Modulation of Mesangial Cell Reactivity by Aberrantly Glycosylated IgA (2000)

The hallmark of primary IgA nephropathy with mesangial IgA deposits (IgAN) is the presence of detectable amounts of IgA in the mesangial area [1]. The pathogenesis of this glomerulonephritis was ascribed to the accumulation of IgA immune complexes (IgAIC) within glomeruli [2, 3]. This hypothesis offered a unifying explanation of the relationship between infection of the upper airways, mucosal immune responses associated with predominant IgA synthesis and mesangial deposition of IgA aggregates leading to gross hematuria. Although many studies revealed high levels of IgAIC during clinically active phases of IgAN [4, 5, 6], circulating IgAIC were detected in only 30–70% of the patients, even if serial samples were analyzed [7]. Moreover, attempts to identify the antigens inciting IgAN have failed to yield consistent results. In 20–30% of the patients, IgA specific for alimentary antigens has been detected, but often the levels of such antibodies did not correlate with IgAIC levels or disease activity [8]. Efforts to detect alimentary or viral antigens (CMV, HSV, EBV) in glomerular deposits produced inconsistent results as well [9, 10]. In summary, much of the data implicating true IgAIC as the unique mechanism in the pathogenesis of IgAN are inconclusive.Recent attention has been focused on the character of the IgA produced by IgAN patients, particularly IgA1, which is the predominant class deposited in mesangial area of IgAN patients [11]. A defect in immunoregulation apparently leads to the synthesis of structurally abnormal IgA. Our group in Turin was among the first to propose that the binding of IgA to any of a wide variety of molecules (exogenous alimentary antigens or endogenous mesangial components) can be based upon lectinic binding to oligosaccharides on the IgA, regardless of its antigen specificity [12, 13, 14]. Indeed, we showed that a single preparation of patient IgA binds to several distinct targets, including environmental antigens and mesangial matrix component [13, 14], and the binding of IgA to each target proved to be reversible by addition of the competitive sugars. Furthermore, IgA isolated from patients with Schönlein-Henoch nephritis, a disease that closely resembles IgAN, exhibits similar reactivity, and we recently demonstrated saccharide-based binding of IgA to ANCA antigens [15].Human IgA is highly glycosylated [16]. Carbohydrates represent 12.3% in serum IgA and 15% in secretory IgA. IgA1 has two residues of complex N-linked oligosaccharides, and the IgA2 isotype 4 and 5, respectively. The J chain of polymeric IgA has one N-linked carbohydrate chain, and the secretory component has four additional N-linked oligosaccharides. In addition to the N-linked oligosaccharides typically present at the carboxy terminus of all classes of immunoglobulin heavy chain, IgA1 contains five short O-linked oligosaccharide chains. These oligosaccharides are coupled to serine and/or threonine residues which lie in the hinge region connecting the CH1 and CH2 domains, at the junction between the Fab and the Fc portions of the IgA molecule [16]. In patients with IgAN, the circulating IgA exhibits a reduced galactose and/or sialic acid content demonstrated by increased binding to GalNAc-specific lectins (Vicia villosa) or increased asialo chains [17, 18, 19, 20, 21, 22]. This abnormal glycosylation could derive from genetically determined defective activity of β-1–3-galactosyltransferase [23]or be acquired, due to an imbalance in lymphocyte function, with a prevalence of Th2 over Th1 T cell subsets [24].Such aberrantly glycosylated IgA can circulate in a monomeric form or participate in the formation of autoaggregates or true immune complexes. In vitro desialylated or degalactosylated IgA shows a high tendency to autoaggregate, forming macromolecules with a molecular weight similar to IgAIC [24]. Whether bound in immune complexes or in autoaggregates, such aberrantly glycosylated IgA likely escapes clearance by hepatic receptors (ASGPR) for asialoglycoproteins [25]. In addition, by virtue of enhanced lectinic reactivity with the fibronectin, laminin, and collagen within the mesangial matrix [13, 14, 22], abnormally glycosylated IgA deposits in glomeruli more readily than does normal IgA. Finally, mesangial catabolism of aberrantly glycosylated IgA may be diminished [26]. In combination, these factors can lead to heightened accumulation and/or prolonged persistence of IgA deposits within the mesangium. In turn, aberrantly glycosylated IgA shows enhanced interaction with Fcα receptors on mesangial cells (MCs), resulting in increased binding [27, 28]. For these reasons, aberrantly glycosylated IgAs are likely to undergo a preferential deposition in the mesangial area.The finding of an abnormal glycosylation of circulating IgA does not mean that IgAN is a sort of myeloma or amyloid storage disease. IgAN is a glomerulonephritis characterized by MC activation which follows the formation of IgA deposits and leads to increase in cytosolic calcium, cell contraction, proliferation and release of mediators. The synthesis of a variety of cytokines (IL-6, PDGF, IL-1, TNF-α, TGF-β), vasoactive factors (prostaglandins, thromboxane, leukotrienes, endothelin, PAF, NO) or chemokines (MCP-1, IL-8, MIP-1, RANTES) is enhanced [29, 30, 31, 32].Over the last years we focused particular attention on the modulation of MC reactivity by aberrantly glycosylated IgA [33, 34, 35, 36, 37]. To this aim we investigated the biological effects of in vitro deglycosylated IgA and of selected glycoforms isolated from patients with IgAN.Desialylated (deSia) IgAs were prepared [33, 34, 35, 36, 37]by treating IgA from healthy subjects with neuraminidase and then isolating fractions not reacting with Sambucus nigra lectin column, hence deficient in terminal sialic acid (Neu5Ac) linked with galactose residues. deSia and degalactosylated (deSia/deGal) IgAs were obtained by treating deSia IgA with β-galactosidase and then removing the residual terminal galactose with Ricinus communis column. The effects of enzymatic hydrolysis of Neu5Ac and Gal to generate deSia and deSia/deGal IgA were confirmed by reciprocal changes in the binding of the treated IgA to Peanut and to V. Villosa lectins.Different glycoform of IgA were isolated in sera from IgAN patients and controls by affinity chromatography using columns of lectins immobilized on Sepharose [36]. The glycoproteins were eluted by the corresponding sugars: Canavalia ensiformis (ConA) specific for α-mannose (Man) group; Triticum vulgare (WGA) specific for N-acetylglucosamine (GlcNAc) group; Glycine maximus (SBA) specific for N-acetylgalactosamine (GalNAc) group; Ulex europaeus (UEA-I) specific for αL-fucose (Fuc), Limulus polyphemus (LPA) specific for sialic acid (Neu5Ac)α2,6GalNAc group. The eluted peaks were fractionated in HPLC to isolate 250- to 400-kD glycoproteins and the IgA content measured.IgAN patients showed, in comparison to controls, increased levels of IgA glycoforms provided with higher affinity for SBA, indicating increased exposure of GalNAc and increased binding to LPA, suggesting increased exposure of sialic acid bound to GalNAc (Neu5Ac-α2,6GalNAc). The prevalence of these IgA glycoforms in sera of IgAN patients is in agreement with the hypothesis of deficient galactosylation of the molecule. Moreover, IgAN patients showed, in comparison to controls, increased levels of IgA glycoforms with affinity to ConA, indicating an increased exposure of Man, suggesting the concomitant defect in N-glycosylation.In vitro prepared deSia IgA and deSia/deGal IgA and IgA glycoforms from IgAN patients and controls rich in terminal GalNAc or Neu5Ac2,6,GalNAc or Man were incubated with cultured MCs to investigate the possible modulations of some cell functions.We focused at first on MC integrin expressions and on their modulation by aberrantly glycosylated IgA [33]. Signal transduction by MC integrins regulates cell growth and survival, extracellular matrix production and organization [38, 39]. Glycation is known to be critical in the expression of adhesion molecules. Cultured MC express ανβ3 and some ανβ1 [38, 40]. We observed that ανβ3 was upregulated by matrix components, including fibronectin or type IV collagen, and also by aberrantly glycosylated IgA and by free sugars [40]. The subunit αν was modulated by sugars including Man (N-linked with asparagine residues in the Fab region) in the side chains of the IgA molecule and the O-linked chain sugars of the hinge region. In fact GalNAc significantly increased αν expression, while sialic acid (Neu5Ac), which covers all the sugar chains, depressed it. In vitro deSia IgA and deSia/deGal IgA enhanced ανβ3 expression on cultured MCs as well as IgA from IgAN patients with high exposure of internal sugars, suggesting that truncation of terminal sugars of IgA glycan chains upregulates the expression of these receptors of matrix components [33].To gain insight into the glomerular capillary repair mechanisms in IgAN, we focused on VEGF-A and NO. We examined whether VEGF-A and NO production by MCs could be modulated by aberrantly glycosylated IgA [34, 35]. RT-PCR experiments demonstrated a significant downregulation of VEGF-A mRNA in MCs incubated with aberrantly glycosylated IgA prepared in vitro by means of glycosidases and with IgA glycoforms isolated from IgAN patients. This resulted in a decreased release of VEGF-A in the culture medium. Conversely, deSia IgA and even more deSia/deGal IgA significantly enhanced NOS activity of MCs – detected as production of [3H] citrulline from [3H]arginine – in comparison to native IgA. Northern blot analysis demonstrated an upregulation of inducible iNOS mRNA by aberrantly glycosylated IgA. No significant modulation of constitutive cNOS mRNA was found. The NOS activity significantly increased after incubation with abnormal glycoforms of IgA from IgAN patients, particularly serum fractions rich in IgA bearing Neu5-2,6,GalNAc residues and to a lesser extent those rich in Man and GalNAc. Depression of VEGF-A production induced by aberrantly glycosylated IgA was mediated by NO, since the phenomenon was completely reversed by the NOS inhibitor Nω-nitro-L-arginine methyl ester.In conclusion, we found a negative control of VEGF-A synthesis in MCs by aberrantly glycosylated IgA, mediated by enhanced iNOS activity. Both increased iNOS activity and depressed VEGF-A synthesis could impair the vascular repair and favor sclerosis in IgAN.deSia IgA and deSia/deGal IgA significantly depressed the MC proliferation rate (assessed by [3H]thymidine incorporation) [36].Similarly, the IgA glycoform from IgAN patients highly expressing GalNAc or Neu5Ac-2,6,GalNAc significantly depressed the MC proliferation rate, while that rich in Man was ineffective.Apoptosis was assessed by detecting the DNA fragmentation and the expression of the apoptosis-related tumor suppressor protein p53. Apoptotic cells were quantified by counting the number of positive cells with the terminal uridine nick 3′ end labeling (TUNEL) method, while the expression of p53 was assessed by flow cytometry. In vitro prepared aberrantly glycosylated IgA significantly enhanced apoptosis.The apoptosis was also significantly enhanced by IgA glycoforms isolated in IgAN patients presenting increased exposure of internal sugars in comparison to healthy controls, as detected by the increased percentage of positive cells at the TUNEL test and higher expression of the tumor suppressor protein p53 [36].Complement is a crucial mediator of renal injury in experimental models of IgAN [41], and signs of alternative complement pathway activation have been demonstrated in patients with IgAN and Henoch-Schönlein purpura nephritis [42]. The process has been considered to be antigen mediated [43]. IgA depleted of carbohydrates shows the same increased fixation of C3b [44]as interfacially aggregated IgA. Therefore, the conformational changes should promote complement activation.We investigated the complement-fixing capability of aberrantly glycosylated IgA by means of a monoclonal antibody (MoAb 13/15) directed against a neoepitope on human activated C3–C3b, iC3b and C3dg (kindly provided by Prof. O. Goetze) [37]. Aberrantly glycosylated IgA fixed the C3 neoantigen of activated C3 more efficiently than native IgA. This activation was carried by selective alternative complement pathway activation. Comparing the C3-fixing capacity in IgA glycoform isolated from IgAN patients and controls, we found no difference in IgA exposing Man, the typical sugar of the N-linked residues, while GalNAc- and Gal-exposing glycoforms, characteristic of the O-linked side chains, were significantly more active for complement activation in patients than in controls [IgAN Club Meeting, The Netherland, 1998].Moreover, we demonstrated that C3 fixation by aberranlty glycosylated IgA further enhanced the NOS activity of cultured MCs, showing a multiplying effect of the activation induced by deSia/deGal IgA [37].Our studies have added further support to the hypothesis of an altered glycosylation of circulating IgA molecules in patients with IgAN and reported a series of modulation of the MC activity consequent to the contact with abnormal IgA glycoforms.Aberrantly glycosylated IgA prepared in vitro as well as IgA glycoforms isolated from IgAN patients were able to fix complement, increase integrin expression, enhance NOS activity and decrease VEGF-A synthesis, meanwhile depressing cell proliferation and increasing the apoptotic rate.The significance of our results must be considered in the complicate network of factors which interplay during development and progression of IgAN. IgAN is a renal disease characterized by MC proliferative changes which leave space for sclerotic lesions during the progression of renal damage. Accordingly, different mediators concurrently enhance either MC proliferation or apoptosis/sclerosis. The timely sequence of expression of VEGF-A, PDGF, IL-6, bFGF, and various other cytokines, like IL-1 and TNFα, are crucial in determining the amounts of NO produced and the functional consequences on cell proliferation/apoptosis with various effects on the course of disease progression.In conclusion, not only we confirmed the presence in patients with IgAN of circulating IgA with altered glycosylation pattern, but we demonstrated that these properties can modulate the reactivity of MCs which could play a role in the development and progression of IgAN.We are grateful to Prof. Yasuhiko Tomino, Division of Nephrology, Department of Medicine, Juntendo University School of Medicine, Tokyo, Japan.Presented in part at the 6th Tokyo International Seminar on IgA Nephropathy 1999.