Charge-dependent binding of polymeric IgA1 to human mesangial cells in IgA nephropathy

BACKGROUND: IgA nephropathy (IgAN) is characterized by raised serum IgA1 and predominant mesangial IgA1 deposits of polymeric nature. The mechanism of polymeric IgA1 (pIgA1) deposition in the kidney mesangium is poorly understood in IgAN. It has been suggested that increased sialic acid content and anionic charge of the pIgA1 molecules may be operational in the IgA1 deposition in human mesangial cells (HMCs). The present study examined the binding of pIgA1 with different surface charges to HMCs. The binding characteristics of IgA1 to HMCs in the presence of polycation (poly-L-lysine) or polyanion (heparin) were also investigated. METHODS: IgA1 was purified in sera from patients with IgAN and from healthy controls by jacalin affinity chromatography. IgA1 was further separated into pIgA1 and monomeric IgA1 (mIgA1) by fast protein liquid chromatography (FPLC). pIgA1 or mIgA1 with different net charges on their surface were resolved by ion exchange chromatography (IEC) with a Mono Q column. The binding characteristics of pIgA1 and mIgA1 to HMCs in the presence or absence of polycation or polyanion were examined by flow cytometry. RESULTS: In patients with IgAN, the absolute amount of mIgA1 and pIgA1 is significantly higher than that of healthy controls (P < 0. 001). There was significant increase in binding of pIgA1 from patients with IgAN to HMC and cell lysate. pIgA1 that interacted strongly with the ion exchanger also bound more to HMCs when compared with IgA1 interacted weakly with the ion exchanger (P < 0. 001). The anionic charged pIgA1 from patients was significantly higher than that of healthy controls (P < 0.001). Preincubation with poly-L-lysine increased the binding of pIgA1 to HMCs. The binding of pIgA1 to HMCs was decreased by preincubation with heparin. CONCLUSIONS: The binding of IgA to HMCs is charge dependent. Polymeric IgA with the highest net negative charge binds more to HMCs. Preincubation with polyanion decreased the binding of polymeric IgA to HMCs. These results suggest an important role for anionic charge in IgA1 deposition onto the kidney mesangial cells.     

Mesangial deposition of J chain-linked polymeric IgA in IgA nephropathy

Renal biopsy specimens from 8 patients with IgA nephropathy (Berger's disease) were examined immunocytochemically at the light and electron microscopic levels with peroxidase-labeled antibodies to IgA, IgM, J chain and secretory component. In 2 of the 8 specimens heavy deposits of IgA, but no IgM, were found in the mesangium. After acid-urea treatment of these tissue sections, J chain, a subunit of polymeric immunoglobulins, was identified in a distribution similar to that of IgA. In the remaining 6 specimens, small amounts of IgM in addition to denser deposits of IgA and J chains were found. We conclude that the IgA deposits in at least some patients with Berger's disease consist of IgA polymers linked by J chain.     

Clinical significance of polymeric and monomeric IgA complexes in patients with IgA nephropathy

IgA nephropathy is currently considered an immune complex (IC) disease. However, though several groups have demonstrated the presence of IgA-IC in the sera of patients by various techniques, a correlation with clinical activity of the nephropathy has not always been found. Since these assays detect (simultaneously) polymeric and monomeric IgA-IC, the pathogenicity of these two classes of complexes could not be established. In this work, we have studied in 66 patients with IgA nephropathy the existence and significance of such IC, by means of a technique described in our laboratory, based on the specific binding of secretory component for polymeric IgA. Furthermore, IgG-ICs were also determined by the standard Raji cell assay in ELISA. The prevalence of these complexes was as follows: Multimeric (polymeric and monomeric) IgA-ICs were detected in 55% of 66 patients studied, polymeric IgA-ICs in 30%, monomeric IgA-ICs in 39%, and IgG-ICs in 46%. The intermittency of all these complexes was clearly noted in sequential examinations. A significant correlation (P less than .025) with hematuria was only found with polymeric IgA-IC, but not with multimeric IgA-IC, monomeric IgA-IC, or IgG-IC. Polymeric IgA-ICs were more frequently observed at the initial phases of the disease. Analytical ultracentrifugation showed that polymeric IgA-IC was of larger size than monomeric IgA-IC. The major pathogenicity of polymeric IgA-IC is in agreement with the finding of this immunoglobulin at the mesangial level in patients and animals with IgA nephropathy.     

Specific binding of circulating IgA antibodies in patients with IgA nephropathy

Detection of circulating IgA antibodies which are specific in patients with IgA nephropathy is described. Freeze and thawed extracts of pharyngeal cells obtained from patients with IgA nephropathy, other glomerular diseases, and healthy adults were cultured with fibroblasts such as Vero or Hel cells at 37 degrees C for 2 weeks. Serum samples were obtained from these patients and healthy adults. The cultured fibroblasts were fixed on slide glasses, and then incubated with the serum samples from the same or other patients with IgA nephropathy. The cells were stained with FITC-labeled heavy-chain specific anti-human IgA antiserum and then examined with a fluorescent microscope. It was demonstrated that the IgA antibodies in sera obtained from patients with IgA nephropathy or HSP nephritis were bound with the nuclear regions of such fibroblasts. It was suggested that IgA antibodies in sera could be bound with some antigenic substances which were transferred from pharyngeal cells of patients with IgA nephropathy to fibroblasts in vitro.     

Elevated production of polymeric and monomeric IgA1 by the bone marrow in IgA nephropathy

In bone marrow cultures of 15 patients with primary IgA nephropathy we found significantly (P = 0.02) increased synthesis of both monomeric and polymeric IgA1 compared to 23 controls, by using high performance liquid chromatography (HPLC) fractionation of culture supernatants. The relative contribution of polymeric to total IgA1 produced was not different for the two groups. Two-color immunofluorescence studies of the percentage of bone marrow IgA1 plasma cells able to bind secretory component in vitro showed no difference between patients and controls. In the sera of patients with primary IgA nephropathy the relative contribution of IgA1 polymers to total IgA1 was also similar to controls. These results indicate that in IgA nephropathy, the increased IgA production in the bone marrow is restricted to the IgA1 subclass. The production of both monomeric and polymeric IgA1 is increased in patients during a quiescent phase of the disease.     

Characteristics of patients with coexisting IgA nephropathy and membranous nephropathy

Background: Coexistence of IgA nephropathy (IgAN) and membranous nephropathy (MN) in the same patient is rare. Few studies have reported the clinical and pathological features of patients with combined IgAN and MN (IgAN–MN).

Methods: The clinico-pathological features, levels of galactose-deficient IgA1 (Gd-IgA1) and autoantibodies against M-type transmembrane phospholipase A₂ receptor (anti-PLA₂R) in sera were compared among IgAN–MN, IgAN, and MN patients.

Results: Twenty-six patients with biopsy-proven IgAN–MN were enrolled. The mean age at biopsy was 43.6?±?15.9?years, and 65.4% were male. Proteinuria and estimated glomerular filtration rate (eGFR) levels in patients with IgAN–MN were similar to that of MN patients. Compared with the IgAN patients, IgAN–MN patients showed a higher median proteinuria level (4.3 vs. 1.2?g/day, p?2, p?p?=?.801). Percentage of IgAN–MN patients with detectable serum levels of anti-PLA₂R was lower than that of MN patients (38.5% vs. 68.6%, p?=?.011).

Conclusions: IgAN–MN patients display similar clinical features to MN patients and milder pathological lesions than IgAN patients. IgAN–MN patients have similar levels of Gd-IgA1 to those of IgAN patients, and a lower proportion of anti-PLA₂R than MN patients.     

Mesangial expression of angiotensin II receptor in IgA nephropathy and its regulation by polymeric IgA1

BACKGROUND: Enhanced gene expression for the renin-angiotensin system (RAS) is detected in glomerular mesangial cells in IgA nephropathy (IgAN). Preliminary studies showed a reduced glomerular gene expression of angiotensin II subtype 1 receptor (AT1R), suggesting a regulatory response to high intrarenal angiotensin II (Ang II) concentration in IgAN. METHODS: We examined the effect of polymeric IgA1 (pIgA1) from patients with IgAN on the expression of Ang II receptors in cultured human mesangial cells (HMC). RESULTS: Polymeric IgA1 from patients with IgAN down-regulated the expression of AT1R in HMC in a dose-dependent manner. When similar experiments were conducted with addition of an angiotensin-converting enzyme inhibitor (captopril) or an AT1R antagonist (losartan), there was a significant increase in the expression of AT1R. Blockade of Ang II with captopril or losartan alone resulted in a stepwise increase of AT1R in cultured HMC. Down-regulation of Ang II subtype 2 receptor (AT2R) was not observed in HMC cultured with pIgA1 from patients with IgAN. The acute suppressive effect of pIgA1 from IgAN on the expression of AT1R was confirmed in HMC incubated with IgA isolated from 15 IgAN patients, 15 healthy subjects, and other glomerulonephritides control subjects. Reduced glomerular expression of AT1R (but not AT2R) was also demonstrated in renal biopsies from patients with IgAN. CONCLUSION: Our findings demonstrate an altered AT1R expression in HMC in response to raised intrarenal Ang II in IgAN. Our in vitro studies also support that an imbalance of AT1R and AT2R activity in HMC following exposure to pIgA plays a significant pathogenetic role in the inflammatory injury in IgAN.     

Differential glycosylation of polymeric and monomeric IgA: a possible role in glomerular inflammation in IgA nephropathy

IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA1 (pIgA1) and complement. Complement activation via mannose-binding lectin and the lectin pathway is associated with disease progression. Furthermore, recent studies have indicated a possible role for secretory IgA. IgAN is associated with abnormalities in circulating IgA, including aberrant O-linked glycosylation. This study characterized and compared functional properties and N-linked glycosylation of highly purified monomeric IgA (mIgA) and pIgA from patients with IgAN and control subjects. Total serum IgA was affinity-purified from patients (n = 11) and control subjects (n = 11) followed by size separation. pIgA but not mIgA contained secretory IgA, and its concentration was significantly higher in patients with IgAN than in control subjects. Both in patients with IgAN and in control subjects, IgA binding to the GalNAc-specific lectin Helix Aspersa and to mannose-binding lectin was much stronger for pIgA than for mIgA. Furthermore, binding of IgA to mesangial cells largely was restricted to polymeric IgA. Binding of pIgA to mesangial cells resulted in increased production of IL-8, predominantly with IgA from patients with IgAN. Quantitative analysis of N-linked glycosylation of IgA heavy chains showed significant differences in glycan composition between mIgA and pIgA, including the presence of oligomannose exclusively on pIgA. In conclusion, binding and activation of mesangial cells, as well as lectin pathway activation, is a predominant characteristic of pIgA as opposed to mIgA. Furthermore, pIgA has different N-glycans, which may recruit lectins of the inflammatory pathway. These results underscore the role of pIgA in glomerular inflammation in IgAN.     

Contribution of macromolecular IgA1 to IgA abnormality in IgA nephropathy

To evaluate the contribution of macromolecular IgA₁ to IgA abnormality in childhood IgA nephropathy, serum samples from 29 healthy children and 26 patients with IgA nephropathy in different age-groups (7–9, 10–12, and 13–15 years) were each separated by sucrose density gradient ultracentrifugation and assayed for IgA₁ using an enzyme-linked immunosorbent assay. IgA₁ in fraction I (sedimentation coefficient >11.4s) was significantly greater in patients 7–15 years of age (median 36.3–57.0 mg/dl) than in the age-matched controls (median 8.8–10.4 mg/dl). IgA₁ in fraction II (11.4–9.3s) was significantly greater in patients 10–15 years of age (median 46.7–52.6 mg/dl) than in the controls (median 27.8–35.5 mg/dl), and IgA₁ in fraction III (<9.3s) was significantly greater in patients 13–15 years of age (median 156.9 mg/dl) than in the controls (median 120.7 mg/dl). The ratio of IgA₁ in fractions I–III was higher in the patients of each age-group (median 0.233–0.314) than in the controls (median 0.067–0.082), while the ratio of IgA₁ in fractions II–III was not significantly high in patients 7–12 years old (median 0.268 to 0.318) compared with the controls (median 0.182–0.264). Thus, IgA abnormality in childhood IgA nephropathy would be better represented by an increase in macromolecular IgA₁ of >11.4s than by an increase in IgA₁ in fractions of 11.4–9.3s or <9.3s. Received: 8 December 1999 / Revised: 2 March 2000 / Accepted: 2 May 2000     

An increased polymeric IgA level is not a prognostic marker for progressive IgA nephropathy.

BACKGROUND: Elution of IgA from renal biopsies of patients with primary IgA nephropathy (IgAN) has suggested that mesangial IgA deposits are mainly multimeric in nature. This macromolecular IgA consists of dimeric and polymeric IgA and may be derived from the circulation. In children with IgAN, circulating macromolecular IgA levels correlate with bouts of macroscopic haematuria, but in adults a correlation with disease activity is less clear. Therefore, we have designed a novel method to assess the levels of polymeric IgA (pIgA) in sera from patients and controls. METHODS: A novel precipitation assay using recombinant CD89 was developed to measure pIgA. Polymeric IgA levels were measured in serum samples obtained from healthy volunteers (n = 21) and patients with IgAN (n = 51). Subsequently, serum pIgA levels were correlated with clinical parameters of disease. RESULTS: Serum pIgA levels were significantly increased in patients with IgAN. However, pIgA concentrations relative to total IgA were significantly lower in sera of patients with IgAN. No correlation was found between serum pIgA levels and clinical parameters of IgAN, such as decline of glomerular filtration rate, haematuria or proteinuria. CONCLUSIONS: Although absolute levels of serum pIgA are increased in patients with IgAN as compared with controls, levels of pIgA relative to total serum IgA are lower. No significant correlation was found between serum concentrations of pIgA and clinical parameters of disease. These data support the notion that it is not the size alone, but the physicochemical composition of the macromolecular IgA that is the key factor leading to mesangial deposition.     

The glomerular response to IgA deposition in IgA nephropathy

Compelling evidence points to a role for IgA receptors in the pathogenesis of IgA nephropathy. The soluble form of the type I IgA receptor (FcalphaRI or CD89) forms complexes with IgA that can be found in patients' serum and that initiate the disease in CD89 transgenic mice. A nonclassic IgA receptor, identified as the transferrin receptor (TfR), is highly expressed in patients' mesangium and colocalizes with IgA deposits. TfR preferentially binds polymeric IgA1 complexes, but not monomeric IgA1 or IgA2. The TfR-IgA1 interaction is dependent on carbohydrate moieties because hypoglycosylated IgA1 has superior binding to TfR than normally glycosylated IgA1. Polymeric IgA1 binding enhances mesangial cell TfR expression and results in cell proliferation and inflammatory and profibrogenic cytokine and chemokine production, suggesting a pivotal role in mesangial cell proliferation, matrix expansion, and recruitment of inflammatory cells. We propose that, as a second event, activation of the classic, FcRgamma-associated transmembrane FcalphaRI expressed on circulating myeloid leukocytes takes place. FcalphaRI/gamma2 cross-linking in human FcalphaRI transgenic animals promotes disease progression by enhancing leukocyte chemotaxis and cytokine production, and IgA immune complexes from IgA nephropathy patients induce FcalphaRI-dependent cell activation. This review therefore details the functional consequences of IgA/receptor interactions and discusses proposed mechanisms to explain the development and chronicity of the disease.     

IgA nephropathy: association of a history of macroscopic hematuria episodes with increased production of polymeric IgA

From the clinical point of view patients with IgA nephropathy present two main symptoms. Some patients have episodes of macroscopic hematuria, either isolated or recurrent, while others present only asymptomatic urinary abnormalities. Although these differences could be due to age-related, geographical or other unknown reasons, in this paper we studied whether patients with these two clinical manifestations differ in some IgA immunological aspects, described occasionally as abnormal in this disease. The following results were obtained: patients having a history of macroscopic hematuria episodes (n = 29) had a significant increase in the percentage of blood polymeric-IgA-producing cells (68 +/- 12%) and in the T cells with IgA Fc-receptors (T alpha cells; 14.5 +/- 3%) in relation to those having only asymptomatic urinary abnormalities (n = 13; 48 +/- 11 and 11.8 +/- 3%, respectively). However, there was no difference in the two groups of patients in relation to the serum IgA levels, percentage of IgA-bearing cells, in vitro synthesis of IgA, T-cell subpopulation and generation of Con A-IgA suppressor cells. Since these patients did not differ in the age of the apparent onset of the disease, in age at the moment of the study or in the activity of the disease determined by hematuria, our results afford an immunological support suggesting the existence of two subgroups of patients with IgA nephropathy.     

Detection of IgA1-dominant immune complexes in peripheral blood polymorphonuclear leukocytes by double immunofluorescence in patients with IgA nephropathy

The amounts of IgA1- and/or IgA2-dominant immune complexes included in peripheral blood polymorphonuclear cells (PMN) were determined by the double immunofluorescence technique in patients with IgA nephropathy. The aim of the present study was to determine the prevalence of IgA1-and/or IgA2-dominant immune complexes phagocytized by PMN in patients with IgA nephropathy. 5 patients with IgA nephropathy and 10 healthy adults were examined. It was demonstrated that the percentages of IgA1 with C3 cytoplasmic inclusion bodies were significantly increased compared with those of IgA2 with C3 cytoplasmic inclusion bodies in patients with IgA nephropathy. It was suggested that IgA1-dominant immune complexes are phagocytized by peripheral blood PMN in patients with IgA nephropathy.     

Role of macromolecular IgA in IgA nephropathy

Primary IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis, leading to progressive renal failure in almost one third of the patients. The disease is characterized by mesangial deposits of IgA. The pathogenesis of IgAN remains incompletely understood. The basic abnormality of this disorder lies within the IgA immune system rather than in the kidney. Elevated levels of IgA and IgA-containing complexes are found in sera of most patients with IgAN, but increased levels alone are not sufficient to develop IgAN. Therefore abnormal physicochemical properties of circulating IgA, such as size, charge, and glycosylation may play a role. This is supported by the presence of altered glycosylation of serum and mesangial IgA in patients with IgAN. Although the precise origin and nature of the mesangial IgA deposits are still uncertain, they contain at least in part macromolecular IgA, which may be derived from circulating IgA-containing complexes. Recently, novel insights have been obtained in the molecular composition of circulating high-molecular-weight IgA, which might include complexes with underglycosylated IgA1 and IgA-CD89 complexes. In this review various aspects of macromolecular IgA in relation to IgAN will be discussed.     

Reduced binding of immunoglobulin A (IgA) from patients with primary IgA nephropathy to the myeloid IgA Fc-receptor, CD89

Background. Primary IgA nephropathy (IgAN) is associated with elevated levels of circulating IgA and is characterized by deposition of primarily IgA1 in the renal mesangium. It has not yet been clarified which mechanisms govern the deposition of IgA1 in the mesangium. One of the factors which may play a role in trapping of IgA in the mesangial area is the interaction of IgA with specific IgA receptors (Fc&agr;R, CD89) on the mesangial cells. Methods. In the present study IgA derived from patients with IgAN and controls was investigated for its interaction with human CD89, expressed on the surface of the murine B cell line IIA1.6. Results. IgA binding to Cd89 expressing cells was specific, concentration dependent and binding of dIgA and pIgA occurred in a more efficient fashion than that of mIgA. IgA binding to CD89 directly from serum of patients compared to controls showed no significant difference. However these experiments are affected by differences in IgA concentration and combinations of different sizes of IgA. Using purified fractions of mIgA, dIgA, and pIgA isolated from serum, a significantly reduced binding of mIgA to CD89 from patients compared to controls was observed. Finally, the binding of aIgA2 to CD89 was less inhibited using mIgA from patients with IgAN compared to controls. Conclusions. The reduced binding of mIgA to CD89 seems to contradict a direct role for CD89 in deposition of IgA. However reduced binding of mIgA to CD89 may affect IgA clearance, leading to higher serum IgA. Furthermore, since it has been demonstrated that mIgA can interfere with binding of di- and pIgA deposition in the mesangial area.