Introduction

Inhalation of cigarette smoke causes respiratory inflammation even in healthy smokers, and long-term smoking causes various respiratory diseases. Of these diseases, chronic obstructive pulmonary disease (COPD) has had the most impact on public health worldwide [1]. In patients with COPD, forced expiratory volume in 1 s (FEV₁) is progressively reduced with the development of this disease. Imbalances of proteases/antiproteases and oxidants/antioxidants were thought to be major mechanisms underlying the development of COPD, but accumulating evidence has recently suggested that systemic inflammation due to the leakage of mediators from sites of local inflammation plays an important role in its pathogenesis [2].

In addition, autoimmunity is associated with the pathogenesis of pulmonary diseases such as COPD [3]. For instance, lymphoid follicles containing B cells accumulate in the airways of patients with COPD [4], and Nunez et al. reported that the anti-tissue antibodies such as mitochondrial, liver-kidney microsomal smooth muscle, and parietal gastric cell antibodies are associated with the degree of airflow limitation in COPD patients [5]. Therefore, autoantibody-induced airway damage may contribute to the pathogenesis of the disease. In particular, the involvement of autoimmune mechanisms is suggested by the persistence of airway inflammation after cessation of cigarette smoking [6] as well as by the pathogenesis of COPD in non-smoking populations [7].

The prevalence of antinuclear antibody (ANA) in the general population is 13.8% in the United States and 26% in Japan [8,9]. We previously reported spirometric values for a healthy Japanese population ≥40 years of age who participated in an annual health check [10], noting that the prevalence of microalbuminuria, a marker of vascular endothelial damage, was significantly greater in the group measuring positive for antibodies to several connective tissue disease autoantigens (ACTDA), as measured using an enzyme immunoassay (EIA) method, than in the ACTDA-negative group [11].

It has been reported that there is no relationship between cigarette smoking and ANA positivity [7,9]. In contrast, the involvement of cigarette smoking in the development of autoimmune diseases has been suggested [12]. If this is true, cigarette smoking may cause airway inflammation by dual pathways: direct impairment by the toxicity of smoke which triggers the inflammatory response, and indirect impairment by the production of autoantibodies which triggers the inflammation in the airway epithelial and/or endothelial cells. In COPD, pulmonary endothelial dysfunction is an important feature of its pathogenesis [13,14].

To date, the association of antinuclear antibodies with spirometric values in the general population has not been elucidated. In this study, we cross-sectionally and longitudinally investigated the relationship between ACTDA values and spirometric parameters in a general Japanese population.

Methods

Results

Relationships between ACTDA and spirometric measures

We investigated the relationship between log ACTDA value and spirometric measures (Figure 1). As shown in Figure 1, there were significant correlations with FEV₁% predicted, FEV₁/FVC and MMF% predicted in males (Figure 1C, Figure 1E, and Figure 1G) but not with FVC% predicted in males or with FVC% predicted, FEV₁% predicted, FEV₁/FVC and MMF% predicted in females (Figure 1A, Figure 1B, Figure 1D, Figure 1F, and Figure 1H). The relationships of log ACTDA to pulmonary functions in individuals with ACTDA ≥ 20 and ≥ 40 have been summarized in Tables 7 and 8, respectively. The correlation of ACTDA ≥ 20 with % predicted FEV₁ and % predicted MMF was significant in men but not in women (Table 7). However, the correlation of ACTDA ≥ 40 with spirometric parameters was not significant in men or women, probably because the number of individuals with ACTDA ≥ 40 was not large enough to demonstrate relationships between ACTDA and spirometric values (Table 8). Next, we evaluated whether these correlations were still significant after adjustment for Brinkman index using multiple linear regression analysis. This analysis revealed that log ACTDA value was significantly associated with % predicted FEV₁ and % predicted MMF, independently of Brinkman index, but not with % predicted FVC and FEV₁/FVC (Table 9). Multiple linear analyses were performed among individuals with ACTDA ≥ 20 and ≥ 40; however, probably because of insufficient numbers of these individuals, significant relationships between log ACTDA and pulmonary functions were not observed (data not shown).

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Figure 1. Correlations between logarithmically-transformed value of antibodies to several connective tissue disease autoantigens (ACTDA) and spirometric measures in all subjects in the study population.

Graphs show the relationships between annual decline in spirometric parameters (A and B, % predicted FVC; C and D, % predicted FEV₁; E and F, FEV₁/FVC; G and H, % predicted MMF) and serum ACTDA value relative to sex (male: A, C, E, and G; female: B, D, F and H). A significant relationship of log ACTDA value was observed with FEV₁, FEV₁/FVC, and MMF in male subjects (A, b = -2.51, P = 0.124, β = -0.04; B, b = -1.88, P = 0.134, β = -0.04; C, b = -4.71, P = 0.014, β = -0.06; D, b = -1.86, P = 0.168, β = -0.03; E, b = -2.52, P = 0.001, β = -0.07; F, b= -0.42, P = 0.457, β = -0.02; G, b = -11.49, P = 0.005, β = -0.07 and H, b= -3.63, P = 0.218, β = -0.03).

ACTDA values were not available for 10 subjects. n = 1498 and 1746 male and female subjects, respectively.

b, partial regression coefficient; β, standard partial regression coefficient; FVC, forced vital capacity; FEV₁, forced expiratory volume in 1 s; MMF, maximal midexpiratory flow.

https://doi.org/10.1371/journal.pone.0081678.g001

	Men (n = 191)				Women (n = 372)
	b	SE	P	β	b	SE	P	β
FVC% predicted	-15.51	9.3	0.097	-0.12	-0.53	4.43	0.905	0.01
FEV1% predicted	-23.73	10.15	0.021	-0.17	-0.49	4.84	0.919	-0.01
FEV1/FVC	-10.08	5.19	0.053	-0.14	-1.2	2.07	0.563	-0.03
MMF% predicted	-41.3	19.91	0.04	-0.15	-10.08	11.02	0.361	-0.05

Table 7. Relationships of logarithmically-transformed values of antibodies to several connective tissue disease autoantigens (ACTDA) to pulmonary functions in ACTDA-positive individuals: univariate linear regression analyses (ACTD ≥ 20).

Univariate linear regression analyses in ACTDA-positive individuals; explanatory variable, log ACTDA value; dependent variable, each spirometric parameter

Abbreviations: b, partial regression coefficient; β, standard partial regression coefficient; FVC, forced vital capacity; FEV₁, forced expiratory volume in 1 s; MMF, maximal midexpiratory flow

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	Men (n = 18)				Women (n = 59)
	b	SE	P	β	b	SE	P	β
FVC% predicted	9.03	35.3	0.801	0.06	11.2	16.13	0.491	0.09
FEV1% predicted	20.4	46.83	0.669	0.11	2.76	16.94	0.871	0.02
FEV1/FVC	11.9	24.36	0.632	0.12	-6.74	7.43	0.368	-0.1
MMF% predicted	-6.4	67.99	0.926	-0.02	-29	32.89	0.382	-0.1

Table 8. Relationships of logarithmically-transformed values of antibodies to several connective tissue disease autoantigens (ACTDA) to pulmonary functions in ACTDA-positive individuals: univariate linear regression analyses (ACTD ≥ 40).

Univariate linear regression analyses in ACTDA-positive individuals; explanatory variable, log ACTDA value; dependent variable, each spirometric parameter

Abbreviations: b, partial regression coefficient; β, standard partial regression coefficient; FVC, forced vital capacity; FEV₁, forced expiratory volume in 1 s; MMF, maximal midexpiratory flow

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Explanatory v. (dependent v.)	b	SE	P	β
BI (FVC% predicted)	-0.005	0.001	<0.0001	-0.14
Log ACTDA (FVC% predicted)	-1.8	1.028	0.08	-0.03
BI (FEV1% predicted)	-0.009	0.001	<0.0001	-0.22
Log ACTDA (FEV1% predicted)	-2.56	1.143	0.025	-0.04
BI (FEV1/FVC)	-0.004	0	<0.0001	-0.2
Log ACTDA (FEV1/FVC)	-0.413	0.523	0.43	-0.01
BI (MMF% predicted)	-0.019	0.002	<0.0001	-0.2
Log ACTDA (MMF% predicted)	-5.796	2.473	0.019	-0.04

Table 9. Multiple linear regression analyses for the relationships between Brinkman index, antibodies to several connective tissue disease autoantigens (ACTDA) value, and spirometric measures in this study population.

Multivariate linear regression analyses in all study subjects; explanatory variable (v.), BI and log ACTDA value; dependent v., each spirometric parameter

FVC% predicted, FEV₁% predicted and MMF% predicted were standardized values by age, sex, and height (Reference #10). FEV₁/FVC was adjusted for age and sex.

Abbreviations: b, partial regression coefficient; β, standard partial regression coefficient; BI, Brinkman index; FVC, forced vital capacity; FEV₁, forced expiratory volume in 1 s; MMF, maximal midexpiratory flow

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Discussion

In the present study, we demonstrated an inverse relationship between spirometric measures and ACTDA value in male subjects participating in an annual health check-up. The relationships between ACTDA value and FEV₁, and MMF remained significant after adjustment for Brinkman index. In male never-smokers, the prevalence of ACTDA positivity was significantly greater in subjects with more severe airway obstruction (% predicted FEV₁ < the median value) than in normal subjects and subjects with less severe airway obstruction (% predicted FEV₁ ≥ the median value), although it is possible that the difference in percentage of male never-smokers positive for ACTDA may be attributable to the difference in the mean age of each group.

Accumulating evidence has suggested the involvement of autoimmunity in the pathogenesis of COPD. In addition, some autoimmune diseases cause restrictive lung disorders, namely, interstitial pneumonias [23]. However, to date, the relationship between pulmonary function and autoimmunity has not been shown in a healthy population. This report is the first to demonstrate this significant relationship, which suggests the involvement of an autoimmune mechanism in the impairment of pulmonary function, especially in obstructive lung disorders. As shown in Table 9, standard partial regression coefficient values of log ACTDA were smaller than those of the Brinkman index, but significant in relation to FEV1 and MMF. In addition, the positivity of ACTDA was high in the male never-smokers with severe obstruction (Table 11). Therefore, in some subjects with airflow obstruction, autoimmunity may be involved in its pathogenesis.

The precise causes and diagnoses of the pulmonary dysfunction/restrictive lung disorders were not clear in the present study because this study was not hospital-based research and such information was not available. There may be various causes of restrictive pulmonary disorders in Japan, and the prevalence of interstitial pneumonias is low [24]. Some portion of this population may have contracted interstitial pneumonias due to autoimmune diseases. However, there were no significant relationship between log ACTDA and FVC% predicted in the univariate linear regression analysis (Figure 1, Tables 7 and 8) and multivariate linear regression analysis (Table 9). In addition, the prevalence of positive ACTDA in restrictive lung disorder was similar to that in normal subjects (Table 10). Thus, we could not confirm a significant relationship between ACTDA level and FVC, the spirometric parameter for restrictive lung disorder, as we could for the relationship between ACTDA and FEV₁, the spirometric parameter for obstructive lung disorder.

It is reasonable to assume that inhalation of cigarette smoke induces impairment of pulmonary function. In this study, we demonstrated a significant positive relationship between ACTDA value and Brinkman index in healthy male subjects, although the habit of smoking did not related to the level of ACTDA (Tables 1 and 4) or its positivity (data not shown). In addition, inverse associations between logarithmically transformed ACTDA value and % predicted FEV₁ and MMF were demonstrated independently of Brinkman index. These results suggest that cigarette smoking does not initiate ACTDA positivity but enhances serum ACTDA levels, and that this elevation of ACTDA plays a significant role in the impairment of pulmonary function in male smokers independent of the direct toxic effect of cigarette smoke on pulmonary cells. In females, these correlations were not observed. A lower rate of cigarette smoking and lower Brinkman index in women than men may be a part of the reason for this difference between the sexes.

A primary cause of obstructive disorders may be COPD or bronchial asthma. In Japan, the prevalence of COPD and bronchial asthma is estimated from 8% to 10% and from 1% to 3% in adults, respectively [25,26]. Bronchial asthma is a comorbidity in allergic granulomatous angiitis (AGA), in which elevation of anti-neutrophil cytoplasmic antibody is seen, although there are no clear links between bronchial asthma and other autoimmune diseases. In addition, the prevalence of AGA is very low [27]. However, the prevalence of positive ANA is higher among asthmatic patients than among healthy subjects, suggesting that the involvement of an autoimmune mechanism in some asthmatic patients [28]. COPD is recognized as a chronic inflammatory disease [29]. The pathogenesis of COPD has traditionally been attributed to the involvement of alveolar macrophages, neutrophils, and CD8 T cells [30]. Recently, the involvement of adaptive immunity has been suggested by several investigators [4,31]. In particular, the presence of ANA and organ-specific antibodies in COPD patients has also been shown by several investigators [5,7,32-34].

In contrast to the positive relationship between ACTDA value and Brinkman index in the present study, we demonstrated that the prevalence of ACTDA positivity was greater in male never-smokers with relatively severe airflow obstruction. Of 501 male never-smokers, 41 subjects had airflow obstruction (31, obstructive; 10 mixed), and 8 of those subjects were positive for ACTDA. The prevalence of ACTDA positivity was greater in male never-smokers with the mixed pulmonary disorder than in subjects with other spirometric patterns. Autoimmunity has been suggested to be involved in the progression of COPD after cessation of cigarette smoking [6]. However, to date, there is little evidence supporting the involvement of autoantibodies in non-smoking COPD patients [7]. In this study, the prevalence of positive ACTDA was greater in male never-smokers with more severe airflow obstruction. This suggests the involvement of autoimmunity, not only in the pathogenesis of smoking-related pulmonary dysfunction but also in that of obstructive pulmonary diseases in male non-smokers. Subjects with mixed disorder and subjects with more severe airflow obstruction were older than subjects in other groups. Higher prevalence of positive ACTDA in the subjects in these groups is merely due to the higher mean ages of these groups. However, subjects in the greater ACTDA group had lower % predicted FVC and FEV₁ in males aged 65 and older, while the mean ages did not differ among groups that were determined by the level of ACTDA (Table 3). Log ACTDA was significantly associated with % predicted FEV₁ and MMF, even if age and sex were included as covariates in the multiple linear regression analysis shown in Table 9 (data not shown), suggesting that ACTDA is a factor associated with FEV₁ and MMF that is independent of age. Thus, although we could not eliminate the possibility that age difference affected the positivity of ACTDA in Tables 10 and 11, the association between greater ACTDA positivity and respiratory dysfunction in male never-smokers is still thought to be possible. The progression of pulmonary impairment induced by autoimmunity in male never-smokers may be slow, and it may take very long time to become apparent as spirometric dysfunction. In female never-smokers, a significantly high prevalence of positive ACTDA was not observed in the subjects with pulmonary dysfunction. The precise reasons for this difference between males and females are unknown, but higher baseline levels of ACTDA in female subjects may hide the relationships between ACTDA levels and respiratory functions.

Severity of airflow obstruction is usually evaluated by GOLD classification [1], and when we compared the difference in ACTDA positivity using GOLD classification, such as GOLD1 (% predicted FEV₁ ≥ 80) vs. GOLD2–4 (% predicted FEV₁ < 80), this statistical significance was diminished (P = 0.06). However, there appears to be no clear scientific support for setting the boundary between GOLD1 (mild airflow limitation) and GOLD2 (moderate airflow limitation) at a % predicted FEV₁ of 80% because there is no prognostic difference between them [35]. In addition, GOLD classification does not adequately reflect the individual severity of COPD with respect to dyspnea, health-related quality of life, exercise capacity, and frequency of exacerbation [36]. Therefore, we did not find the GOLD classification to be the optimal way to classify the population in the present study.

As mentioned earlier, the fact that the present study was not hospital based presents several limitations, including the lack of information regarding patients’ (i) history of autoimmune disease; (ii) findings on chest radiography; (iii) current medications, including immunosuppressive agents such as corticosteroids; and (iv) socio-economic status, which has the potential to create sampling bias. Furthermore, we did not measure ANA levels by IIF using HEp-2 cells, which is used in clinical environments to confirm the presence of ANA. The MESACUP ANA ELISA test performed in this study uses a mixture of only 9 antigens recognized by autoantibodies in systemic rheumatic diseases. In contrast, antibodies to hundreds to thousands of antigens are detected in a standard immunofluorescence ANA screening test. Only a small subset of individuals positive for ACTDA by immunofluorescence have autoantibodies to some of the 9 antigens used in MESACUP ANA EIA. In the future, IIF measurement of ANA and disease-specific antibodies should be evaluated in subjects with airflow obstruction without a history of cigarette smoking.

Because this study was performed in a sample of the Japanese population, it is still unknown if this relationship between ACTDA and respiratory function would exist for other ethnicities. ANA levels are reported to be twice as high in Japan than in the USA. Genetic backgrounds including human leukocyte antigens; and lifestyles, such as diet and smoking habit, are different in each ethnicity. Therefore, further studies are needed to confirm this correlation between autoimmunity and respiratory functions for other ethnicities.

In the longitudinal analysis, although significant associations between decline in FVC and FEV1 and ACTDA were observed, these associations seem to be weak because statistical significance did not remain after adjustment for Brinkman Index. The number of subjects participating in the longitudinal study (n= 147) might not have been large enough to achieve statistical significance in this assay. In the future, studies using larger numbers of subjects may be needed to evaluate the relationships between longitudinal changes in spirometric values and autoimmunity.

In conclusion, we have demonstrated inverse relationships between ACTDA value and spirometric parameters in healthy male subjects participating in an annual health checkup, suggesting the involvement of an autoimmune mechanism in the development of pulmonary dysfunction.

Acknowledgments

We thank Taiko Aita, Emiko Nakamura, and Eiji Tsuchida for their excellent technical assistance. We also thank the following contributors: Michiko Nishiwaki (Saiseikan Yamagata City Hospital, Yamagata, Japan), Toshihiro Wada (Saiseikan Yamagata City Hospital, Yamagata, Japan), Jun-Ichi Machiya (Nihonkai General Hospital, Sakata, Japan), Noriyuki Hirama (NHO Yamagata National Hospital, Yamagata, Japan), Noriaki Takabatake (Tohoku Central Hospital, Yamagata, Japan), and Makoto Sata (National Cerebral and Cardiovascular Center, Osaka, Japan). We also thank Toshiro Tango (Center for Medical Statistics, Tokyo, Japan) for evaluating the statistical analyses.