2.7 mmol/L CaCl2 and 1% bovine serum albumin (BSA)] on ice for one hour [28] Buffer B without CaCl2 supplement also was used in some experiments, as described in the text In the inhibition experiments, MC were preincubated for one hour with inhibitors on ice, before radiolabeled IgA1 was added and incubated for another one hour Following extensive washing with buffer B, cells were lysed with 0.4 mL 0.3 N NaOH and the radioactivity of the lysate was determined in a ␥ spectrometer Cell count was determined with cells released by trypsin/ ethylenediaminetetraacetic acid (EDTA) treatment using a hemocytometer Catabolic degradation of internalized proteins Mesangial cells and HepG2 in tissue culture flasks were incubated with radiolabeled proteins (⬃1 ␮g of ASOR, AS-IgA1, or immune complexes) in mL buffer B at 37⬚C for four hours Radioactivity was determined in (a) incubation medium, (b) cells washed and released by trypsinization, and (c) released cells after additional trypsinization to remove protein bound on cell surface Catabolic degradation was expressed as percentage of total radioactivity not precipitable by 10% trichloroacetic acid (TCA; from incubation medium or cell lysate, as specified in the text) [28] Protein in the cell lysate was determined spectrophotometrically using a BioRad assay (BioRad, Hercules, CA, USA) with BSA as the standard To determine molecular masses of proteins catabolically degraded, MC were incubated in mL HEPES buffer, pH 7.3 containing 1% BSA with 10 ␮g 125I-labeled IgA1 at 37⬚C for 16 hours The cells were washed with 467 HEPES buffer, lysed with a polyacrylamide gel electrophoresis loading buffer containing 2% SDS, and analyzed by SDS-polyacrylamide gel electrophoresis (SDSPAGE), using 1.5 mm thick vertical slab gels (20 ⫻ 20 cm) with to 20% polyacrylamide gradient [28] The gels were fixed, frozen with dry ice, and sliced with a gel slicer The radioactivity in 1-mm sections was measured by a gamma spectrometer Confocal microscopy Mesangial cells grown on a microscope slide were incubated with TRITC-labeled IgA1 (Mce; 10 ␮g) overnight at 4⬚C, extensively washed with RPMI medium containing 0.5% FCS and then incubated at 37⬚C for four hours, followed by incubation at room temperature for two hours Imaging was performed on a Leica DMIRBE inverted epifluorescence/Nomarski microscope outfitted with Leica TCS NT Confocal optics The system is equipped with UV, argon ion, krypton ion, and helium/neon lasers for imaging in a wide range of blue, red, and far-red fluorescence The laser was set to optimal TRITC excitation wavelength to observe the internalized TRITCIgA1 Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA isolated from the cells (MC, HepG2, and U937) with RNA-Stat-60 reagent (Tel-Test, Friendswood, TX, USA) was used for RT-PCR HepG2 and U937 cells served as controls for ASGP-R and CD89 expression, respectively First-strand cDNA synthesis was performed at 42⬚C for 15 minutes, followed by 37⬚C for 45 minutes using murine leukemia virus reverse transcriptase and oligo(dT)16 as the primer The cDNA was amplified using the following primers that we designated based on the sequences of the correspond ing genes submitted to GeneBank [30, 31, 44] for Fc␣ receptor (CD89): CD89F 5⬘-AGCACGATGGACCCCAAACA GA-3⬘; CD89R 5⬘-CTGCCTTCACCTCCAGGTGTT-3⬘, and the following primers for H1 and H2 genes encoding the two ASGP-R subunits: H1F 5⬘-CTGGACAAT GAGGAGAGTGAC-3⬘; H1R 5⬘-TTGAAGCCCGTC TCGTAGTC-3⬘; H2F CCTGCTGCTGGTGGTCATC TG-3⬘; H2R 5⬘-CCCATTTCCAAGAGCCATCAC-3⬘ A pair of primers specific to ␤-actin (F 5⬘-TTCCAGCC TTCCTTCCTGG-3⬘; R 5⬘-TTGCGCTCAGGAGGAG CAA-3⬘) was used as a control PCR was performed in a DNA thermal cycler using 94⬚C melting, 60⬚C annealing, and 72⬚C extension temperatures for 35 cycles PCR amplicons were analyzed on 2% NuSieve 3:1 agarose gels RESULTS Binding of IgA1 and IgA1-containing CIC to MC To determine binding characteristics of IgA1, MC were incubated for one hour on ice with various concentra- 468 Novak et al: Mesangial cells and IgA-containing complexes Fig Binding of 125I-labeled IgA1 (Mce) myeloma protein (䊏) and 125 I-labeled IgA1 (Mce) with partially degalactosylated and desialylated O-linked glycans ( ) to mesangial cells (MC) MC were incubated for one hour on ice with ␮g radiolabeled proteins Cells were washed and the bound radioactivity determined as described in the Methods section tions of radiolabeled pIgA1 (Mce) myeloma protein Consistent with earlier reports, the binding of the pIgA1 to MC was dose-dependent and saturable The binding of radiolabeled pIgA1 was inhibited by 68% using a 150fold excess of unlabeled pIgA1 Analyses of IgA1 binding to MC using a Scatchard plot (not shown) suggested a single population of receptors with approximately ⫻ 105 binding sites per cell and Ka 4.79 ⫻ 106 mol/L⫺1 The calculations were based on the assumption that pIgA1 (Mce) was predominantly polymeric, as judged from its elution profile on a calibrated size-exclusion high pressure liquid chromatography (HPLC) column (TSK 5000) Furthermore, we assumed that each receptor bound only one IgA1 molecule Because the mesangial immune deposits in IgAN patients contain aberrantly glycosylated (Gal-deficient O-linked glycans) IgA1 [18, 19], we examined MC binding of Gal-deficient IgA1 myeloma protein The binding of IgA1 that was modified by treatment with neuraminidase and ␤-galactosidase (that removes preferentially ␤1,3 bound Gal) was more than twofold greater compared with the unmodified control (Fig 1) In sera of IgAN patients, however, the aberrantly glycosylated IgA1 [16, 45–48] is not free, but rather is complexed with IgG (or IgA1) in CIC [5, 6, 16] To prepare IgA1containing CIC, serum from an IgAN patient was fractionated using size-exclusion chromatography Fractions with Gal-deficient IgA1 were identified with GalNAcspecific lectin (HAA) in ELISA (Fig 2) The HAAreactive fractions (CIC of molecular mass 700 to 900 kD), designated as CIC containing Gal-deficient IgA1, were pooled and used in further experiments Binding of radiolabeled Gal-deficient pIgA1 was inhibited by unlabeled Gal-deficient pIgA1 (Fig 3A), but Fig Isolation of Gal-deficient IgA1 containing CIC from serum of an IgAN patient Serum (0.5 mL) was fractionated on a Superose column (0.9 ⫻ 60 cm), 0.25 mL fractions were collected and analyzed by ELISA for IgA (䉭), IgG (䊐), and reactivity with HAA lectin (䊉; specific for terminal GalNAc, thus reacting with Gal-deficient IgA1) Fractions containing CIC with aberrantly glycosylated IgA1 (molecular mass about 700 to 900 kD) were pooled and used in the experiments not by the Fab fragment of IgA1 (that contained a portion of the hinge region) Surprisingly, CIC containing aberrantly glycosylated IgA1 appeared to be better inhibitors than uncomplexed monomeric or polymeric IgA1 (Fig 3A) The same amount of uncomplexed IgA1 (Galdeficient or normally glycosylated) had no significant inhibitory effect (data not shown), suggesting that properties such as spatial organization of IgA1 in CIC may play a role in MC binding By comparing the inhibitory activity of Fab and Fc portions of IgA1, respectively, we concluded that IgA1 bound to a MC IgA receptor by its Fc portion (Fig 3B) This is consistent with our finding that both IgA subclasses (IgA1 and IgA2) bound to MC (data not shown) The results in the prior section suggested better binding of IgA1 in CIC compared with free IgA1 To verify this finding, sera from three IgAN patients were fractionated using size-exclusion chromatography on a calibrated Superose column and analyzed for IgA, and for reactivity with HAA The fractions corresponding to mIgA and to IgA complexed in CIC were incubated with MC grown on microscope slides After three hours of incubation at 4⬚C, MC were washed with phosphate-buffered saline (PBS) and stained with TRITC-conjugated F(ab⬘)2 fragment of anti-human IgA antibody and examined with a fluorescence microscope MC incubated with CIC showed strong IgA binding, while MC incubated with uncomplexed IgA exhibited only background-level fluorescence Novak et al: Mesangial cells and IgA-containing complexes 469 Fig Inhibition of 125I-labeled Gal-deficient pIgA1 (Mce) binding to MC MC grown to 80% confluence in a 24-well plate were pre-incubated with inhibitors for one hour on ice and then about ␮g radiolabeled protein was added Cells were washed and the bound radioactivity was determined as described in Methods (A) Control is no inhibitor, and inhibitors are Gal-deficient pIgA1 Mce (100 ␮g), mIgA from serum of an IgAN patient (⬃50 ␮g), and Gal-deficient-IgA1-containing CIC from serum of an IgAN patient (⬃1 ␮g) (B) Control is no inhibitor, and inhibitors include Gal-deficient pIgA1 Mce (100 ␮g), Fab fragment of IgA1 (100 ␮g), and Fc fragment of IgA1 Mce (100 ␮g) These results indicated that IgA1 bound to MC through the Fc part of its molecule because IgA1 or its Fc fragment, but not its Fab fragment, inhibited IgA binding Furthermore, abnormalities of O-linked glycans of IgA1 and spatial organization of IgA1 molecules clustered in CIC influenced binding to MC, favoring IgA in CIC over uncomplexed IgA Catabolism and internalization of IgA1 by MC To detect potential degradation products of cell-associated and internalized IgA1, the radiolabeled protein was added to MC and incubated for 16 hours; the cells were then washed, lysed, and the lysate was analyzed by SDS gel electrophoresis under non-reducing conditions (Fig 4) Generation of protein fragments smaller than 30 kD was observed after incubation with MC, indicating catabolic degradation of the 125I-labeled IgA1 This finding is consistent with decreased TCA-precipitable radioactivity To visualize internalization of IgA1 by MC, we incubated MC grown on a microscope slide with TRITClabeled IgA1 The cells were then fixed and observed with a confocal laser scanning microscope Many MC showed intracellular fluorescent vesicles, which indicated internalization of IgA1 (Fig 5) Internalization and catabolism of AS-IgA1, ASOR, and SC by HepG2 and MC Asialoglycoprotein receptor has been reported as one of the receptors responsible for binding of IgA1 to MC [21] To verify this report, we incubated 125I-labeled ASIgA1 with MC and HepG2 cells HepG2 cells express Fig Catabolism of 125I-labeled IgA1 (Mce) by MC About 10 ␮g 125 I-labeled protein was added to the cells in tissue culture flask and incubated in mL HEPES buffer with 1% BSA at 37⬚C for 16 hours The cells were then washed, and lysed using 2% SDS buffer and the lysate was analyzed by SDS gel electrophoresis under non-reducing conditions (solid line) The distribution of radioactivity in the gel was determined by assaying the radioactivity of 1-mm sections of the gel in a gamma counter Control 125I-labeled IgA1 (dotted line) was electrophoresed in parallel Migration of standards is shown by arrows ASGP-R [28, 29] and served as a positive control The cell cultures were then washed, treated with trypsin to release cells from the flasks and surface-bound radiolabeled protein from cells After further washing, the cells were lysed with NaOH The internalized protein was de- 470 Novak et al: Mesangial cells and IgA-containing complexes Fig Confocal laser scanning photomicrograph of IgA1 internalized by a MC MC grown on a microscope slide were incubated with TRITC-labeled IgA1 (Mce; 10 ␮g) overnight at 4⬚C, extensively washed with RPMI 1640 medium containing 0.5% FCS and incubated at 37⬚C for four hours, followed by incubation at room temperature for two hours A single MC is shown with fluorescent vesicles in the cytoplasm Bar depicts 20 ␮m tected as radioactivity in the trypsin-treated and washed cells AS-IgA1 was internalized by both cell cultures (Fig 6) Under the same conditions, HepG2 internalized sixfold more AS-IgA1 per mg cell protein than did MC To estimate the kinetics of catabolic degradation of internalized proteins, percentage of the radiolabeled protein not precipitable with TCA was determined HepG2 cells catabolized 47% of the internalized AS-IgA1 compared to 39% for MC Unlike with HepG2, the binding and internalization of AS-IgA1 by MC was not Ca2⫹-dependent (data not shown) Earlier studies demonstrated that ASGP-R on hepatocytes or HepG2 cells is responsible for rapid internalization and catabolic degradation of ASOR [28] We investigated whether ASOR also is internalized and catabolized by MC 125I-labeled ASOR incubated with HepG2 and MC was internalized 144-fold more effectively by HepG2 than MC (Fig 6) Likewise, the catabolic degradation was more effective in HepG2 cells Furthermore, it was also reported that HepG2 internalize and catabolize SC [28] Therefore, we compared catabolism of SC and IgA1 in MC (Table 1) More than 99% of original 125I-labeled SC incubated with MC remained intact, while the original 125I-labeled IgA1 was partially (about 13%) catabolically degraded during the four-hour incubation These data demonstrated that SC, but not IgA1, escaped catabolic degradation by MC Therefore, ASGP-R is missing or nonfunctional on MC Binding and internalization of IgA1-containing CIC by HepG2 and MC Studies described above indicated that IgA1-containing CIC bound to MC more effectively than free IgA1 Novak et al: Mesangial cells and IgA-containing complexes Fig Internalization of 125I-labeled ASOR and 125I-labeled asialo(AS) IgA1 (Mce) by HepG2 and MC About ␮g of each radiolabeled protein was added to the cells in tissue culture flask and incubated in mL buffer B at 37⬚C for four hours Cells were washed and radioactivity measured after trypsinization to release surface-bound molecules Results represent an average from experiments conducted in triplicates 471 Fig HepG2 and MC cell-associated (bound and internalized) 125 I-labeled Gal-deficient IgA1-containing CIC isolated from serum of an IgAN patient (䊏) and CIC from a healthy control ( ) About ␮g aliquots of the radiolabeled proteins were added to the cells in tissue culture flasks and incubated in mL buffer B at 37⬚C for four hours Cells were washed before the cell-associated radioactivity was measured Results are averages from experiments conducted in triplicates Table Catabolism of radioiodinated secretory component (SC) and IgA1 (Mce) by human mesangial cells TCA precipitable protein % Before incubation After incubation SC IgA1 (Mce) 93.9 93.0 95.6 82.8 One-microgram aliquots of the SC or IgA1 proteins were added to MC in cultivation flasks and incubated for hours at 37⬚C Then, the supernatant was collected, precipitated with TCA, and the intact protein (TCA-precipitable radioactivity measured using gamma counter) was expressed as % of TCA-precipitable radioactivity The experiment was conducted in triplicate To examine the possible role of liver cells and ASGP-R in binding and processing of these CIC, we compared the binding and catabolism of these CIC by MC and HepG2 Gal-deficient IgA1-containing CIC were purified from serum of an IgAN patient and control CIC were isolated from serum of a healthy volunteer using size-exclusion chromatography [6] These CIC (molecular mass about 700 to 900 kD) were radioiodinated, and incubated with MC and HepG2 MC bound and internalized more protein from the IgAN-CIC than from control CIC On the other hand, HepG2 cells bound less protein from the IgAN-CIC (Fig 7) We hypothesized that the lower degree of internalization of IgAN-CIC by hepatoma cells may be due to the presence of GalNAc-specific IgG [6] that bound to IgA1 and thus prevented IgA1 hinge region O-glycans [32] or Fc glycans [49, 50] from binding to ASGP-R on HepG2 cells To test this hypothesis, we prepared in vitro 125I-labeled Gal-deficient IgA1 in a free form and bound to GalNAc-specific IgG and used HepG2 cells to assess the effect on internalization of the radiolabeled IgA1 Complexing IgG with the IgA1 reduced the binding and internalization by HepG2 cells (Fig 8) Fig Internalization of a complex of IgG-Gal-deficient pIgA1 (䊏) and free Gal-deficient pIgA1 ( ) by human hepatoma cell line HepG2 125 I-labeled degalactosylated IgA1 was incubated with purified human IgG with anti-GalNAc activity Free IgA1 was separated from the IgGIgA1 complexes by size-exclusion chromatography on Superose column The fractions containing IgG bound to the radiolabeled IgA1 were detected by capture radioimmunoassay using Protein-G-coated Removawell strips and gamma-counter detection The proteins were incubated with HepG2 cells at 37⬚C for three hours, then the cells were washed, treated with trypsin, and the radioactivity was measured with a gamma counter and expressed per mg of cell protein In summary, these experiments indicated that MC bound and internalized IgA1-containing CIC via a receptor different from ASGP-R MC bound more effectively the CIC from an IgAN patient than that from a healthy control Furthermore, CIC from an IgAN patient were less efficiently internalized by hepatoma cells (HepG2) than control CIC The IgG bound to Gal-deficient IgA1 apparently masks the binding sites on IgA1 glycans from ASGP-R or interferes with an efficient internalization 472 Novak et al: Mesangial cells and IgA-containing complexes Fig RT-PCR of Fc␣R (CD89) transcripts in MC and U937 cells Total RNA was reversetranscribed and the cDNA was PCR-amplified with CD89-specific primers The amplicons were separated on 2% agarose gel and photographed under UV light Lane 1, molecular size standards; lanes 2-5, ␤-actin RT-PCR in MC; lane 6, ␤-actin RT-PCR in U937; lane 7, RT-PCR of Fc␣R transcripts in U937 with three signals detected that correspond to the a.1, a.2, and a.3 splicing variants; lanes 8-11, RT-PCR of mRNA from MC grown under various conditions failed to reveal any CD89specific signal (lanes 10, 11, MC were supplemented with insulin-like growth factor; lanes 9, 10, 5% glucose was added to the storage medium) CD89 and ASGP-R mRNA expression in MC To determine a possible involvement of CD89 in binding IgA to MC, RT-PCR was used to examine whether the CD89 gene is transcribed in MC Total RNA isolated from MC grown with, or without, insulin-like growth factor and from U937 cells served as templates for RT followed by PCR amplification with CD89-specific primers The results did not indicate the presence of CD89 mRNA in MC, although all three major splicing products, a.1, a.2, and a.3, (observed as PCR amplicons of about 0.9 kb, 0.85 kb, and 0.62 kb, respectively) of CD89 mRNA were detected in samples from CD89-positive U937 cells (Fig 9) Adding insulin-like growth factor (known to alter gene expression in MC [51] and used in experiments by others [20, 52, 53]) to the growth medium or glucose [54] to the storage medium did not induce expression of CD89 (Fig 9) Furthermore, we determined whether MC express ASGP-R cDNA prepared from total RNA from MC and HepG2 cell cultures served as templates for RTPCR with two sets of primers specific for H1 and H2 subunits of the ASGP-R MC did not yield any specific signals, while samples from HepG2 contained RNA for both ASGP-R subunits, detected on an agarose gel as bands of about 0.6 kb and 0.5 kb, respectively (data not shown) In summary, MC expressed a receptor that bound IgA1 and IgA1-containing CIC, but this receptor did not exhibit properties of Fc␣R (CD89) or ASGP-R Greater binding affinity of this receptor for Gal-deficient IgA1containing CIC compared with uncomplexed IgA may explain mesangial deposition of these CIC in IgAN DISCUSSION IgA deposits in the glomerular mesangium in IgAN are apparently derived from CIC [2, 4, 5, 8, 10, 15, 17]; however, the nature of antigens and ensuing CIC is unknown The evidence suggesting that the mesangial im- mune deposits originate from CIC includes: (a) IgA1, but not IgA2, is present in CIC in the circulation of most IgAN patients [4, 5] and in their mesangial deposits [55]; (b) shared idiotypic determinants are expressed on CIC and in mesangial deposits [56], however, without a disease-specific idiotype [57]; (c) Gal-deficient IgA1 is present in CIC [6, 16, 45–48] and mesangial deposits [18, 19] in IgAN; and (d) Gal-deficient IgA1 is also found in the circulation of Henoch-Schoănlein purpura patients, but only in those with clinical nephritis [58] We have postulated that aberrant glycosylation of the hinge region of some IgA1 molecules of IgAN patients exposes antigenic determinant(s) comprised of GalNAc linked to Ser or Thr of the polypeptide chain [16] The Gal-deficient IgA1 is, in turn, recognized by naturally occurring antibodies (IgG or IgA1 specific for GalNAc) that form CIC [6, 16], some of which deposit in the mesangium Indeed, two groups have detected highly undergalactosylated IgA1 in the kidney mesangial cells of IgAN patients [18, 19] While binding of human IgA1 to human and rat MC has been well documented [20, 24, 26, 27, 53], there had been no such study with CIC containing undergalactosylated IgA1 isolated from sera of IgAN patients This study compared uncomplexed Gal-deficient IgA1 and CIC containing Gal-deficient IgA1 for the binding, internalization, and catabolism by human MC Intact IgA1 or the Fc portion but not its Fab fragment inhibited binding of IgA1 to MC This finding indicates that IgA1 bound to MC through the Fc portion of the molecule MC bound asialo-agalacto-IgA1 better than normally glycosylated IgA1 [59, 60] Results of inhibition experiments indicated that CIC from IgAN patients bound to MC more efficiently than complexes from healthy controls, or than normally glycosylated IgA1 or asialo-agalacto-IgA1 Interestingly, binding of CIC to MC was partially inhibited by normally glycosylated IgA1 or asialo-agalacto-IgA1 but only marginally by IgG These Novak et al: Mesangial cells and IgA-containing complexes findings underscore the importance of IgA receptor(s) for binding of IgA1-CIC and are consistent with observations that IgG receptors are significantly expressed only after MC activation or stimulation [61] Our study showed for the first time that: (a) the IgAN CIC containing Gal-deficient IgA1 bound to MC more efficiently than uncomplexed IgA; (b) a greater amount of CIC from an IgAN patient bound to MC than CIC from a healthy control; and (c) a novel IgA Fc receptor was important for CIC binding to MC These findings suggest a direct role for aberrant IgA1 glycosylation in the formation of CIC and their binding to MC Preliminary experiments suggested reduced binding of CIC from an IgAN patient to HepG2, implying that these CIC may more easily escape hepatic catabolism This characteristic may be one of the factors responsible for increased circulating IgA1 levels in IgAN patients While MC in vitro bind IgA1 in a saturable manner and the binding is inhibited by an excess of unlabeled IgA1 [20, 21, 24, 26, 27, 52], the nature of the receptor(s) has remained controversial Several IgA receptors have been identified on human cells: ASGP-R on hepatocytes [28, 32]; pIgR on epithelial cells [62]; Fc␣R (CD89) on monocytes, neutrophils, and eosinophils [29, 44, 63, 64]; CD71 (transferrin receptor) [65]; and Fc␣/␮ receptor [66, 67] The pIgR [26] and surface-bound galactosyltransferase can be excluded as possible candidates because the bound proteins are not catabolically degraded [68–75] and pIgR binds polymeric but not monomeric IgA Asialoglycoprotein receptor is a hetero-oligomer of two homologous subunits, H1 and H2, encoded by separate genes [30, 31] ASGP-R on hepatocytes binds and internalizes some glycans or glycoproteins with terminal Gal and GalNAc residues The internalized proteins are then catabolically degraded [28] ASOR and IgA1 are excellent probes for ASGP-R as they are efficiently bound, internalized, and catabolized by human hepatocytes and the hepatoma cell line HepG2 [28, 29, 76] To examine the postulated presence of ASGP-R on human MC [21], we compared binding, internalization, and catabolism of radiolabeled AS-IgA1 and ASOR by MC in primary culture and by a HepG2 cell line Our experiments showed that only HepG2 bound, internalized, and catabolized both AS-IgA1 and ASOR, while MC bound and degraded AS-IgA1, but not ASOR This finding was consistent with our observation that only HepG2 cells exhibited mRNAs encoding the two ASGP-R subunits Therefore, it was unlikely that MC, under the conditions of our experiments, expressed ASGP-R Other investigators recently reached the same conclusion [26] Fc␣R (CD89) is a glycoprotein expressed on myeloid cells that binds both IgA subclasses [36] Some investigators have postulated that this receptor accounts for IgA1 binding to MC [20, 22, 23] In contrast to earlier reports 473 [20, 22, 23], we did not detect its mRNA in MC from normal kidney tissue or commercial sources Concordant with our results, other recent studies also failed to detect CD89 on human MC [24–27] Insulin-like growth factor was used by others as a supplement in the culture medium [20] and, therefore, we also tested whether this growth factor would induce CD89 expression However, no induction was detected Fc␣R (CD89) has several isoforms that originate from alternative splicing [77] Because some reports have shown conflicting data about the expression of CD89 on MC, we considered that an expressed variant of CD89 may be a splicing version that was not detected by the primers Therefore, we designed primers that could detect spliced variants, but found no such transcripts in MC The reasons some investigators detected CD89 are unclear, but may include differences in cultivation techniques, contamination with other cell types (such as macrophages) known to express CD89, or valid induction of CD89 or its variant [27] Two other receptors have recently emerged as novel candidate IgA receptors on MC: CD71 (transferrin receptor) [65] and Fc␣/␮ receptor [66, 67] We confirmed the expression of CD71 mRNA by proliferating MC in culture (not shown) However, MC bind both IgA subclasses [27], while CD71 binds only IgA1 [65] MC bind pIgA1 better than mIgA [27], in contrast to the strong preference of CD71 for mIgA1 [65] Furthermore, CD71 is expressed by many proliferating cells, some of which not bind IgA1 Obviously the issues concerning the role of CD71 on MC in binding Gal-deficient IgA or IgA-CIC remain to be addressed The other newly described candidate, Fc␣/␮ receptor, appears to be transcribed by MC in vitro [67] The expression of this receptor by MC or in renal tissue and its role in binding IgA-CIC remain to be investigated, although IgM does not inhibit IgA1 binding to MC [27] This binding pattern may be explained if the variant of the Fc␣/␮ receptor expressed by MC preferentially binds IgA Clearly, the properties of this receptor need to be investigated A well-defined system, such as COS-7 cells expressing the receptor [67], may be appropriate for such a task In conclusion, we detected binding, internalization, and catabolism of IgA1 and IgA1-containing CIC by human MC, which was apparently independent of CD89 and ASGP-R While IgA1 is likely to bind to a single population of receptors, CIC can interact with MC in a more complex manner that may involve other Ig-specific or complement receptors However, the hallmark of IgAN is mesangial deposition of IgA1, and because purified IgA1 was shown to bind to MC, it is very likely that a specific, not yet biochemically characterized, IgA receptor is involved Its similarity to a recently described novel IgA receptor on human intestinal epithelial cells [78] and the role of other IgA receptors [65, 67] remain to 474 Novak et al: Mesangial cells and IgA-containing complexes be investigated Understanding the nature of the interaction of IgA1 and its mesangial receptor may not only explain how IgA1 and IgA1-containing CIC deposit in glomeruli and cause IgAN, but may also provide a theoretical basis for developing disease-specific therapeutic inhibitors 17 18 19 ACKNOWLEDGMENTS This work was supported by grants DK 49358, DK 57750, and DK61525 from the National Institutes of Health The authors express their appreciation to Ms R Brown, Ms R Kulhavy, and Ms C Barker for technical assistance and Ms L.R Brewer and Dr K Matousovic for critically reading the manuscript 20 21 22 Reprint requests to Dr Jan Novak, Department of Microbiology, University of Alabama at Birmingham, 845 19th Street S, BBRB 734, Birmingham, Alabama 35294, USA E-mail: Jan_Novak@microbio.uab.edu 23 REFERENCES 24 Julian BA, Waldo FB, Rifai A, Mestecky J: IgA nephropathy, the most common glomerulonephritis worldwide A neglected disease in the United States? 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IgA induced activation of human mesangial cells: Independent of Fc␣R1 (CD 89) Kidney Int 54:837–847, 1998 Westerhuis R, Van Zandbergen G, Verhagen NA, et al: Human mesangial cells in culture and... proteins and regulation of IGFBP3 in human mesangial cells Kidney Int 49:1071–1078, 1996 Gomez-Guerrero C, Gonzalez E, Hernando P, et al: Interaction of mesangial cells with IgA and IgG immune... some of which deposit in the mesangium Indeed, two groups have detected highly undergalactosylated IgA1 in the kidney mesangial cells of IgAN patients [18, 19] While binding of human IgA1 to human