**3. Autoantibodies against apoptosis-related molecules**

Our previous reports indicated that autoantibodies against molecules related to apoptosis, Fas and caspase-8, were found in SIL [26–28]. These molecules may be expressed when cells in the body progressed to apoptosis in physiological as well as pathological situations.

Regarding anti-caspase-8 auto-Ab, although HV and cases comprised a different series from the aforementioned volunteers and cases, anti-caspase-8 auto-Ab was detected in 70% of HV, 62% of SIL, 90% of SSc and 60% of SLE cases, using four fragments of caspase-8 protein [26]. As a result, the positivity was not unique to autoimmune diseases and SIL. It was easily detected even in sera of HV. The report that revealed these positivities for anti-caspase-8 auto-Ab examined the epitope mapping. The epitopes were widely spread from the death effector domain to caspase regions and there was no specific epitope expressed in specific disease types such as SSc, SLE, SIL or HV [26].

The anti-Fas auto-Ab was also found in SIL cases [28]. This was detected as 23.1% in SIL, 53.3% in SLE and 46.7% in SSc, but not detected in HV. For the anti-Fas auto-Ab, epitope mapping was also performed and there was no special site, with epitopes being widely spread from

(dominant) and Tregs (less) will occur.

**Figure 2.** Anti-Fas auto-Ab was found in ca. 25% of SIL. The function of anti-Fas auto-Ab was examined using sister human myeloma cell lines, KMS-12PE and KMS-12BM. Only Fas-expressing KMS-12PE proceeded onto apoptosis when cultured with sera from SIL which revealed the highest titer for anti-Fas auto-Ab. Since anti-Fas auto-Ab seems to be functional, regulatory T cells (Tregs) in SIL with this auto-Ab may fall into apoptosis, given the higher expression of Fas in Tregs from SIL compared to responder T cells (Tresps) from SIL or Tregs from HV. As a result, an imbalance of Tresps

Autoantibodies in Silicosis Patients: Silica-Induced Dysregulation of Autoimmunity

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The evaluation of SSc showed that anti-CENP-B and anti-Scl-70 Abs were typical autoantibodies. Anti-CENP-B Ab is usually thought to be found in SSc cases with a type of localized skin lesion. On the other hand, SSc cases positive for anti-Scl-70 Ab are regarded as a generalized type with diffuse and extensive skin lesions [30–32]. Our results shown in **Figure 1C** (anti-CENP-B Ab) and **1D** (anti-Scl-70 Ab), for both Abs, demonstrated that there were clear breaks between positive and negative (close to levels of HV) cases in SSc. Regarding SIL, anti-CENP-B Ab was significantly higher than that in HV with the highest case whose titer was just as high as the positive case in SSc [29]. However, there was no case that showed higher

Thus, the clinical evaluation of anti-CENPN Ab in SIL was performed [29]. There was no correlation with other immunological or respiratory parameters in SIL such as titer of ANA, immunoglobulin (Ig) G, Ig A, Ig M, age, radiological classification of SIL (PR: profusion ratio), exposure years, percentage vital capacity (VC), forced expiratory volume 1.0 (SEC) (FEV1.0 (%)) or forced expiratory flow at 25% of vital capacity divided by body height (V25/H) except positive for anti-Scl-70 Ab titers, although anti-Scl-70 titers were similar to the those of HV. Factor analysis was performed using these immunological and respiratory clinical parameters [29]. As a result, anti-CENP-B Ab was found to contribute to the second and fourth factors. Factor 2 (17.7% contribution ratio) comprised the titer indices of anti-CENP-B and Scl-70 Abs, Ig G and age, all with positive values. This factor is understood as an immunological factor with aged patients showing a tendency for higher antibodies and Ig G. The fourth factor with a 13.2% contribution ratio was formed by the titer index of anti-CENP-B Ab with a negative value, the anti-Scl-70 autoantibody with a positive value, in addition to the Ig A level with a positive value. As found in the analyses of individual correlations, the titer index of anti-Scl-70 Ab and Ig A showed a positive correlation. This fourth factor indicated that even though the titer index of anti-Scl-70 autoantibody was located in the range of HV, among these titers, there is a correlation with Ig A and this tendency was the opposite of that observed for the titer index of anti-CENP-B auto-Ab. Thus, even with lower levels of titers, higher SIL cases with anti-CENP-B or anti-Scl-70 Ab differed as both Abs were divided in subtypes of SSc. Taken together, both Abs, especially anti-CENP-B (as well as anti-SS-A Ab), may indicate a pre-clinical status for forthcoming manifestations of

anti-Scl-70 Ab in this series of SIL cases [29].

138 Autoantibodies and Cytokines

autoimmune disease in SIL [29].

types such as SSc, SLE, SIL or HV [26].

**3. Autoantibodies against apoptosis-related molecules**

Our previous reports indicated that autoantibodies against molecules related to apoptosis, Fas and caspase-8, were found in SIL [26–28]. These molecules may be expressed when cells in the body progressed to apoptosis in physiological as well as pathological situations.

Regarding anti-caspase-8 auto-Ab, although HV and cases comprised a different series from the aforementioned volunteers and cases, anti-caspase-8 auto-Ab was detected in 70% of HV, 62% of SIL, 90% of SSc and 60% of SLE cases, using four fragments of caspase-8 protein [26]. As a result, the positivity was not unique to autoimmune diseases and SIL. It was easily detected even in sera of HV. The report that revealed these positivities for anti-caspase-8 auto-Ab examined the epitope mapping. The epitopes were widely spread from the death effector domain to caspase regions and there was no specific epitope expressed in specific disease

**Figure 2.** Anti-Fas auto-Ab was found in ca. 25% of SIL. The function of anti-Fas auto-Ab was examined using sister human myeloma cell lines, KMS-12PE and KMS-12BM. Only Fas-expressing KMS-12PE proceeded onto apoptosis when cultured with sera from SIL which revealed the highest titer for anti-Fas auto-Ab. Since anti-Fas auto-Ab seems to be functional, regulatory T cells (Tregs) in SIL with this auto-Ab may fall into apoptosis, given the higher expression of Fas in Tregs from SIL compared to responder T cells (Tresps) from SIL or Tregs from HV. As a result, an imbalance of Tresps (dominant) and Tregs (less) will occur.

The anti-Fas auto-Ab was also found in SIL cases [28]. This was detected as 23.1% in SIL, 53.3% in SLE and 46.7% in SSc, but not detected in HV. For the anti-Fas auto-Ab, epitope mapping was also performed and there was no special site, with epitopes being widely spread from the cysteine-rich domain (CRD) in extracellular sites to the death domain in intracellular sites. However, in contrast to anti-caspase-8 auto-Ab (caspase-8 is an intracellular molecule), anti-Fas-auto-Ab can bind to the Fas molecule which is present on the cell surface and, if this auto-Ab is functional, cells presenting Fas/death receptor may be induced toward apoptosis. Thus, we examined whether anti-Fas auto-Ab is functional, whereby it can cause cell death and growth inhibition in Fas-expressing cells [28]. For this purpose, two myeloma cell lines established in our laboratory, called KMS-12PE and KMS-12BM, were employed which were sister cell lines derived from the same Japanese myeloma patients [33]. KMS-12PE was derived from an earlier stage of patients and from pleural effusion, while KMS-12BM was derived from the terminal stage and from bone marrow. Interestingly, Fas expression was higher in KMS-12PE, but very scant in KMS-12BM [28, 33]. Thus, we incubated both cell lines with sera from SIL which showed the highest titer for anti-Fas auto-Ab. As a result, KMS-12PE progressed to apoptosis, but 12BM did not [28]. From these analyses, anti-Fas auto-Ab functions to induce apoptosis against Fasexpressing cells. From our previous study [34], it was found that Tregs in SIL expressed higher levels of Fas molecules compared to Tregs derived from HV. Taken together, if SIL patients possessed anti-Fas auto-Ab in their serum, Tregs may easily proceed to apoptosis and be reduced [34]. The imbalance of Tregs and Tresps (dominant Tresps and less Tregs) is a typical situation that induces the occurrence of autoimmune disorders. Thus, functional anti-Fas auto-Ab is a key molecule involved in dysregulation of autoimmunity (**Figure 2**).
