A novel cytoplasmic compartment referred to as GW bodies was initially identified using human autoantibodies to a 182 kDa protein named GW182. GW bodies are small, generally spherical, cytoplasmic domains that vary in number and size in several mammalian cell types examined to date. Based on our earlier studies, GW bodies were proposed to be cytoplasmic sites for mRNA storage and/or degradation. In the present study, immunogold electron microscopy identified electron dense structures of 100-300 nm diameter devoid of a lipid bilayer membrane. These structures appeared to comprise clusters of electron dense strands of 8-10 nm in diameter. By costaining with CENP-F and PCNA, and employing a double-thymidine block to synchronize HeLa cells, GW bodies were observed to be small in early S phase and larger during late S and G2 phases of the cell cycle. The majority of GW bodies disassembled prior to mitosis and small GW bodies reassembled in early G1. The analysis of GW bodies in two experimental models of cell proliferation using reversal of 3T3/serum-starvation and concanavalin A stimulation of mouse splenocytes and T cells, revealed that proliferating cells contained larger, brighter, and more numerous GW bodies as well as up to a fivefold more total GW182 protein than quiescent cells. In vitro gene knockdown of GW182 led to the disappearance of GW bodies demonstrating that GW182 is a critical component of GW bodies. The incremental expression of the GW182 protein in cells induced to proliferate and the cyclic formation and breakdown of GW bodies during mitosis are intriguing in view of the notion that GW bodies are specialized centers involved in maintaining stability and/or controlling degradation of mRNA.

To gain insight into the mechanisms of autoantibody induction, sera from 40 patients with systemic lupus erythematosus (SLE) were tested by ELISAs for antibody binding to denatured individual histones, native histone-histone complexes, histone-DNA subnucleosome complexes, three forms of chromatin, and DNA. Whole chromatin was the most reactive substrate, with 88% of the patients positive. By chi-square analysis, only the presence of anti-(H2A-H2B), anti-[(H2A-H2B) -DNA], and antichromatin were correlated with kidney disease measured by proteinuria > 0.5 g/d. SLE patients could be divided into two groups based on their antibody-binding pattern to the above substrates. Antibodies from about half of the patients reacted with chromatin and the (H2A-H2B) -DNA subnucleosome complex but displayed very low or no reactivity with native DNA or the (H3-H4)2-DNA subnucleosome complex. An additional third of the patients had antibody reactivity to chromatin, as well as to both subnucleosome structures and DNA. Strikingly, all sera that bound to any of the components of chromatin also bound to whole chromatin, and adsorption with chromatin removed 85-100% of reactivity to (H2A-H2B)-DNA, (H3-H4)2-DNA, and native DNA. Individual sera often bound to several different epitopes on chromatin, with some epitopes requiring quaternary protein-DNA interactions. These results are consistent with chromatin being a potent immunogenic stimulus in SLE. Taken together with previous studies, we suggest that antibody activity to the (H2A-H2B) -DNA component signals the initial breakdown of immune tolerance whereas responses to (H3-H4)2-DNA and native DNA reflect subsequent global loss of tolerance to chromatin. (J. Clin. Invest. 1994. 94:184-192.)

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Autoantibodies reacting with chromatin and its components, histones and DNA, are characteristic of the human autoimmune diseases SLE and drug-induced lupus, but the mechanisms of their induction remain unknown. Serial serum samples collected over short intervals from lupus-prone MRL/MPlpr/lpr and BXSB mice were tested by ELISA on chromatin and its substructures to characterize the initial autoimmune response to these antigens. Direct binding studies demonstrated that the early autoantibodies recognized discontinuous epitopes on native chromatin and the (H2A-H2B)-DNA subnucleosome. As the immune response progressed, native DNA and other chromatin constituents generally became antigenic. Based on adsorption studies and IgG subclass restriction, antibodies to native DNA were more related to chromatin than to denatured DNA. The kinetics of autoantibody appearance and the Ig class distribution were similar to the kinetics and distribution seen in antibodies induced by immunization with an exogenous T-dependent antigen. These results are most consistent with the view that autoantibodies reacting with chromatin are generated by autoimmunization with chromatin, and antibodies to native DNA are a subset of the wide spectrum ofantichromatin autoantibodies. (J. Clin. Invest. 1993. 91:1687-1695

Serum deoxyribonuclease I (DNase I) activity in systemic lupus erythematosus (SLE) patients was shown to be lower than that of healthy laboratory personnel, rheumatoid arthritis, and scleroderma patients (P less than 0.001). The decrease in DNase I activity in SLE sera was not due to the effect of various autoantibodies or to heat labile DNase I inhibitor. A relationship between serum DNase I activity and active SLE was demonstrated. Patients with active lupus nephritis had the lowest levels of enzymatic activity.