Antibodies to Nucleosomes Chromatin

Antibodies to nucleosomes have had a "comeback" in the last few years. This has to do with both the clinical utility of these antibodies in the diagnosis of SLE and drug-induced lupus (DIL) and new evidence suggesting that nucleosomes may be major candidate autoimmunogens in lupus. Since it is generally accepted that anti-nucleosome antibodies cause the LE cell phenomenon, they were actually among the first autoantibodies discovered. Of note, autoantibodies against individual components of nucleosomes, i.e., DNA or histone, cannot induce LE cell formation. For decades, the LE cell test introduced in 1948 was one of the most common immunological tests performed in clinical laboratories to diagnose SLE. Different names for anti-nucleosome antibodies have caused some confusion. They have been referred to as anti-DNP or anti-sNP antibodies in older publications and as anti-nucleosome, anti-chromatin, and anti-(H2A-H2B)-DNA antibodies in more recent articles [31-33].

The nucleosome is the fundamental unit of chromatin. It consists of a core particle composed of an octamer with two copies each of histones H2A, H2B, H3, and H4, around which is wrapped helical DNA with approximately 146 base pairs (bp) [34, 35]. Chromatin, the native complex of histones and DNA found in the cell nucleus of eukaryotes, is comprised of approximately 40% DNA, 40% histones, and 20% non-histone proteins, RNA, and other macro-molecules. The periodic arrangement of histones along the DNA gives chroma-tin a "beads-on-a string" appearance in electron micrographs. The "beads" can be isolated by digesting the linker DNA between them with micrococcal nucle-ase, yielding nucleosomes. Thus, polynucleosomes and chromatin are identical [33].

Anti-nucleosome/chromatin antibodies are defined as antibodies that react with the portion of histones exposed in chromatin/nucleosome, the structure of DNA found in chromatin/nucleosome, or an epitope comprised of the native histone-DNA complex. Specifically excluded are antibodies that react with nonhistone proteins, with epitopes on histones buried in chromatin, and with histone-DNA structures, such as A, C, and Z forms not present in chromatin. Thus, some but not all DNA-reactive antibodies have anti-nucleosome reactivity [31-33].

The new strategy in lupus research is to define a broad anti-nucleosome antibody family, including nucleosome-specific antibodies (anti-nucleosome antibodies without anti-dsDNA and anti-histone reactivities), anti-nucleosome antibodies with anti-dsDNA reactivity (bona fide anti-dsDNA antibodies), and anti-

nucleosome antibodies with anti-histone reactivity (bona fide anti-histone antibodies) [31].

Anti-nucleosome antibodies are clinically important for several reasons. The nucleosome is emerging as one of the major nuclear autoantigen targets, and 70-80% of SLE patients are anti-nucleosome antibody positive [36-41]. Independent studies have shown that the contribution of anti-dsDNA and anti-histone antibodies to serum reactivity against nucleosomes in SLE patients is only 2530% at most [36, 37]. One-third of SLE sera studied have high anti-nucleosome activity and little if any anti-dsDNA or anti-histone reactivity [36, 37, 41]. The level of the anti-nucleosome antibody titer correlates with the level of disease activity [39, 42-46]. This finding was also observed in SLE patients negative for anti-dsDNA antibodies [43, 44]. Anti-nucleosome antibodies are also associated with lupus nephritis [36, 39, 42-45], which is not surprising considering the bulk of evidence suggesting that they contribute to the pathogenesis of lupus nephritis [47-50]. An increase in IgG3 anti-nucleosome titers was observed during SLE flare-ups, and this increase was found to be closely associated with active nephritis. IgG1 anti-nucleosome antibody titers tended to correlate inversely with SLE disease activity [45]. One group found an association between anti-nu-cleosome antibodies and lupus psychosis [42].

A very recent study showed that measurement of anti-nucleosome antibodies can help to predict the development of SLE in patients with primary antipho-spholipid syndrome (PAPS). The authors followed 18 PAPS patients (15 female, three male) for a mean 11 years to evaluate the potential for SLE development. When PAPS was diagnosed, nine patients were positive for anti-nucleosome antibodies, and six of them developed clinical manifestations of SLE. In contrast, none of the patients who were anti-nucleosome-negative developed SLE [51].

Almost all patients with procainamide-induced lupus, half of those with qui-nidine-induced lupus, and some with hydralazine-induced lupus were positive for anti-nucleosome antibodies [52]. Most procainamide patients without drug-induced lupus were negative for anti-nucleosome antibodies. A few other drugs infrequently cause drug-induced lupus, and these patients often demonstrate anti-nucleosome activity [53].

In some studies, a high percentage of patients with systemic sclerosis were positive for anti-nucleosome antibodies [32, 41]. This was surprising because scleroderma patients do not exhibit a typical ANA immunofluorescence pattern when tested using HEp-2 cells. Methodological reasons for anti-nucleosomes reacting with scleroderma sera include the use of DNA reconstituted with the denatured antigens H2A and H2B, use of whole chromatin containing residual to-poisomerase I protein as the antigen, and use of an inappropriate cutoff. When H1-stripped chromatin or nucleosome core particles are used as the antigen with a cutoff that properly distinguishes between positive and negative, virtually no patients with systemic sclerosis should test positive for anti-nucleosome antibodies [53-55]. In one study, 50% of 36 patients with autoimmune hepatitis type I were anti-chromatin positive, while 5-13% of patients with other liver diseases were positive [56].

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