Detection by Flow Cytometry of Anti-DNA Autoantibodies and Circulating DNA Immune Complexes in Lupus Erythematosus

Nisen Abuaf,Chantal Desgruelles,Hidayeth Rostane,Mohamed Moumaris,Camille Frances,Emilie Bellec,Faïza Boussa-Khettab

doi:10.1155/2019/6047085

Abstract

A new method for the detection by flow cytometry of anti-double-stranded DNA antibodies and of circulating immune complexes (IC) containing endogenous DNA (IC-eDNA) is described. From each serum sample, two samples were taken, one was used to detect IC-eDNA. The other to detect anti-DNA antibodies was incubated with calf thymus DNA. ICs were isolated by polyethylene glycol precipitation or by cryoprecipitation, after which immunoglobulins were labeled with FITC-conjugated anti-human globulin. Serum samples from 63 systemic lupus erythematosus (SLE) patients, 32 incomplete lupus, and 87 control patients were tested. Detection of anti-dsDNA antibodies by flow cytometry had a diagnostic sensitivity and specificity almost comparable to routine tests, the fluorescent enzyme immunoassay EliA™-dsDNA test, and the ultrasensitive Crithidia luciliae indirect immunofluorescence test. In 21 (33%) out of 63 SLE serum samples, IC-eDNA was detected. In these samples, free anti-dsDNA antibodies were hardly detectable or undetectable by flow cytometry or by routine tests. When anti-DNA antibodies are neutralized by endogenous DNA and can no longer be detected by routine tests, the serologic diagnosis and the follow-up of relapses in patients with SLE is compromised. To overcome this obstacle, we propose an accessible solution: the detection of circulating IC-eDNA by flow cytometry.

Highlights

Antibodies to double-stranded DNA are the serological hallmark of systemic lupus erythematosus (SLE)
We developed a sensitive Flow cytometry (FCM)-based assay that evaluates the amount of circulating anti-dsDNA antibodies and of the circulating immune complexes (IC)-endogenous DNA (eDNA)
DNA was stained by ethidium bromide (EB) (Figure 1(a))

Summary

Introduction

Antibodies to double-stranded DNA (anti-dsDNA) are the serological hallmark of systemic lupus erythematosus (SLE). The increase in the titer of anti-dsDNA antibodies concomitantly with decreased levels of complement proteins C1q, C3, and C4 is frequently associated with acute exacerbations of the disease because anti-dsDNA antibodies can mediate tissue inflammation by the formation of immune complexes (ICs) with endogenous DNA (IC-eDNA). According to the Systemic Lupus International Collaborating Clinics classification criteria (SLICC), a biopsy confirmed nephritis compatible with SLE associated with anti-dsDNA antibodies is sufficient for SLE diagnosis [2]. Numerous studies demonstrated the ability of ICs to effectively activate Toll-like receptors (TLR) and induce interferon (IFN) production [3, 4]. Work in several murine models suggests an important role for the NLRP3 inflammasome in mediating lupus nephritis [7]

Methods

Results

Conclusion