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

54 Apoptosis and Medicine

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

Immunization of normal BALB/c mice with Herp elicited anti-dsDNA Abs and caused glomerular IgG deposition [62]. However, urinary protein level did not increase and overt nephritis did not develop. The pathological changes in the kidneys in Herp-immunized

Nucleosomes, which are major autoantigens in SLE, are exposed at the apoptotic cell surface [66,67]. Anti-nucleosome Abs are present in SLE at a rate of more than 50% and they have been linked to lupus nephritis [68]. Nucleosomes and histones are present in glomerular deposits [69]. Nucleosomes bind to glomerular endothelial cells and serve as targets for antinucleosome Abs [70]. Therefore, a portion of anti-nucleosome Abs may be involved in lupus nephritis [71]. However, even oligonucleosomes are much less effective than Herp in inducing anti-nucleosome Abs as well as anti-dsDNA Abs [62]. Therefore, to reproduce overt lupus nephritis, BALB/c mice were immunized with Herp followed by immunization with oligonucleosomes. In this procedure, both anti-dsDNA Ab and anti-nucleosome Abproducing clones induced by Herp may be able to recognize oligonucleosomes easily. The production of anti-dsDNA Abs and glomerular IgG deposition were observed in all mice. In addition, overt nephritis with significant proteinuria occurred in one mouse (Figure 2). Although further investigations are in progress to define the mechanisms, it was speculated that (i) the Herp-induced anti-dsDNA Abs efficiently bound to nucleosomes and formed pathogenic immune complexes, and (ii) affinity maturation and epitope spreading of Herp-

**Figure 2.** Overt nephritis in a BALB/c mouse immunized with Herp followed by immunization with oligonucleosomes. Left: Periodic acid Schiff (PAS) staining. Right: Immunofluorescence staining with

cells may become anti-Herp/dsDNA cross-reactive Abs, i.e., initial anti-dsDNA Abs.

**4.3. Antigenicity of Herp for anti-dsDNA Ab production in mice** 

BALB/c mice went no further than silent lupus nephritis.

induced anti-dsDNA Abs occurred by nucleosomes.

fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG Ab.

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