**4. Anti-dsDNA Abs**

## **4.1. Brief historical aspects of anti-dsDNA Abs**

SLE is characterized by the production of a variety of autoantibodies. Especially, antidsDNA Abs are the most characteristic of SLE and contribute to the pathogenesis of lupus

nephritis. In general, anti-dsDNA Abs are specific for SLE and the anti-dsDNA Abs titer is closely correlated to the activity of lupus nephritis [35]. A proportion of anti-dsDNA Abs are directly involved in immune complex-mediated glomerulonephritis [36]. Thus, the trigger of anti-DNA response may be closely related to the pathogenesis of SLE. However, mammalian native dsDNA is not immunogenic, suggesting that DNA itself does not act as a triggering or driving antigen [37]. The origin of anti-DNA Abs is a long-standing enigma.

Cell Death and Anti-DNA Antibodies 53

well as lupus glomerular deposits [55-58]. The intravenous infusion of IgG isotype anti-DNA Abs expressing O-81 Id also caused glomerular IgG deposition in SCID mice [59]. The VH region of O-81 Ab contains many somatic mutations [60]. Similarly, the VH regions of O-81 Id-positive B cells in patients with SLE were shown to already contain somatic mutations [61]. These observations prompted us to explore the triggering Ags for human

**Figure 1.** The expression of Herp in peripheral blood mononuclear cells (PBMCs) or the cells in a cervical lymph node (LN) from a patient who developed SLE and had yet to receive treatment.

F(ab') 2 anti-mouse IgG Ab (KPL, Gaithersburg, MD) for 1 h at room temperature [63].

system in lymph nodes where Ag recognition occurs (Figure 1).

[Methods] The cells were fixed in 50% acetone/50% methanol for 20 min at –20°C and blocked with 5% normal goat serum and 3% BSA in PBS overnight at 4°C. The cells were then incubated with HT2 mouse monoclonal IgG1 anti-Herp Ab or mouse IgG1 as an isotype control for 1 h at room temperature followed by incubation with FITC-conjugated goat

We found that the O-81 Ab specifically cross-reacts with human homocysteine-induced endoplasmic reticulum protein (Herp) [62]. Anti-dsDNA Abs purified from the sera of SLE patients bound to Herp, and anti-Herp Abs purified from the sera of SLE bound to dsDNA [62]. The production of Herp is induced by endoplasmic reticulum (ER) stress. The PBLs from subjects in active SLE, especially at the time of onset or flare-up of the disease, tended to show Herp expression [62]. The expression of Herp was also observed in the lymph node of an untreated patient with active SLE, indicating that Herp can be exposed to the immune

nephritogenic anti-DNA Abs using the O-81 Ab.

Anti-dsDNA responses can be evoked by dsDNA with the aid of a carrier, such as the 27 amino acid nucleic acid-binding Fus1 peptide [38], polyoma BK virus large T Ag [39], or DNaseI-dsDNA complex [40] which have been shown to induce production of anti-dsDNA Abs in mice, suggesting a possible role of excess amounts of DNA – protein complex in disruption of tolerance to DNA. Nucleosomes have been suggested as possible Ags responsible for triggering of anti-dsDNA Abs [41,42]. Crude nucleosomes or crude histones [41] have been shown to induce production of anti-dsDNA Abs in mice. Mononucleosomereactive Th clones augment the production of IgG autoantibodies to dsDNA, histones, and histone – DNA complex. However, immunization of SNF1 mice with pure mononucleosomes did not elicit production of IgG anti-dsDNA Abs [43]. HMGB1 – nucleosome complexes derived from apoptotic cells, but not HMGB1-free nucleosomes, elicited IgG anti-dsDNA Abs in BALB/c mice although their titer was not high, suggesting that adjuvants such as HMGB1 are necessary to break tolerance to dsDNA in nonautoimmune mice [44].

Another possible mechanism is molecular mimicry. Some mouse or human monoclonal anti-DNA Abs have been shown to cross-react with non-nucleic acid self-Ags, such as extracellular matrix protein HP8 [45], heterogeneous nuclear ribonucleoprotein A2 [46], NR2 glutamate receptor [47], -actinin [48,49], ribosomal protein S1 [50], and phospholipids, including cardiolipin [51]. However, it is not yet known whether these molecules can elicit anti-DNA responses.

The peptide, DWEYSVWLSN, is recognized by the R4A mouse monoclonal anti-dsDNA Ab [52]. Immunization with this peptide elicited anti-dsDNA Ab production and caused deposition of IgG in glomeruli in normal mice [53]. These observations indicate that a nonnucleic acid Ag can elicit production of anti-DNA Abs and cause renal disorder in normal animals. However, no proteins containing this peptide sequence have been reported to date.

It should be noted that immunization with recombinant EBNA-1 protein elicited anti-EBNA-1 Abs that cross-react with dsDNA, suggesting molecular mimicry between the viral antigen and dsDNA [54]. However, nephritogenicity of the anti-EBNA-1/dsDNA Abs has not been reported.
