**4.2. Cross-reactive antigen of the O-81 human nephritogenic anti-DNA mAb**

We prepared human monoclonal anti-DNA Ab, O-81, which binds strongly to singlestranded DNA (ssDNA) and moderately to dsDNA, and demonstrated that the O-81 idiotype (Id) is distributed among IgG anti-DNA Abs of circulating immune complexes as 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 nephritogenic anti-DNA Abs using the O-81 Ab.

52 Apoptosis and Medicine

autoimmune mice [44].

anti-DNA responses.

not been reported.

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.

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 non-

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

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

**4.2. Cross-reactive antigen of the O-81 human nephritogenic anti-DNA mAb** 

We prepared human monoclonal anti-DNA Ab, O-81, which binds strongly to singlestranded DNA (ssDNA) and moderately to dsDNA, and demonstrated that the O-81 idiotype (Id) is distributed among IgG anti-DNA Abs of circulating immune complexes as

**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.

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

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

Excessive ER stress is known to induce apoptosis [64,65]. Herp can be exposed on apoptotic blebs of ER stress-induced apoptotic cells [62]. Many apoptotic cells expressing Herp were observed in the peripheral blood mononuclear cells (PBMCs) of patients with active SLE, but not normal control subjects [62]. This observation is compatible with those reported previously [11]. These results suggest that Abs against Herp on ER stress-induced apoptotic cells may become anti-Herp/dsDNA cross-reactive Abs, i.e., initial anti-dsDNA Abs.

Cell Death and Anti-DNA Antibodies 55

[Methods] Five 6-week-old female BALB/c mice were immunized intraperitoneally with 100 g of Herp on days 0 and 10 and 50 g of Herp on day 20, followed by immunization with 10 g of oligonucleosomes on days 30, 40, and 50. Preparation of Herp and oligonucleosomes was described previously [62]. Fresh-frozen tissue sections 4 m thick were fixed in 100% acetone for 10 min at 4°C and blocked with 5% normal goat serum and 3% BSA in PBS overnight at 4°C. Sections were stained with FITC-conjugated goat F(ab')2

To examine whether Herp can be an antigen for anti-dsDNA Ab production in humans, ELISPOT was performed using PBMCs (representative cases are shown in Figure 3). The number of spots increased when the PBMCs were incubated with Herp, but not with dsDNA, in 4 of 6 untreated active SLE patients (Figure 3A); in 2 of these 4 positive cases, a few spots were observed even in wells without stimulation (Figure 3B). The remaining two cases showed no spots (Figure 3C). On the other hand, no spots were detected in the PBMCs from nine treated active SLE patients, eight inactive SLE patients, and five normal control subjects (data not shown). These results suggest that Herp can stimulate anti-dsDNA antibody-producing clones but this stimulation is cancelled by immunosuppressive therapy. [Methods] Approximately 1106 PBMCs in 20% fetal calf serum (FCS)-supplemented RPMI 1640 (20% FCS-RPMI 1640) were cultured for 5 days with or without 2 g/mL Herp, or 10 g/mL dsDNA. For preparation of dsDNA, calf thymus DNA (Invitrogen, Carlsbad, CA) was pretreated with S1 nuclease (Takara Bio, Otsu, Japan) to remove single-stranded DNA (ssDNA) according to the manufacturer's instructions. Multiscreen 96-well filtration plates (Millipore, Billerica, MA) were coated with 10 mg/mL protamine overnight at 4°C, and washed with PBS followed by coating with 10 g/mL dsDNA in PBS for 2 h at room temperature. Following blocking with 20% FCS-RPMI 1640, the cultured PBMCs in 20% FCS-RPMI were plated at 1×105 cells/well and cultured for 24 h. After washing the cells with PBS, goat alkaline phosphatase-conjugated anti-human IgG antibodies (diluted 1:10000; Sigma-Aldrich, St. Louis, MO) were added and the wells were incubated for 1 h at room temperature. Following a further wash, the spots were visualized using NBT-5-bromo-4-

The mechanism involved in the production of anti-ssDNA Abs has yet to be elucidated. As ssDNA can have multiple conformational epitopes and all Abs that bind to ssDNA are called anti-ssDNA Abs, these Abs are highly heterogeneous and display low disease specificity. However, the susceptibility of lupus-inducing drugs to anti-ssDNA Ab production is very high. In such cases, there may be a unique mechanism of anti-ssDNA Ab production, as the chemical structures and pharmacological actions of lupus-inducing drugs are known to be highly diverse [72,73]. As higher risk drugs include procainamide and hydralazine, which inhibit DNA methylation, hypomethylation may be one of the causes of

anti-ssDNA Ab production, but its precise mechanism remains unknown [74-76].

anti-mouse IgG Ab (KPL) for 1 h at room temperature.

chloro-3-indolyl phosphate substrate (Sigma-Aldrich).

**5. Anti-single-stranded DNA (ssDNA) Abs** 

**4.4. Antigenicity of Herp for anti-dsDNA Ab production in humans** 
