Correlation of serum anti‐DNA topoisomerase I antibody levels with disease severity and activity in systemic sclerosis

Abstract

Objective. To investigate correlations between serum levels of topoisomerase I-specific antibody (antitopo I) and clinical features of systemic sclerosis (SSc), including disease severity (the total skin score [TSS]) and disease activity.Methods. Using highly sensitive enzyme-linked immunosorbent assays, we measured the levels of antitopo I antibody, including total IgG, individual IgG subclasses, and IgA, and analyzed their correlations with the TSS in 59 patients with SSc, all of whom had diffuse cutaneous involvement. Serial serum samples were obtained from 11 of these patients.Results. The titers of anti-topo I antibody, including IgG and IgA, were positively correlated with the TSS, a measure of SSc disease severity. In 8 of the 11 patients from whom serial serum samples were obtained, changes in the levels of both IgG and IgA, when detectable, paralleled changes in the TSS. In 3 patients, an increasing anti-topo I IgG level preceded an increase in the TSS. The level of each IgG subclass also correlated with and tended to parallel the TSS. The patients with very active disease had higher mean IgG (P < 0.001) and IgA (P < 0.05) titers than did those with inactive disease.Conclusion. Serum levels of anti-topo I antibody correlate positively with disease severity and disease activity in SSc.

Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death

“…We used the fluorogenic substrate-based assay Magic Red MR-(FR) 2 , which allows the localization of cathepsin L activity in live cells. To augment the specificity of this localization, we simultaneously exposed the L929 cells to the cathepsin B inhibitor CA-074 to avoid nonspecific cleavage of the fluorogenic substrate by cathepsin B.…”

“…There is evidence that autoantibody responses to topo I in SSc are driven by cryptic determinants and that a very specific combination of fragmented forms of topo I and the type of APCs that process these forms may be involved in breaking tolerance to topo I in the early stages of development of this disease (1,2,7,41). It is conceivable that immunogenic fragmented forms of topo I could arise in vivo via caspase-or cathepsin-mediated cleavage of the protein during apoptosis or secondary necrosis.…”

“…Cathepsin L activity was detected using the fluorogenic substrate Magic Red MR-(FR) 2 . Briefly, cultured L929 cells were exposed for 30 minutes to the cathepsin L substrate MR-(FR) 2 , rinsed twice with medium, and directly examined under the fluorescence microscope.…”

“…Briefly, cultured L929 cells were exposed for 30 minutes to the cathepsin L substrate MR-(FR) 2 , rinsed twice with medium, and directly examined under the fluorescence microscope. Cells were counterstained with Hoechst 33342 for nuclear visualization.…”

“…Cells were counterstained with Hoechst 33342 for nuclear visualization. CA-074 (20 M), a specific inhibitor of cathepsin B (21), was added to cells to increase the specificity of the cleavage of the MR-(FR) 2 substrate by cathepsin L.…”