**2. Anti-CENP-B and Scl-70 autoantibodies**

The clinical evaluation of anti-centromere protein B (CENP-B) and scleroderma (Scl)-70 autoantibodies in SIL patients was performed and reported [29].

**Figure 1** shows the titers of anti-nuclear antibody (ANA), anti-Sjögren's-syndrome-related antigen A (SS-A) antibody (Ab), anti-CENP-B and anti-Scl-70 (also known as anti-topoisomerase I) Abs in healthy volunteers (HV), SIL and SSc. All subjects were Japanese. 19 HV [median age = 46.0 years old (y.o.); mean ± standard deviation (SD) = 44.8 ± 8.6 y.o.; male:female (M:F) = 8:11], 20 SIL [median age = 73.5 y.o.; mean ± SD = 74.9 ± 5.4; male:female (M:F) = 19:1] and 25 SSc [median age = 65.0 y.o.; mean ± SD = 62.3 ± 12.1; male:female (M:F) = 3:22] were included in the study. All SIL were brickyard workers in Bizen City, Okayama prefecture, Japan, and were diagnosed according to the ILO 2000 guideline for pneumoconiosis. They were clinically followed in Kusaka Hospital or Hinase Uragami Iin according to Japanese law regarding the medical care of pneumoconiosis patients. The amount of free silica inhaled by these patients was estimated as high as 40 to 60% as determined from the work environment. These individuals did not show any symptoms of autoimmune diseases such as sclerotic skin, Raynaud's phenomenon, facial erythema or arthralgia. The SSc patients were diagnosed and monitored by the Department of Dermatology, Kawasaki Medical School Hospital, Kurashiki, Japan [29].

As shown in **Figure 1A**, investigation of the titers of ANA in HV, SIL and SSc revealed that a few SIL cases showed a higher titer of ANA, but there was no statistical significance. Not surprisingly, most of the SSc cases showed significantly higher ANA (compared to HV and SIL). Interestingly, titers of anti-Sjögren's-syndrome-related antigen A (SS-A) in SIL and SSc were higher than those of HV (**Figure 1B**). SS-A may be detected not only in Sjögren's syndrome, but also in other autoimmune diseases such as SSc and SLE. However, it may be interesting to note that SIL without any symptoms related to autoimmune diseases exhibited a higher titer for anti-SS-A Ab. Although clinical evaluation of anti-SS-A Ab in SIL has not been investigated, it is worth mentioning that SIL showed a pre-clinical status for autoimmune diseases as indicated by various epidemiological studies [9–17].

pneumoconiosis [5, 6], which is defined as lung inflammation and fibrosis with scarring in the form of nodules in the middle to upper lungs. Although various clinical types such as acute, progressive and chronic SIL are distinguished depending on the exposed dosage of silica particles and duration, patients clinically exhibit dyspnea, fatigue, cough, chest pain and other pulmonary symptoms. There are several typical pulmonary complications such as pulmonary tuberculosis, tuberculous pleurisy, pneumothorax, bronchiectasis and lung cancer [7, 8]. In addition to these lung complications, it is well known that the condition of SIL patients is often complicated with autoimmune diseases. The classical disease is known as Caplan's syndrome, complicated with rheumatoid arthritis (RA) [9]. The initial description reported by Caplan involved 51 cases among coal miners. Thereafter, many epidemiological reports revealed high odds ratios for the occurrence of RA in SIL [10, 11]. Furthermore, other autoimmune diseases such as systemic sclerosis (SSc) [12, 13], systemic lupus erythematosus (SLE) [14, 15] and anti-neutrophil cytoplasmic antibody (ANCA) positive vasculitis/nephritis [16, 17]

We have been studying the direct effects of silica particles on human lymphocytes, especially responder T (Tresp) and regulatory T (Treg) cells [18–20], as well as investigating autoantibodies found in SIL [21–28]. In this chapter, clinical evaluation, epitope search and functional assays of several autoantibodies found in SIL are described and mechanistic analyses of T cells

The clinical evaluation of anti-centromere protein B (CENP-B) and scleroderma (Scl)-70 auto-

**Figure 1** shows the titers of anti-nuclear antibody (ANA), anti-Sjögren's-syndrome-related antigen A (SS-A) antibody (Ab), anti-CENP-B and anti-Scl-70 (also known as anti-topoisomerase I) Abs in healthy volunteers (HV), SIL and SSc. All subjects were Japanese. 19 HV [median age = 46.0 years old (y.o.); mean ± standard deviation (SD) = 44.8 ± 8.6 y.o.; male:female (M:F) = 8:11], 20 SIL [median age = 73.5 y.o.; mean ± SD = 74.9 ± 5.4; male:female (M:F) = 19:1] and 25 SSc [median age = 65.0 y.o.; mean ± SD = 62.3 ± 12.1; male:female (M:F) = 3:22] were included in the study. All SIL were brickyard workers in Bizen City, Okayama prefecture, Japan, and were diagnosed according to the ILO 2000 guideline for pneumoconiosis. They were clinically followed in Kusaka Hospital or Hinase Uragami Iin according to Japanese law regarding the medical care of pneumoconiosis patients. The amount of free silica inhaled by these patients was estimated as high as 40 to 60% as determined from the work environment. These individuals did not show any symptoms of autoimmune diseases such as sclerotic skin, Raynaud's phenomenon, facial erythema or arthralgia. The SSc patients were diagnosed and monitored by the Department of Dermatology, Kawasaki Medical School Hospital,

As shown in **Figure 1A**, investigation of the titers of ANA in HV, SIL and SSc revealed that a few SIL cases showed a higher titer of ANA, but there was no statistical significance. Not surprisingly, most of the SSc cases showed significantly higher ANA (compared to HV and SIL). Interestingly,

have been reported in case reports and epidemiological investigations.

exposed to silica particles are conducted.

136 Autoantibodies and Cytokines

Kurashiki, Japan [29].

**2. Anti-CENP-B and Scl-70 autoantibodies**

antibodies in SIL patients was performed and reported [29].

**Figure 1.** Comparison of titers for anti-nuclear antibody (ANA), anti-SS-A antibody (Ab), anti-CENP-B Ab and anti-Scl-70 Ab among healthy volunteers (HV), silicosis cases (SIL) and patients with systemic sclerosis (SSc). Except for ANA, titers are shown as logarithmic values. Statistical significance was examined using the student T-test and p < 0.05 was defined as significant. All titers were measured using a multiplex ELISA kit for ANA.

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

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 autoimmune disease in SIL [29].
