Anti-DNA-IgM Favors the Detection of NET-Associated Extracellular DNA

Abstract

During inflammatory responses, neutrophils enter the sites of attack where they execute various defense mechanisms. They (I) phagocytose microorganisms, (II) degranulate to release cytokines, (III) recruit various immune cells by cell-type specific chemokines, (IV) secrete anti-microbials including lactoferrin, lysozyme, defensins and reactive oxygen species, and (V) release DNA as neutrophil extracellular traps (NETs). The latter originates from mitochondria as well as from decondensed nuclei. This is easily detected in cultured cells by staining of DNA with specific dyes. However, in tissues sections the very high fluorescence signals emitted from the condensed nuclear DNA hamper the detection of the widespread, extranuclear DNA of the NETs. In contrast, when we employ anti-DNA-IgM antibodies, they are unable to penetrate deep into the tightly packed DNA of the nucleus, and we observe a robust signal for the extended DNA patches of the NETs. To validate anti-DNA-IgM, we additionally stained the sections for the NET-markers histone H2B, myeloperoxidase, citrullinated histone H3, and neutrophil elastase. Altogether, we have described a fast one-step procedure for the detection of NETs in tissue sections, which provides new perspectives to characterize neutrophil-associated immune reactions in disease.

Neutrophil Extracellular Traps Drive Dacryolithiasis

“…In addition, we employed stainings for NE, citH3, and MPO together with DNA to differentiate between neutrophils and NETs within MPC-Ds. A recent publication by Wang et al reports the superiority of the anti-DNA-IgM antibody in detecting loose, decondensed DNA, such as that in the patches of NETs [25]; we used this novel method to detect extracellular DNA in dacryoliths. We found neutrophils on the inside of the tissue and NETs on the rim of the tissue, i.e., NE colocalizing with the DNA.…”

“…Neutrophils are well-known for forming NETs, whose primary function is to trap and kill various pathogens. To detect NETs, antibodies against granular proteins, such as NE and myeloperoxidase (MPO), as well as citrullinated, decondensed chromatin, using citrullinated histone H3 (citH3) are commonly used in combination with various DNA intercalating dyes, such as DAPI, Sytox Green, Hoechst 33342, and PI [24,25]. In the context of the immune system, NETs are "double edged swords", meaning they can act pro-or antiinflammatories [26], depending on the density of neutrophil infiltration [27].…”