Exposure of HEp-2 Cells to Stress Conditions Influences Antinuclear Antibody Reactivity

Abstract

This study of stress-related antinuclear antibody (ANA) reactivity was undertaken with the objective of improving clinical ANA testing. ANA was determined by parallel enzyme-linked immunosorbent assays of crude nuclear protein antigen extracted from HEp-2 cells either grown under optimal conditions (providing nonstress ANA antigen) or exposed to stress (providing stress ANA antigen). The stress stimuli used were gamma radiation (causing DNA damage) and a hypertonic environment (causing apoptosis). Signs of stress-related ANA reactivity were seen among connective tissue disease (CTD) patients (including patients with systemic lupus erythematosus; mixed CTD; calcinosis, Reynaud's phenomenon, esophageal motility disorders, sclerodactyly, and telangiectasia; scleroderma; and Sjögren's syndrome): 11% showed stress-positive ANA (i.e., a significantly stronger ANA reactivity with the extract from stressed cells), whereas 21% showed a markedly weaker reaction with the stress antigen. In contrast, among ANA screening patient sera, with no diagnosis of CTD, the fraction showing stress-positive ANA was higher (7 to 8%, depending on the type of stress) than among those showing a lower reactivity with stress antigen (1.5 to 2.5%). Only one serum among 89 (1%) tested sera from healthy individuals showed a stress-related ANA reaction. This demonstration of stress-related ANA suggests a means to improve the performance of clinical ANA testing.

Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests

“…This two-stage approach comprises the following benefits: (a) highly sensitive screening of the most frequent and clinically relevant non-organ-specific AABs, (b) optimal combination with other assay techniques for the downstream differentiation of AAB reactivities based on the IIF pattern detected and the diagnosis suspected, (c) assessment of clinically relevant AABs without the need for further testing (for example, anti-centromere AABs), and (d) evaluation of AABs detectable only by IIF in case of unknown autoantigenic targets or non-available commercial assays [ 12 - 14 ]. Due to the key position of ANA screening in the serological diagnosis of systemic rheumatic diseases, consistent reproducibility and high quality of HEp-2 cell-based IIF assays are required [ 8 , 15 , 16 ]. However, the visual and therefore subjective evaluation of cell-based IIF assays complicates the standardized and reproducible evaluation of HEp-2 cell assays.…”

Interkit Reproducibility of the Indirect Immunofluorescence Assay on HEp-2 Cells Depends on the Immunofluorescence Reactivity Intensity and Pattern

“…The heterogeneity and lack of standardization in the preparation of kits by manufacturers contribute to the discrepancy of results obtained using different HEp-2-IFA kits. Previous studies provide an experimental technical basis to explain the inconsistency of results between different HEp-2 kits, pointing out that cell fixation and permeabilization protocols are capable of modifying the structure and composition of cell compartments, the size of nuclei and nucleoli, and the availability of epitopes for recognition by autoantibodies (40)(41)(42)(43)(44).…”

Ro60 and La ribonucleoproteins become self-aggregated by cell stress

“…By stress the transcription of heat shock proteins (HSP) is activated; and HSP play a role in degradation, refurbishing and reactivation of damaged proteins (25). The stress induces changes of the cytoskeleton which is of particular importance because the actin-based cytoskeleton is a sensitive monitor of extra cellular stimuli (26,27). Is well established that stress increases the availability of intracellular antigens on cell surface, in consequence the autoantibodies trigger better antigens produced under cellular stress (28,29), present results confirm this notion, and suggest that the Ro and La redistribution is associated to the cytoskeleton modification; another interesting observation suggest that Ro and La suffer conformational changes and aggregates form transitory complexes with the cytoskeleton proteins through HSP70.…”