Figure 1.

Overexpression of tumor necrosis factor α (TNFα) amplifies autoantibody production and serum citrulline. A, Sera from 5-month-old TNFα-overexpressing (TNF) mice and wild-type (WT) littermates were subjected to a multiplex assay to detect autoantibodies against citrullinated (Cit) and native antigens. The median fluorescence intensity of individual serum samples is displayed in a heatmap of antigens against which antibodies are present at higher levels in sera from TNFα-overexpressing mice compared to sera from WT mice (false discovery rate <0.1% by Significance Analysis of Microarrays). B and C, Native (N) and citrullinated (C) vimentin (Vim), histone H2A, and histone H2B were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with sera from WT and TNFα-overexpressing mice to detect autoantibodies against native and citrullinated antigens. Representative blots are shown in B, with the mean ± SEM of the band density shown in C (n = 8 for citrullinated histones and n = 6 for citrullinated vimentin). * = P ≤ 0.05 by Wilcoxon matched pairs signed rank test. D, Desalted sera from 5-month-old TNFα-overexpressing and WT mice were subjected to the color development reagent (COLDER) assay to quantify citrulline. Graph depicts the mean ± SEM relative absorbance. Samples were tested in duplicate (n = 5). ** = P < 0.01 by paired t-test.