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. Author manuscript; available in PMC: 2019 Dec 1.
Published in final edited form as: Arthritis Rheumatol. 2018 Oct 20;70(12):1946–1958. doi: 10.1002/art.40587

Figure 5.

Figure 5

RA subject-derived recombinant antibody:citrullinated-antigen immune complexes stimulate macrophages to produce TNF-α. Plate-bound immune complexes were formed by coating wells with a citrullinated antigen (H2B, H2A or PAD) followed by incubation with recombinant antibodies representing persistent lineages from A, Subject 1 or B, Subject 8. Monocyte-derived macrophages were added to each well, and after 24 hrs, supernatants harvested and TNF-α levels measured using ELISA. A portion of macrophages were pre-incubated with the TLR4 inhibitor TAK-242 (TLR4B) and FcγRII inhibitor anti-CD32 clone IV.3 (FcRB) prior to addition to the plate. LPS was used as a positive control, and cells alone with buffers as negative controls. The same controls are displayed in multiple panels for comparison. TNF-α levels were compared between antibodies observed at different timepoints within the same lineage, and differences in levels produced in the presence of inhibitors were compared to IC alone. ** = P < 0.001; * = P < 0.05 by two-way ANOVA, Tukey correction. Cells incubated with citrullinated proteins without IC were also compared to cells alone. † = P < 0.05 by one-way ANOVA, Tukey correction. Representative data presented as mean ± SEM, with similar trends observed in two independent experiments.