IgA Structure Variations Associate with Immune Stimulations and IgA Mesangial Deposition

Abstract

IgA1 mesangial deposition is the hallmark of IgA nephropathy and Henoch-Schönlein purpura, the onset of which often follows infections. Deposited IgA has been reported as polymeric, J chain associated, and often, hypogalactosylated but with no information concerning the influence of the IgA repertoire or the link between immune stimuli and IgA structure. We explored these issues in the α1KI mouse model, which produces polyclonal human IgA1 prone to mesangial deposition. Compared with mice challenged by a conventional environment, mice in a specific pathogen-free environment had less IgA deposition. However, serum IgA of specific pathogen-free mice showed more galactosylation and much lower polymerization. Notably, wild-type, α1KI, and even J chain-deficient mice showed increased polymeric serum IgA on exposure to pathogens. Strict germfree conditions delayed but did not completely prevent deposition; mice housed in these conditions had very low serum IgA levels and produced essentially monomeric IgA. Finally, comparing monoclonal IgA1 that had different variable regions and mesangial deposition patterns indicated that, independently of glycosylation and polymerization, deposition might also depend on IgA carrying specific variable domains. Together with IgA quantities and constant region post-translational modifications, repertoire changes during immune responses might, thus, modulate IgA propensity to deposition. These IgA features are not associated with circulating immune complexes and C3 deposition and are more pertinent to an initial IgA deposition step preceding overt clinical symptoms in patients.

Keywords: IgA; IgA deposition; IgA nephropathy; Immunology and pathology; transgenic mouse.

Figure 1.

IgA1 mesangial deposits in adult α1KI mice housed in CIS facilities. (A) Kidney section stained with FITC–conjugated anti–human IgA (green). (B) Electron microscopy of kidney section: white arrow points to IgA deposits. (C) Electron microscopy with immunogold labeling of human IgA. (D) Confocal microscopy after double immunofluorescence to detect human IgA in green and endothelial cells (CD31+ cells) in red. (E) Confocal microscopy of kidney section stained with FITC–conjugated anti–human IgA antibody (green) and AlexaFluor568–labeled antipodocin antibody (red). (F) Kidney section stained with hematoxylin and eosin. Pictures are representative of all of the glomeruli found in kidney sections of n>10 mice.

Figure 2.

IgA production and deposition vary with housing conditions. Influence of environmental stimuli on (A) mesangial IgA deposition and (B) plasma IgA levels in 6-week-old and 3-month-old α1KI mice housed in GF, SOPF, or CIS facilities. Homogeneous groups of five to six age-matched mice housed in the same conditions were studied. Deposits were detected on kidney sections by fluorescence microscopy using FITC–conjugated anti–human IgA. Representative pictures are shown. IgA was measured in plasma by ELISA (mean±SEM; Kruskall–Wallis test followed by Bonferroni test). **P<0.01.

Figure 3.

Influence of environmental stimuli on the monomeric-to-polymeric ratio of circulating IgA in groups of young (6–8 weeks old) and adult (3–4 months old) mice housed in GF, SOPF, or CIS facilities. (A) Plasma proteins were separated under nonreducing conditions, blotted, and stained with HRP–linked anti–human IgA. (B) Blots from two α1KI and two α1KI J−/− mice were stained with anti–mouse J–chain antiserum. (C) Blots from α1KI J−/− mice housed in SOPF and CIS facilities. (D) Blots from wild-type (WT) mice housed in SOPF and CIS facilities. Molecular mass scale (M_r) is expressed in kilodaltons. Data are representative of multiple experiments (n=4–8).

Figure 4.

Kinetics of plasma IgA levels and IgA deposition in glomeruli of 1-week-old to 3-month-old α1KI and α1KI J−/− mice. (A) IgA was measured by ELISA in mice housed in CIS facilities (means±SEM of n=4–8 mice; Kruskall–Wallis test followed by Bonferroni test). *P<0.05; **P<0.01. (B) IgA deposits (green) by fluorescence microscopy on representative kidney sections from groups of four to eight mice housed in CIS facilities. (C) IgA kidney deposits compared on representative kidney sections from groups of 6-week-old α1KI J−/− mice housed under either SOPF or CIS conditions.

Figure 5.

IgA glycosylation varies with housing conditions. Analysis of (A) N-acetylneuraminic acid and (B) exposed GalNac in plasma IgA from 6-week-old and 3-month-old α1KI, α1KI J−/−, and wild-type (WT) mice housed in GF, SOPF, or CIS conditions. Glycosylation was quantified by lectin assay using MA or HAA lectins. For each sample, OD of lectin assay was normalized using OD of IgA ELISA. Results are expressed as means±SEMs of n=4–9 α1KI or α1KI J−/− animals and n>10 WT mice (Mann–Whitney test or Kruskall–Wallis test followed by Bonferroni test). *P<0.05; **P<0.01; ***P<0.001.

Figure 6.

Analysis of IgA deposition in the kidney of immunodeficient mice 2 hours after injection of human IgA1 mAb. Clones 1–10 were injected in two mice each. Ctrl− is a negative control after injection of saline. (A) IgA deposits in kidney sections labeled with FITC–conjugated anti–human IgA (green) and AlexaFluor568-linked antipodocin (red). (B) Quantification of IgA staining by image analysis. Results are means±SEMs of n=20 glomeruli (Kruskall–Wallis test followed by Bonferroni test). ***P<0.001. (C) Confocal microscopy imaging of IgA (green) and endothelial CD31+ cells (red) in kidney sections from a mouse administered IgA1 mAb6. A and C are representative of all of the glomeruli analyzed in sections of n=2 mice.

Figure 7.

In vivo deposited IgA in RAG-2/γc–deficient mice after intraperitoneal grafting of α1KI hybridomas 1, 5, 6, 9, and 10. Each hybridoma was injected in two mice, which were euthanized after tumor development. Ctrl− stands for negative control with ungrafted littermates. (A) Immunofluorescence microscopy on kidney sections stained with FITC–conjugated anti–human IgA (green) and AlexaFluor568-linked antipodocin (red). (B) Quantification of IgA staining by image analysis. Results are means±SEMs of n=10 glomeruli (Kruskall–Wallis test). ***P<0.001. (C) Plasma IgA was measured by ELISA.

Figure 8.

Biochemical features of purified IgA1 mAbs. (A) Detection of J chain by Western blot. (B) Analysis of monomeric (mIgA) and polymeric (pIgA) forms by Western blot after SDS-PAGE in nonreducing conditions. (C) Glycosylation of IgA1 mAbs that deposit or do not deposit 2 hours after injection. Clones are identified with their number next to the corresponding data. N-acetylneuraminic acid and GalNac were analyzed using MA and HAA lectins, respectively. Results are expressed as means±SEMs of n=3–7 clones (Mann–Whitney test).

Figure 9.

Calculated pI of combined VH and Vκ CDR regions from α1KI IgA1 hybridomas. Amino acid sequences of VH and Vκ CDR from hybridomas 1–10 were combined to calculate pI; α1KI hybridomas yielding (circles) or not yielding (squares) IgA deposits are shown.

Figure 10.

Circular dichroism analysis of IgA1 mAbs. (A) Far-ultraviolet spectra of six mAbs acquired at 20°C with a path length of 0.2 cm on a spectropolarimeter. IgA1 mAb1 and IgA1 mAb6 yield IgA deposition, and the others do not. (B) Thermal unfolding analysis of six IgA1 mAbs. Heat denaturation temperatures are indicated on the right.

Figure 11.

Western blot analysis of trypsin digestion products. Nine IgA1 mAbs were submitted to 10% SDS-PAGE after various digestion times (0–10 hours) by trypsin. Western blot was performed to reveal IgA determinants using HRP–linked goat anti–human IgA. (Left panel) mAb2, mAb4, mAb7, mAb8, mAb9, and mAb10 yield no IgA deposits. (Right panel) mAb6, mAb1, and mAb5 yield deposits.

Figure 12.

Role of mAb V regions in kidney deposition. IgA1 mAb6 or its IgG1 switched variant was injected in immunodeficient mice 2 hours before euthanasia. IgA1 and IgG1 were revealed on kidney sections with FITC-labeled (green) antisera against either (upper panel) human IgA or (lower panel) IgG. Staining was also with AlexaFluor568 anti–mouse podocin (red). Injection of saline provided a negative control; α1KI and CH1− mice provided controls for the glomerular deposition of human IgA and γ-chains, respectively. Pictures are representative of hundreds of glomeruli observed in kidney sections (two or more mice for each condition). Ab, antibody; Ctrl+, control; i.v., intravenous.