Abstract
IntroductionWhile protective plasma cells (PCs) are an important part of the individual’s immune defense, autoreactive plasma cells such as dsDNA-specific plasma cells contribute to the pathogenesis of autoimmune diseases like systemic lupus erythematosus (SLE). However, the research on dsDNA-specific plasma cells was restricted to the ELISpot technique, with its limitations, as no other attempt for identification of dsDNA-reactive plasma cells had been successful.MethodsWith improved fluorochrome labeling of dsDNA, removal of DNA aggregates, and enhanced blocking of unspecific binding, we were able to specifically detect dsDNA-reactive plasma cells by immunofluorescence microscopy.ResultsVia this novel technique we were able to distinguish short-lived (SLPCs) and long-lived (LLPCs) autoreactive plasma cells, discriminate dsDNA-specific plasma cells according to their immunoglobulin class (IgG, IgM, and IgA) and investigate autoreactive (dsDNA) and vaccine-induced ovalbumin (Ova) plasma cells in parallel.ConclusionsThe detection of autoreactive dsDNA-specific plasma cells via immunofluorescence microscopy allows specific studies on pathogenic and protective plasma cell subsets and their niches, detailed evaluation of therapeutic treatments and therefore offers new possibilities for basic and clinical research.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0811-2) contains supplementary material, which is available to authorized users.
Highlights
While protective plasma cells (PCs) are an important part of the individual’s immune defense, autoreactive plasma cells such as double-stranded deoxyribonucleic acid (dsDNA)-specific plasma cells contribute to the pathogenesis of autoimmune diseases like systemic lupus erythematosus (SLE)
Immunofluorescence staining of dsDNA-reactive plasma cells for histologic analysis Detecting dsDNA-specific PCs via immunofluorescence microscopy has not been successful so far due to demanding technical challenges of staining with labeled dsDNA that lead to no or unspecific staining. We could achieve this task by establishing a protocol which combined several blocking and staining conditions. dsDNA-reactive PCs were identified by Ig light chain kappa (IgL) staining and binding of fluorochrome-labeled dsDNA, while PCs reactive against other antigens were singularly positive for IgL (Fig. 1a and Additional file 1: Figure S1)
A strong signal from PCs producing dsDNA-binding antibodies was acquired in the SLE mouse model strain (NZB/W) while in the non-autoimmune mouse strain (C57BL/6) or mice with experimental autoimmune myasthenia gravis (EAMG)—a disease that is not associated with anti-dsDNA antibodies—no to few positive signals were found (Additional file 1: Figure S1)
Summary
While protective plasma cells (PCs) are an important part of the individual’s immune defense, autoreactive plasma cells such as dsDNA-specific plasma cells contribute to the pathogenesis of autoimmune diseases like systemic lupus erythematosus (SLE). While the first are mainly associated with flares the latter are Winter et al Arthritis Research & Therapy (2015) 17:293 which might be differently modified by therapeutic treatments [16]. All these factors influence the protective and pathogenic humoral memory, disease progression and therapeutic success. The method should enable a distinction of dsDNA LLPCs and SLPCs, investigation of dsDNA PCs of different immunoglobulin (Ig) classes and the evaluation of therapeutic treatments
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