Journal: PLoS ONE
Article Title: Critical Role of Kupffer Cell CD89 Expression in Experimental IgA Nephropathy
Figure Lengend Snippet: Decreased clearance of IgAN patient IgA by CD89-expressing Kupffer cells. (A) IgA, purified from a normal donor and from a patient with IgAN, was stained with Cy3-anti-human IgA Ab, and subsequently incubated with Kupffer cells from CD89 transgenic mice at a concentration of 1 μg/mL and a temperature of 37°C. After 1-h-incubation, intracellular vesicles containing IgA were diminished when IgAN patient-derived IgA endocytosis was assessed and compared with normal human IgA. (B) sCD89-IgA complexes in cell supernatant of (A) were detected by ELISA using A3 and anti-mouse IgA mAb, using 1 μg/mL human IgA binding to 5 μg/mL recCD89 as an internal control. The sCD89-IgA complexes were detected in the Kupffer cell supernatant after 1h of incubation with either control IgA or patient IgA. (C) Decreased clearance of 10 μg IgAN patient-derived IgA in C57BL/6-Tg mice when compared to normal human IgA. A total of 10 μg of each of the proteins was injected into the tail vein of C57BL/6-Tg (n = 5 per group). At the indicated times after injection, sera were obtained and IgA levels were determined. IgA from IgAN patients was cleared less quickly in Tg mice compared with normal IgA. (D) Induction of mesangial IgA/sCD89 deposits by injecting patient IgA into 6-wk-old C57BL/6-Tg mice. Double staining by anti-human IgA and anti-CD89 in kidney 48 h after constant injection of patient IgA into C57BL/6-Tg mice(n = 4) is shown. Bar = 10μm.
Article Snippet: The mouse IgA, IgG, and IgM or human IgA concentration in mouse serum were determined using mouse IgA, IgG, IgM or human IgA ELISA Kits (Bethyl lab), respectively, according to the manufacturer’s instructions.
Techniques: Expressing, Purification, Staining, Incubation, Transgenic Assay, Mouse Assay, Concentration Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Injection, Double Staining