sCD89–TfR1 interaction favors IgA1 deposition and activates mesangial cells. (A) HMCs were incubated with the IgA1 or IgA1–sCD89 complexes at 4°C, and binding was detected using anti–IgA-FITC. The graph shows the mean of IgA1 binding (±SEM) as a mean fluorescence intensity ratio to background of six independent experiments. (B) Cell surface colocalization of IgA1 and CD89 on HMCs after incubation with IgA1–sCD89 complexes on ice. Wheat germ agglutinin (WGA) was used as cell membrane marker. Bars, 2 µm. (C) Detection of IL-8, IL-6, and TNF in the supernatant of HMCs stimulated for 48 h with BSA, IgA1, and IgA1–sCD89 complexes. n = 6 experiments. (D) Cytokine production after stimulation of TfR1 miRNA–treated HMCs with IgA1–sCD89 complexes for 8 h. n = 3 experiments. HMCs were infected with lentivirus bearing either TfR1 miRNA or irrelevant miRNA. (C and D) Error bars indicate SEM. (E) mRNA levels of MIP-2, IL-6, and TNF normalized to GAPDH mRNA levels in kidney cortex of WT, α1KI, and α1KI-CD89Tg mice as detected by quantitative PCR. Bars represent the mean. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (using the Mann-Whitney U test).