**4. Other autoantibodies and silicosis associated with autoimmune diseases**

Some reports have identified anti-desmoglein auto-Ab in SIL [21, 22]. Thus, SIL showed various auto-Abs against ANA, Scl-70, CENP-B, SS-A, Fas and caspase-8. How are these various auto-Abs manifested in SIL without any autoimmune symptoms? As mentioned above, Tregs may be reduced in SIL, especially SIL with anti-Fas auto-Ab. Therefore, what about Tresps? If the imbalance defined by dominant Tresps and less Tregs is important for the onset of autoimmune diseases, what kinds of alterations were found in Tresps derived from SIL?

We found that there were many T cell activation markers in SIL, such as higher soluble interleukin (IL)-2 receptor [34], higher program death (PD)-1 expression in Tresps (T helper (Th) 4 cells) as well as Tregs [35], and an *in vitro* assay showed that Tresps expressed CD69 as the earliest activation marker of T cells when peripheral blood mononuclear cells (PBMCs) were cultured with silica particles [36]. In addition to this evidence of chronic activation of Tresps, there were many inhibitors of Fas-mediated apoptosis present in SIL serum, for example, soluble Fas (sFas) [37] and Fas-alternatively spliced variants (lacking the transmembrane domain, but maintaining the Fas-ligand binding domain) [38]. Additionally, PBMCs from SIL showed higher decoy receptor 3 (DcR3; which acts similar to sFas binding with trail-apoptosis induced at the extracellular area) expression compared to PBMCs from HV [39] (**Figure 3**). Taken together, Tresps in SIL are stimulated and survive longer by inhibition of Fas-mediated apoptosis. These Tresps can encounter various self-antigens and force B cells to produce auto-Abs [18–20].

**Figure 3.** In addition to various auto-Abs found in SIL, and earlier apoptosis of Tregs in SIL, Tresps in SIL revealed a chronically activated status with CD69 and PD-1 expression as well as higher serum soluble IL-2 receptor. Additionally, Tresps in SIL inhibited Fas-mediated apoptosis by excess soluble Fas and similar molecules such as DcR3. Thus, Tresps in SIL survive longer and encounter various autoantigens. Moreover, the imbalance between Tresps and Tregs may be

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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**).

**diseases**

140 Autoantibodies and Cytokines

**4. Other autoantibodies and silicosis associated with autoimmune** 

immune diseases, what kinds of alterations were found in Tresps derived from SIL?

can encounter various self-antigens and force B cells to produce auto-Abs [18–20].

Some reports have identified anti-desmoglein auto-Ab in SIL [21, 22]. Thus, SIL showed various auto-Abs against ANA, Scl-70, CENP-B, SS-A, Fas and caspase-8. How are these various auto-Abs manifested in SIL without any autoimmune symptoms? As mentioned above, Tregs may be reduced in SIL, especially SIL with anti-Fas auto-Ab. Therefore, what about Tresps? If the imbalance defined by dominant Tresps and less Tregs is important for the onset of auto-

We found that there were many T cell activation markers in SIL, such as higher soluble interleukin (IL)-2 receptor [34], higher program death (PD)-1 expression in Tresps (T helper (Th) 4 cells) as well as Tregs [35], and an *in vitro* assay showed that Tresps expressed CD69 as the earliest activation marker of T cells when peripheral blood mononuclear cells (PBMCs) were cultured with silica particles [36]. In addition to this evidence of chronic activation of Tresps, there were many inhibitors of Fas-mediated apoptosis present in SIL serum, for example, soluble Fas (sFas) [37] and Fas-alternatively spliced variants (lacking the transmembrane domain, but maintaining the Fas-ligand binding domain) [38]. Additionally, PBMCs from SIL showed higher decoy receptor 3 (DcR3; which acts similar to sFas binding with trail-apoptosis induced at the extracellular area) expression compared to PBMCs from HV [39] (**Figure 3**). Taken together, Tresps in SIL are stimulated and survive longer by inhibition of Fas-mediated apoptosis. These Tresps

**Figure 3.** In addition to various auto-Abs found in SIL, and earlier apoptosis of Tregs in SIL, Tresps in SIL revealed a chronically activated status with CD69 and PD-1 expression as well as higher serum soluble IL-2 receptor. Additionally, Tresps in SIL inhibited Fas-mediated apoptosis by excess soluble Fas and similar molecules such as DcR3. Thus, Tresps in SIL survive longer and encounter various autoantigens. Moreover, the imbalance between Tresps and Tregs may be enhanced.
