Abstract: SA-PO593
IgA1 from Sera of Patients with IgA Nephropathy, but Not Purified Monomeric or Polymeric IgA1, Associates with Cultured Primary Human Mesangial Cells and Induces Cellular Signaling
Session Information
- Glomerular Diseases: Immunology, Inflammation - II
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Authors
- Huang, Zhi qiang, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Hall, Stacy D., University of Alabama at Birmingham, Birmingham, Alabama, United States
- Julian, Bruce A., University of Alabama at Birmingham, Birmingham, Alabama, United States
- Novak, Jan, University of Alabama at Birmingham, Birmingham, Alabama, United States
Background
Circulating immune complexes (CIC) containing galactose-deficient IgA1 (Gd-IgA1) bound by Gd-IgA1-specific autoantibodies play a key role in the pathogenesis of IgA nephropathy (IgAN). Using a model of cultured primary human mesangial cells (hMC), we have previously shown that Gd-IgA1-containing CIC are biologically active, as they activate multiple protein-tyrosine kinases, lead to ERK1/2 phosphorylation, and stimulate cellular proliferation. In this study, we assessed how purified IgA1 of different molecular forms and serum IgA1 associate with and activate hMC.
Methods
Monomeric and polymeric forms of Gd-IgA1 were isolated from plasma of a patient with multiple myeloma by size-exclusion chromatography. IgA concentrations in serum samples from IgAN patients were measured by ELISA. Primary hMCs were incubated for 15 min at 37°C with 5% sera from IgAN patients or with the corresponding amount of monomeric or polymeric IgA1; hMC without any IgA1 served as a negative control. Cell lysates obtained after the incubation were used for pull-down using antibody specific for integrin β1 followed by protein G agarose. Cell lysates and pull-down samples were subjected to SDS-PAGE/Western blotting to detect IgA and phospho-ERK1/2.
Results
Purified monomeric as well as polymeric Gd-IgA1 exhibited minimal interactions with hMC, as only small amounts of IgA1 appeared in the hMC lysates and no induction of ERK1/2 phosphorylation was observed. In contrast, when sera from IgAN patients were used, we observed more IgA1 in hMC lysates (4 times more compared to monomeric Gd-IgA1, 8 times more compared to polymeric Gd-IgA1) as well as robust ERK1/2 phosphorylation. Preliminary pull-down experiments indicated that a fraction of IgA1 was associated with integrin β1, a previously described hMC receptor for IgA1. Moreover, we found that hMC formed α1β1 and α5β1 integrin complexes.
Conclusion
IgA1 from sera of IgAN patients, but not free monomeric or polymeric Gd-IgA1, associates with hMC and activates hMC. Integrin β1 may be involved in this process and may provide clues for development of future targeted therapy of IgAN.
Funding
- NIDDK Support