**6. Postulated mechanism of anti-DNA Ab generation**

56 Apoptosis and Medicine

**Figure 3.** Herp can stimulate anti-dsDNA Ab-producing B cells in untreated patients with active SLE. The PBMCs were stimulated with Herp or dsDNA. Anti-dsDNA Ab-producing PBMCs were detected with ELISPOT. Three representative cases (A, Case M. S.; B, Case S. S.; C, Case R. S.) are shown. The lowest row is the positive control (P. C.: human serum with anti-dsDNA Abs, diluted 1:200) and

Immunization with Herp elicits production of not only anti-dsDNA antibodies but also antissDNA antibodies in BALB/c mice. Among several anti-Herp mAbs established in our laboratory, the HT4 anti-Herp mAb cross-reacts specifically with ssDNA [77]. The epitope of the HT4 mAb on Herp, EPAGSNR, was identified by screening a synthetic peptide library. The binding of HT4 mAb to the peptide was competitively inhibited by ssDNA. Immunization of the epitope peptide elicited anti-ssDNA Abs in BALB/c mice. Treatment with chlorpromazine, procainamide, and hydralazine induced Herp expression and apoptosis in HeLa cells. These findings suggest that (i) ER stress and apoptosis by drugs and

negative control (N. C.; second antibody only).

Autoimmunity is associated with both genetic predisposition and environmental factors [78]. The monozygotic disease concordance rate ranges from 24% to 57% (and not 100%) for SLE [79]. Most patients with SLE are non-familial sporadic cases. That is, environmental etiologies of SLE may be common. It is well known that environmental factors such as viral infection, UV exposure, chemicals, etc., can trigger clinical onset or flare of SLE [80,81]. However, little is known regarding how those factors elicit anti-DNA antibody production in vivo. These factors, i.e., cell stressors, affect the expression patterns of cellular proteins, resulting in ER stress in some cases.

What is a practical model of this hypothesis? Natural infection with viruses can cause ER stress on a large scale in vivo [80]. ER stress has been shown to increase when viral proteins are produced at high levels, e.g., in virion formation during the active lytic cycle of infection. Epstein–Barr virus (EBV) infection has been suggested to have a causative role in SLE [82,83]. The titers of anti-EBV Abs in SLE patients are higher than those of healthy controls [84]. Kang et al. reported that: (i) patients with SLE had an approximately 40-fold increase in EBV viral load compared with controls; (ii) the frequency of EBV-specific CD69+ CD8+ T cells producing IFN- was higher in patients with SLE than in controls, but the frequency of EBV-specific CD69+ CD4+ T cells producing IFN- was lower in patients with SLE than in controls; and (iii) the EBV viral loads were positively correlated with the frequency of EBVspecific CD69+ CD8+ T cells but inversely correlated with the frequency of EBV-specific CD69+ CD4+ T cells [85]. Larsen et al. reported that EBV-specific CD8+ T cell responses in patients with SLE are functionally impaired [86]. The defective control of latent EBV infection in patients with SLE may result in recurrent reactivation of EBV. In fact, aberrant expression of BZLF1, which is a hallmark of EBV lytic infection, has been detected in the PBMCs of SLE patients [87]. In primary EBV infection, EBV infects tonsillar B cells in which lytic replication occurs, and differentiation of latently EBV-infected B cells to plasma cells in lymphoid tissues is associated with induction of the EBV lytic cycle [88]. Herp is expressed in BZLF1-positive EBV-infected B cells (Figure 4).

[Methods] EBV-transformed B 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 stained with DAPI. The cells were then co-stained with HT4 mouse IgG2a antihuman Herp mAb and mouse IgG1 anti-BZLF1 mAb (Dako, Glostrup, Denmark) for 1 h at room temperature followed by co-staining with rhodamine-conjugated goat anti-mouse IgG2a Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and FITC-conjugated goat antimouse IgG1 Ab (Santa Cruz Biotechnology) for 1 h at room temperature.

ER stress, which is induced by the production of viral proteins, causes EBV lytic replication, resulting in the release of virions and intracellular molecules [89]. The Herp produced in cells entering the lytic phase of EBV infection can be recognized by the immune system in

Cell Death and Anti-DNA Antibodies 59

The results of the present study led to the following hypothesis in which cell stress triggers an anti-DNA response via Herp in normal individuals: ER stress by environmental factors Herp expression recognition by the immune system of minor epitope(s) mimicking ssDNA|or dsDNA anti-Herp/ssDNA or dsDNA cross-reactive Abs (initial anti-ssDNA or anti-dsDNA Abs) anti-DNA Ab – DNA complex, anti-DNA Ab – nucleosome complex ? tissue injury (Figure 5). After the initial production of anti-Herp/DNA Abs, the production of the Abs is stimulated whenever ER stress-induced apoptosis occurs, and the cause of ER stress is not restricted. Repeated cell stress during daily life may strengthen this pathway. Herp is a good candidate as a link between common environmental factors and

As dead cells are not only a source of intracellular antigens but also a source of proinflammatory molecules, it is likely that they play an important role in the generation of nephritogenic anti-dsDNA Abs. Herp was identified as a molecule directly involved in cell stress/death as the cause of anti-dsDNA Ab production. Further investigations are therefore

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needed to clarify the relationship between cell stress/death and the etiology of SLE.

*Department of Rheumatology, Hikarigaoka Spellman Hospital, Japan* 

*Department of Hematology & Rheumatology, Tohoku University Hospital, Japan* 

the etiology of SLE.

**7. Conclusions** 

**Author details** 

**8. References** 

Yasuhiko Hirabayashi

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**Figure 4.** Herp protein is expressed in BZLF1 positive EBV infected B cells.

lymphoid tissues. In addition, EBV-encoded latent membrane protein 2A (LMP2A) induces hypersensitivity to TLR stimulation, leading to activation of autoreactive B cells through the BCR/TLR pathway [90]. Immunization with the membrane fraction of EBV-transformed B cells elicited anti-dsDNA Abs as well as anti-Herp Abs and causes glomerular IgG deposition in BALB/c mice [62]. These observations support the hypothesis that EBV infection may be a trigger of SLE.

**Figure 5.** Hypothetical mechanism of anti-dsDNA Ab induction.

The results of the present study led to the following hypothesis in which cell stress triggers an anti-DNA response via Herp in normal individuals: ER stress by environmental factors Herp expression recognition by the immune system of minor epitope(s) mimicking ssDNA|or dsDNA anti-Herp/ssDNA or dsDNA cross-reactive Abs (initial anti-ssDNA or anti-dsDNA Abs) anti-DNA Ab – DNA complex, anti-DNA Ab – nucleosome complex ? tissue injury (Figure 5). After the initial production of anti-Herp/DNA Abs, the production of the Abs is stimulated whenever ER stress-induced apoptosis occurs, and the cause of ER stress is not restricted. Repeated cell stress during daily life may strengthen this pathway. Herp is a good candidate as a link between common environmental factors and the etiology of SLE.
