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Antiendothelial Cell Antibodies in Patients With COPD
CHEST
Original Research COPD
Antiendothelial Cell Antibodies in Patients With COPD Masato Karayama, MD; Naoki Inui, MD, PhD; Takafumi Suda, MD, PhD; Yutaro Nakamura, MD, PhD; Hirotoshi Nakamura, MD, PhD; and Kingo Chida, MD, PhD
Background: Circulating antiendothelial cell antibodies (AECA) bind to endothelial antigens and induce endothelial cell damage. These antibodies have been detected in patients with collagen vascular diseases and systemic vasculitis. Recently, autoimmune mechanisms and vascular involvement have attracted attention in COPD. This study aimed to investigate the expression of AECA in patients with COPD. Methods: A total of 116 patients with COPD, whose condition was established based on the Global Initiative for Chronic Obstructive Lung Disease criteria, were evaluated. Serum samples were examined for AECA by a cellular enzyme-linked immunosorbent assay using human umbilical vein endothelial cells. In addition, 157 subjects without any clinical or radiologic evidence of COPD or pulmonary disease served as a reference population. Results: The patients with COPD exhibited significantly higher serum AECA concentrations than subjects in the reference population. The expression of AECA was significantly elevated in the patients with COPD, even when compared with that in smokers among the reference population who had similar smoking habits to the patients with COPD but normal spirometry. Conclusions: These findings suggest that an autoimmune component associated with endothelial cell damage is possibly involved in COPD. CHEST 2010; 138(6):1303–1308 Abbreviations: AECA 5 antiendothelial cell antibodies; BSA 5 bovine serum albumin; ELISA 5 enzyme-linked immunosorbent assay; ER 5 enzyme-linked immunosorbent assay ratio; GOLD 5 Global Initiative for Chronic Obstructive Lung Disease; PBS 5 phosphate-buffered saline; VEGF 5 vascular endothelial growth factor
is characterized by chronic airflow limitaCOPD tion and inflammation with neutrophils, macro-
phages, and CD81 lymphocytes1,2 in the small airways and alveoli.3-5 Although the precise mechanisms remain unclear, oxidative stress, protease-antiprotease imbalance, and inflammatory responses to noxious particles and gas mainly caused by smoking are thought to play roles in the development of COPD.3-6 On the other Manuscript received April 1, 2010; revision accepted May 19, 2010. Affiliation: From the Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan. Funding/Support: This study was supported in part by a grant to the Diffuse Lung Diseases Research Group from the Japanese Ministry of Health, Labour and Welfare. Correspondence to: Naoki Inui, MD, PhD, 1-20-1 Handayama, Hamamatsu, Japan 431-3192; e-mail: [email protected] © 2010 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.10-0863
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hand, the fact that not all smokers develop clinically significant COPD3,7,8 suggests that additional determinants are involved in the disease onset. Circulating antiendothelial cell antibodies (AECA) recognize various antigenic determinants on human endothelial cells. Although their target antigen and precise pathogenic role remain unclear, they bind to endothelial cell membrane antigens and induce endothelial cell damage, which leads to vascular injury.9-11 Several studies, including our previous studies, have demonstrated the presence of AECA in patients with collagen vascular diseases, sarcoidosis, and systemic vasculitic diseases.12-16 Recently, autoimmune mechanisms have been recognized as being partly involved in the pathogenesis of COPD.5,17-20 Circulating autoantibodies have been detected in patients with COPD.21,22 In an animal model for the induction of emphysematous changes, endothelial cells23 are one of the target cells. Therefore, we hypothesized that AECA are involved in CHEST / 138 / 6 / DECEMBER, 2010
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COPD because of their possible pathogenic function. The present study aimed to evaluate the expression of AECA against human umbilical vein endothelial cells in patients with COPD.
systemic lupus erythematosus without lung disease was used. As described in previous studies, a sample was judged to be positive when the ER was greater than the mean 1 3 SDs of the reference population according to the Rosenbaum method.12-16 High-Resolution CT Scan Protocol
Materials and Methods Patients and Reference Population A total of 116 consecutive patients who met the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria for COPD were enrolled in this study. None of the patients had experienced a respiratory tract infection or exacerbation of COPD for at least 4 weeks prior to the study. Patients were excluded if they had asthma, collagen vascular disease, sarcoidosis, or other pulmonary diseases. Patients who were under systemic corticosteroid therapy or had pulmonary hypertension also were excluded. Serum samples were collected and stored at -30°C until analysis. In addition, 157 subjects without any clinical, radiologic, or serological evidence of COPD, asthma, collagen vascular diseases, or other pulmonary diseases served as a reference population. The study protocol complied with the Declaration of Helsinki and was approved by our institutional review board. Each patient provided informed consent. Data Collection Clinical data were obtained from medical records. A pulmonary function test was assessed at the time of serum sample collection. For all subjects, lung function (FEV1, FVC, and FEV1/FVC ratio) was measured with a spirometer according to American Thoracic Society recommendations.24
The high-resolution CT scans consisted of 1-mm collimation sections that were obtained at window settings appropriate for viewing the lung parenchyma (window level, -600 Hounsfield units; window width, 1,500 Hounsfield units) with an Aquilion scanner (Toshiba; Tokyo, Japan). The images were assessed using the visual emphysema scoring method as described by Goddard et al.25 In brief, scans were analyzed at three levels (upper [at the superior margin of the aortic arch], middle [at the level of the carina], and lower [at the level of the orifice of the inferior pulmonary vein]) and scored separately for each side of the lungs. The extent of emphysema was graded with a 5-point scale based on the percentage of hypovascular low attenuation areas as follows: 0, no emphysema; 1, ⱕ 25% of the lung was involved; 2, 25% to 50% of the lung was involved; 3, 50% to 75% of the lung was involved; and 4, . 75% of the lung was involved. Total scores were obtained by summing the scores of all six zones. Statistical Analysis The Wilcoxon test and analysis of variance were used for the statistical analyses. Correlations between different parameters were evaluated by Spearman rank correlation test. P , .05 indicated significant differences. All data are expressed as the median (interquartile range) or mean 6 SD. All analyses were performed with statistical software JMP, version 5.0.1J (SAS Institute Japan; Tokyo, Japan).
Results Enzyme-Linked Immunosorbent Assay for the Detection of AECA
Characteristics of Patients With COPD
The enzyme-linked immunosorbent assay (ELISA) was used to detect AECA in serum and was performed as described previously.12-16 In brief, human umbilical vein endothelial cells at passage 3 were seeded in a 96-well flat-bottomed microtiter plate (Nunc; Roskilde, Denmark) at a concentration of 2 3 104 cells/well and allowed to grow to confluence as a monolayer for 2 to 3 days. Cells were fixed with 0.1% glutaraldehyde (Sigma-Aldrich Corp; St Louis, MO) for 10 min at 4°C, and nonspecific binding sites were blocked with phosphate-buffered saline (PBS)-bovine serum albumin (BSA) (Sigma-Aldrich) for 1 h at 37°C. After five washes, 100 mL of serum diluted 1:1,000 in PBS-1% BSA was added to each well in triplicate for 1 h at 37°C. The wells were washed five times with PBS-1% BSA and incubated with horseradish peroxidase-conjugated rabbit F-(ab9)2 antihuman IgG (KPL Protein Research Products; Gaithersburg, MD) diluted 1:800 in PBS-1% BSA for 1 h at 37°C. After five more washes with PBS1% BSA, 3,39,5,59-tetramethylbenzidine (KPL Protein Research Products) was added as a substrate and incubated at room temperature. After 10 min, the enzyme reaction was stopped by the addition of 1-M H2SO4, and optical density was measured at 450 nm using an ELISA reader (BioTek; Winooski, VT). The results were expressed as an ELISA ratio (ER) calculated as (S 2 A)/(B 2 A), where S is the optical density of the sample tested, and A and B are the optical densities of the negative and positive controls, respectively. All assays included at least one positive and one negative control sample on each plate. As a positive control, a highly positive serum sample from a patient with
The characteristics of the 116 patients with COPD are shown in Table 1. All patients, except one, had a smoking history, with a median of 51 pack-years, and 34 (29%) patients were currently smoking. All of the patients had FEV1/FVC ratios of , 70%, and the median predicted FEV1 was 62.4%. The patients were well distributed into the four GOLD stages (stage I, 32 [28%]; stage II, 40 [34%]; stage III, 24 [21%]; stage IV, 20 [17%]). With regard to the treatments for COPD, 42 (35%) patients were treated with single-agent therapy, and 24 were treated with combinations of two or three drugs. Although anticholinergic therapy was widely used for all stages, the use of a long-acting b2-agonist and inhaled corticosteroids increased with advancing disease severity. Ninety-six percent of the patients with COPD showed low attenuation areas on high-resolution CT scan, and most had moderate to severe emphysematous changes. In the reference population, 75 subjects had never smoked (non-COPD never smokers), and 82 had smoking statuses and pack-year histories similar to those of the patients with COPD but who had normal spirometry (non-COPD smokers). As shown in
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Table 1—Characteristics of Patients With COPD COPD
Reference Population
Characteristic
All Cases
Stage Ia
Stage IIa
Stage IIIa
Stage IVa
All Cases
Never Smokers
Smokers
Number Male sex, % Mean age, y Range BMI, kg/m2 Range Smoking status, % Current Former Never Pack-years Range PFT FEV1/FVC ratio IQR FEV1 % predicted IQR
116 91 73 56-88 21.0 13.3-31.0
32 91 73 60-88 22.0c 17.9-29.0
40 93 72 56-85 21.9c 13.3-26.1
24 96 73 56-87 20.4c 16.6-25.2
20 85 74 56-86 17.1 14.0-31.0
157 86 73 50-90 21.1 11.9-29.4
75 77b 72 51-85 20.5 11.9-28.5
82 94 74 50-90 21.3 14.2-29.4
29 70 1 51d 0-150
34 66 0 60 8-125
35 63 2 53 0-100
21 79 0 46 15-150
20 80 0 50 5-125
15 37 48 25 0-105
0.55e 0.42-0.64 62.4e 41.2-82.2
0.64 0.61-0.67 91.0 84.1-97.7
0.60 0.52-0.64 63.6 60.0-73.0
0.40 0.39-0.48 40.9 36.0-46.5
0.35 0.32-0.43 27.1 23.7-35.3
0.82 0.77-0.87 94.0 82.1-112
0 0 100 0
0.83 0.77-0.88 95.1 83.8-111
28 72 0 40 5-105 0.82 0.75-0.87 89.8 80.8-117
Values are expressed as number or median, unless otherwise indicated. IQR 5 interquartile range; PFT 5 pulmonary functional test. Stages according to the Global Initiative for Chronic Obstructive Lung Disease criteria. bP , .001 compared with patients with COPD or smokers in the reference population. cP , .01 compared with patients with COPD patients in stage IV. dP , .0001 for patients with COPD compared with never smokers or all cases in the reference population. eP , .0001 for patients with COPD compared with never smokers, smokers, or all cases in the reference population. a
Table 1, the reference population had similar clinical features of being elderly, a predominance of men, and a lower BMI. The patients with COPD had a significantly more-frequent smoking status (P , .001) and a larger number of pack-years (P , .001) than the reference population. As expected, the patients with COPD showed a significantly lower FEV1 (P , .001), percent-predicted FEV1 (P , .001), and FEV1/FVC ratio (P , .001) than the reference population. Even when compared with the non-COPD smokers, the patients with COPD showed a significantly lower FEV1 (P , .001), percent-predicted FEV1 (P , .001), and FEV1/FVC ratio (P , .001). Expression of AECA in Serum There were no significant correlations of the serum AECA values with age (r 5 20.11; P 5 .166), sex (P 5 .58), and pack-year history (r 5 20.10; P 5 .28) in all subjects. In the present study, the mean serum ER for AECA in the reference population was 0.346 6 0.106, and the cutoff value was determined to be 0.664 by the Rosenbaum method.12 Using this threshold, 35 (31%) of the 116 patients with COPD were considered to be positive for AECA. There were no significant differences in the clinical features, smoking habits, pulmonary function, or emphysema score on high-resolution CT scan between AECA-positive patients and AECA-negative patients with COPD (Table 2). The median ER of AECA was 0.566 (0.490-0.696) in the patients with www.chestpubs.org
COPD, which was significantly higher than that in the reference population (P , .0001) (Fig 1). The serum ERs of AECA in the patients with COPD were Table 2—Characteristics of Antiendothelial Cell Antibodies-Positive and Antiendothelial Cell Antibodies-Negative Patients With COPD Characteristic Patients, No. Sex Male Female Mean age (range), y Smoking status Current smoker Former smoker Never smoker Pack-years (range) PFT FEV1/FVC ratio FEV1 % predicted GOLD stagea I II III IV Emphysema score on CT scanb
AECA-Positive Patients
AECA-Negative Patients P Value
35
81
ns
31 (89) 4 (11) 73 (56-87)
75 (93) 6 (7) 73 (56-88)
ns
11 (31) 23 (66) 1 (3) 50 (0-150)
23 (28) 58 (72) 0 (0) 55 (5-129)
ns
0.55 (0.47-0.67) 63.0 (52.9-87.3) 10 (28) 15 (43) 3 (9) 7 (20) 14 (6-16.5)
0.53 (0.40-0.64) 60.6 (39.0-80.7) 22 (27) 25 (32) 21 (25) 13 (16) 10 (4-16)
ns
ns ns ns ns
ns
Values are expressed as No. (%) or median (IQR), unless otherwise indicated. AECA 5 antiendothelial cell antibodies; GOLD 5 Global Initiative for Chronic Obstructive Lung Disease; ns 5 not significant. See Table 1 legend for expansion of other abbreviations. aBased on GOLD criteria. bThe visual emphysema score based on high-resolution CT scan. CHEST / 138 / 6 / DECEMBER, 2010
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compared with the non-COPD smokers, the ERs for AECA were significantly increased in the patients with COPD (P , .001) (Fig 1).
Discussion
Figure 1. Expression of serum AECA in patients with COPD and the reference population. Horizontal lines, boxes, error bars, and circles represent the median, the 25th and 75th percentiles, the 10th and 90th percentiles, and data apart from the 10th or 90th percentiles, respectively. AECA 5 antiendothelial cell antibodies; ER 5 enzyme-linked immunosorbent assay ratio. *P , .0001 for patients with COPD compared with never smokers, smokers, or all cases in the reference population.
not correlated with age, BMI, smoking history, pulmonary function, emphysema score on high-resolution CT scan, or antinuclear autoantibody levels. Neither the prevalence nor the ERs of AECA differed among the GOLD stages (Fig 2). Even when the comparisons were restricted to the patients with positive AECA expression, the ERs were not correlated with the clinical findings and pulmonary functions (data not shown). We then divided the reference population into two groups according to their smoking habits (Table 1). Interestingly, the 78 non-COPD smokers showed similar median ER values to those in the nonCOPD never smokers group (0.369 [0.290-0.427] and 0.325 [0.258-0.401], respectively) (Fig 1). Even when
Figure 2. Expression of serum AECA in patients with COPD according to the GOLD criteria. GOLD 5 Global Initiative for Chronic Obstructive Lung Disease. See Figure 1 legend for specifics of the box plot and expansion of other abbreviations.
The present study examined the expression of AECA in patients with COPD. We found that patients with COPD had a significantly higher prevalence and level of serum AECA than the reference population. Onethird of the patients with COPD had elevated AECA, whereas none of the subjects in the non-COPD reference population had these antibodies. There were no significant relationships between the expression of AECA and pulmonary function or patient characteristics. In the reference population, smokers and never smokers had similar low AECA expression levels. Interestingly, there has been accumulating evidence that COPD is regarded as having the properties of an autoimmune disease.5,17-20 The expression of autoantibodies has been reported in patients with COPD.21,22 Antielastin antibodies are markedly increased in patients with COPD. Increases in interferon-g and IL-10 secretion after stimulation with elastin peptides also have been demonstrated.21 In this previous study, the authors proposed that cigarette smoking initiated T- and B-cell-mediated immunity against elastin in susceptible individuals. Feghali-Bostwick et al22 reported that the prevalence of circulating antipulmonary airway epithelial cell IgG antibodies was elevated among patients with COPD and that these antibodies had the potential to mediate cytotoxicity against pulmonary epithelial cells in vitro. They further evaluated the antipulmonary artery endothelial cell antibodies using indirect immunofluorescence assays, but the study was very small, targeting only 12 patients with COPD. Recently, Leidinger et al19 established protein macroarrays containing 1,827 immunogenic clones and screened serum samples from patients with COPD. They detected 381 peptide clones that reacted with the COPD serum samples. Surprisingly, 17 of these clones reacted with . 60% of the COPD sera, whereas seven clones reacted with . 90% of the sera. The findings that not all smokers developed clinically significant COPD5,7,8 and some smokers develop COPD even after smoking cessation5,17 suggest that additional determinants are involved in onset of the disease. In this context, it is interesting that we found that AECA were found only in the patients with COPD, whereas AECA expression in the non-COPD smokers, who had similar smoking habits to the patients with COPD but normal spirometry, was not elevated. Although the identities of the antigens that
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induce autoantibodies in patients with COPD remain unknown,21,22 COPD has an autoimmune profile, and antibodies, including AECA, may be associated with its pathogenesis. Cigarette smoke contains approximately 4,700 xenobiotic compounds, some of which are harmful and potentially immunogenic in some cases.5,26 These compounds can precipitate in the lungs and induce adaptive immune responses, including autoantibody production.27 Agustí et al17 proposed the possibility that cigarette smoke itself damages airway cells and creates autoantigens that drive immune and inflammatory responses. Because there is a loss of lung microvessels and significant reductions in capillary length density, COPD has a vascular disease component.20,28 Vascular endothelial growth factor (VEGF) is critical for vascular maintenance and endothelial cell survival.29 The gene and protein expressions of VEGF and VEGF receptor are decreased in lung tissues from patients with COPD,29,30 supporting the idea that the breakdown of a VEGF-dependent maintenance program leads to the death of alveolar endothelial cells.20,30 In in vivo studies, VEGF receptor blockade in adult rats causes lung endothelial cell apoptosis and airspace enlargement.18,31 Interestingly, intraperitoneal injection of human endothelial cells leads to the production of antibodies directed against VEGF receptor and endothelial cells and induces alveolar cell apoptosis and pathologic features similar to COPD.18,20,23 Furthermore, antibodies against human endothelial cells in rats induce apoptosis of endothelial cells in vitro and alveolar airspace enlargement in passively immunized mice. Taken together, these findings indicate that injury and damage to endothelial cells contribute to the pathologic changes in patients with COPD.30 In the present study, we investigated the presence of AECA in serum samples and found that only the patients with COPD had elevated AECA levels. Although further studies are required to clarify the vascular involvement in COPD both pathologically and immunologically, AECA may be involved in COPD development through vascular or endothelial cell injury caused by autoantibodies. There are some limitations to the present study. First, we could not determine the vascular involvement pathologically because no specimens were taken from the patients and reference population. Second, although AECA bind to endothelial cell membrane antigens and induce endothelial cell damage,9-11 their target antigen remains to be investigated. Because the issue of whether AECA actually cause vascular damage or are merely the result of vascular damage remains unclear in humans, further studies, including characterization of the antigens recognized by AECA, will elucidate the precise roles of AECA in the pathophysiology of COPD. www.chestpubs.org
In conclusion, the present study demonstrated that patients with COPD had a significantly higher prevalence and levels of AECA than a reference population, suggesting that an autoimmune component associated with endothelial cell injury is involved in the pathogenesis of COPD. Investigations from the viewpoint of autoimmune mechanisms, including AECA, may provide important information toward a better understanding of COPD and a new therapeutic strategy for this disease. Acknowledgments Author contributions: Dr. Karayama: contributed to the study design; data analysis and interpretation; and critical review, revision, and final approval of the manuscript. Dr Inui: contributed to the study design; data analysis and interpretation; and the drafting, critical review, revision, and final approval of the manuscript. Dr Suda: contributed to the data collection, analysis, and interpretation; and to the drafting, critical review, revision, and final approval of the manuscript. Dr Y. Nakamura: contributed to the data collection, analysis, and interpretation; and to the drafting, critical review, revision, and final approval of the manuscript. Dr H. Nakamura: contributed to the statistical analysis; data analysis and interpretation; and critical review, revision, and final approval of the manuscript. Dr Chida: contributed the study design; data analysis and interpretation; and critical review, revision, final approval of the manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.
References 1. MacNee W. Pathogenesis of chronic obstructive pulmonary disease. Proc Am Thorac Soc. 2005;2(4):258-266. 2. Saetta M, Di Stefano A, Turato G, et al. CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1998;157(3 pt 1): 822-826. 3. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS; GOLD Scientific Committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J Respir Crit Care Med. 2001;163(5):1256-1276. 4. Rabe KF, Hurd S, Anzueto A, et al; Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med. 2007;176(6):532-555. 5. Cosio MG, Saetta M, Agusti A. Immunologic aspects of chronic obstructive pulmonary disease. N Engl J Med. 2009; 360(23):2445-2454. 6. Tuder RM, Yoshida T, Arap W, Pasqualini R, Petrache I. State of the art. Cellular and molecular mechanisms of alveolar destruction in emphysema: an evolutionary perspective. Proc Am Thorac Soc. 2006;3(6):503-510. 7. Fletcher C, Peto R. The natural history of chronic airflow obstruction. BMJ. 1977;1(6077):1645-1648. 8. Rennard SI, Vestbo J. COPD: the dangerous underestimate of 15%. Lancet. 2006;367(9518):1216-1219. CHEST / 138 / 6 / DECEMBER, 2010
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9. del Papa N, Meroni PL, Barcellini W, et al. Antibodies to endothelial cells in primary vasculitides mediate in vitro endothelial cytotoxicity in the presence of normal peripheral blood mononuclear cells. Clin Immunol Immunopathol. 1992; 63(3):267-274. 10. Savage CO, Pottinger BE, Gaskin G, Lockwood CM, Pusey CD, Pearson JD. Vascular damage in Wegener’s granulomatosis and microscopic polyarteritis: presence of anti-endothelial cell antibodies and their relation to anti-neutrophil cytoplasm antibodies. Clin Exp Immunol. 1991;85(1):14-19. 11. Damianovich M, Gilburd B, George J, et al. Pathogenic role of anti-endothelial cell antibodies in vasculitis. An idiotypic experimental model. J Immunol. 1996;156(12):4946-4951. 12. Rosenbaum J, Pottinger BE, Woo P, et al. Measurement and characterisation of circulating anti-endothelial cell IgG in connective tissue diseases. Clin Exp Immunol. 1988;72(3): 450-456. 13. Constans J, Dupuy R, Blann AD, et al. Anti-endothelial cell autoantibodies and soluble markers of endothelial cell dysfunction in systemic lupus erythematosus. J Rheumatol. 2003; 30(9):1963-1966. 14. Salojin KV, Le Tonquèze M, Saraux A, et al. Antiendothelial cell antibodies: useful markers of systemic sclerosis. Am J Med. 1997;102(2):178-185. 15. Inui N, Matsui T, Suda T, Chida K. Anti-endothelial cell antibodies in patients with sarcoidosis. Chest. 2008;133(4): 955-960. 16. Matsui T, Inui N, Suda T, Chida K. Anti-endothelial cell antibodies in patients with interstitial lung diseases. Respir Med. 2008;102(1):128-133. 17. Agustí A, MacNee W, Donaldson K, Cosio M. Hypothesis: does COPD have an autoimmune component? Thorax. 2003; 58(10):832-834. 18. Taraseviciene-Stewart L, Douglas IS, Nana-Sinkam PS, et al. Is alveolar destruction and emphysema in chronic obstructive pulmonary disease an immune disease? Proc Am Thorac Soc. 2006;3(8):687-690.
19. Leidinger P, Keller A, Heisel S, et al. Novel autoantigens immunogenic in COPD patients. Respir Res. 2009;10:20. 20. Voelkel N, Taraseviciene-Stewart L. Emphysema: an autoimmune vascular disease? Proc Am Thorac Soc. 2005;2(1):23-25. 21. Lee SH, Goswami S, Grudo A, et al. Antielastin autoimmunity in tobacco smoking-induced emphysema. Nat Med. 2007; 13(5):567-569. 22. Feghali-Bostwick CA, Gadgil AS, Otterbein LE, et al. Autoantibodies in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2008;177(2):156-163. 23. Taraseviciene-Stewart L, Scerbavicius R, Choe KH, et al. An animal model of autoimmune emphysema. Am J Respir Crit Care Med. 2005;171(7):734-742. 24. American Thoracic Society. Standardization of spirometry, 1994 update . Am J Respir Crit Care Med. 1995;152 (3): 1107-1136. 25. Goddard PR, Nicholson EM, Laszlo G, Watt I. Computed tomography in pulmonary emphysema. Clin Radiol. 1982; 33(4):379-387. 26. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, et al. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest. 2000;106(11):1311-1319. 27. Voelkel NF, Vandivier RW, Tuder RM. Vascular endothelial growth factor in the lung. Am J Physiol Lung Cell Mol Physiol. 2006;290(2):L209-L221. 28. Sopori M. Effects of cigarette smoke on the immune system. Nat Rev Immunol. 2002;2(5):372-377. 29. van der Strate BW, Postma DS, Brandsma CA, et al. Cigarette smoke-induced emphysema: A role for the B cell? Am J Respir Crit Care Med. 2006;173(7):751-758. 30. Voelkel NF, Douglas IS, Nicolls M. Angiogenesis in chronic lung disease. Chest. 2007;131(3):874-879. 31. Kasahara Y, Tuder RM, Cool CD, Lynch DA, Flores SC, Voelkel NF. Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am J Respir Crit Care Med. 2001;163(3 pt 1):737-744.
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Original Research COPD
Antiendothelial Cell Antibodies in Patients With COPD Masato Karayama, MD; Naoki Inui, MD, PhD; Takafumi Suda, MD, PhD; Yutaro Nakamura, MD, PhD; Hirotoshi Nakamura, MD, PhD; and Kingo Chida, MD, PhD
Background: Circulating antiendothelial cell antibodies (AECA) bind to endothelial antigens and induce endothelial cell damage. These antibodies have been detected in patients with collagen vascular diseases and systemic vasculitis. Recently, autoimmune mechanisms and vascular involvement have attracted attention in COPD. This study aimed to investigate the expression of AECA in patients with COPD. Methods: A total of 116 patients with COPD, whose condition was established based on the Global Initiative for Chronic Obstructive Lung Disease criteria, were evaluated. Serum samples were examined for AECA by a cellular enzyme-linked immunosorbent assay using human umbilical vein endothelial cells. In addition, 157 subjects without any clinical or radiologic evidence of COPD or pulmonary disease served as a reference population. Results: The patients with COPD exhibited significantly higher serum AECA concentrations than subjects in the reference population. The expression of AECA was significantly elevated in the patients with COPD, even when compared with that in smokers among the reference population who had similar smoking habits to the patients with COPD but normal spirometry. Conclusions: These findings suggest that an autoimmune component associated with endothelial cell damage is possibly involved in COPD. CHEST 2010; 138(6):1303–1308 Abbreviations: AECA 5 antiendothelial cell antibodies; BSA 5 bovine serum albumin; ELISA 5 enzyme-linked immunosorbent assay; ER 5 enzyme-linked immunosorbent assay ratio; GOLD 5 Global Initiative for Chronic Obstructive Lung Disease; PBS 5 phosphate-buffered saline; VEGF 5 vascular endothelial growth factor
is characterized by chronic airflow limitaCOPD tion and inflammation with neutrophils, macro-
phages, and CD81 lymphocytes1,2 in the small airways and alveoli.3-5 Although the precise mechanisms remain unclear, oxidative stress, protease-antiprotease imbalance, and inflammatory responses to noxious particles and gas mainly caused by smoking are thought to play roles in the development of COPD.3-6 On the other Manuscript received April 1, 2010; revision accepted May 19, 2010. Affiliation: From the Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan. Funding/Support: This study was supported in part by a grant to the Diffuse Lung Diseases Research Group from the Japanese Ministry of Health, Labour and Welfare. Correspondence to: Naoki Inui, MD, PhD, 1-20-1 Handayama, Hamamatsu, Japan 431-3192; e-mail: [email protected] © 2010 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.10-0863
www.chestpubs.org
hand, the fact that not all smokers develop clinically significant COPD3,7,8 suggests that additional determinants are involved in the disease onset. Circulating antiendothelial cell antibodies (AECA) recognize various antigenic determinants on human endothelial cells. Although their target antigen and precise pathogenic role remain unclear, they bind to endothelial cell membrane antigens and induce endothelial cell damage, which leads to vascular injury.9-11 Several studies, including our previous studies, have demonstrated the presence of AECA in patients with collagen vascular diseases, sarcoidosis, and systemic vasculitic diseases.12-16 Recently, autoimmune mechanisms have been recognized as being partly involved in the pathogenesis of COPD.5,17-20 Circulating autoantibodies have been detected in patients with COPD.21,22 In an animal model for the induction of emphysematous changes, endothelial cells23 are one of the target cells. Therefore, we hypothesized that AECA are involved in CHEST / 138 / 6 / DECEMBER, 2010
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COPD because of their possible pathogenic function. The present study aimed to evaluate the expression of AECA against human umbilical vein endothelial cells in patients with COPD.
systemic lupus erythematosus without lung disease was used. As described in previous studies, a sample was judged to be positive when the ER was greater than the mean 1 3 SDs of the reference population according to the Rosenbaum method.12-16 High-Resolution CT Scan Protocol
Materials and Methods Patients and Reference Population A total of 116 consecutive patients who met the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria for COPD were enrolled in this study. None of the patients had experienced a respiratory tract infection or exacerbation of COPD for at least 4 weeks prior to the study. Patients were excluded if they had asthma, collagen vascular disease, sarcoidosis, or other pulmonary diseases. Patients who were under systemic corticosteroid therapy or had pulmonary hypertension also were excluded. Serum samples were collected and stored at -30°C until analysis. In addition, 157 subjects without any clinical, radiologic, or serological evidence of COPD, asthma, collagen vascular diseases, or other pulmonary diseases served as a reference population. The study protocol complied with the Declaration of Helsinki and was approved by our institutional review board. Each patient provided informed consent. Data Collection Clinical data were obtained from medical records. A pulmonary function test was assessed at the time of serum sample collection. For all subjects, lung function (FEV1, FVC, and FEV1/FVC ratio) was measured with a spirometer according to American Thoracic Society recommendations.24
The high-resolution CT scans consisted of 1-mm collimation sections that were obtained at window settings appropriate for viewing the lung parenchyma (window level, -600 Hounsfield units; window width, 1,500 Hounsfield units) with an Aquilion scanner (Toshiba; Tokyo, Japan). The images were assessed using the visual emphysema scoring method as described by Goddard et al.25 In brief, scans were analyzed at three levels (upper [at the superior margin of the aortic arch], middle [at the level of the carina], and lower [at the level of the orifice of the inferior pulmonary vein]) and scored separately for each side of the lungs. The extent of emphysema was graded with a 5-point scale based on the percentage of hypovascular low attenuation areas as follows: 0, no emphysema; 1, ⱕ 25% of the lung was involved; 2, 25% to 50% of the lung was involved; 3, 50% to 75% of the lung was involved; and 4, . 75% of the lung was involved. Total scores were obtained by summing the scores of all six zones. Statistical Analysis The Wilcoxon test and analysis of variance were used for the statistical analyses. Correlations between different parameters were evaluated by Spearman rank correlation test. P , .05 indicated significant differences. All data are expressed as the median (interquartile range) or mean 6 SD. All analyses were performed with statistical software JMP, version 5.0.1J (SAS Institute Japan; Tokyo, Japan).
Results Enzyme-Linked Immunosorbent Assay for the Detection of AECA
Characteristics of Patients With COPD
The enzyme-linked immunosorbent assay (ELISA) was used to detect AECA in serum and was performed as described previously.12-16 In brief, human umbilical vein endothelial cells at passage 3 were seeded in a 96-well flat-bottomed microtiter plate (Nunc; Roskilde, Denmark) at a concentration of 2 3 104 cells/well and allowed to grow to confluence as a monolayer for 2 to 3 days. Cells were fixed with 0.1% glutaraldehyde (Sigma-Aldrich Corp; St Louis, MO) for 10 min at 4°C, and nonspecific binding sites were blocked with phosphate-buffered saline (PBS)-bovine serum albumin (BSA) (Sigma-Aldrich) for 1 h at 37°C. After five washes, 100 mL of serum diluted 1:1,000 in PBS-1% BSA was added to each well in triplicate for 1 h at 37°C. The wells were washed five times with PBS-1% BSA and incubated with horseradish peroxidase-conjugated rabbit F-(ab9)2 antihuman IgG (KPL Protein Research Products; Gaithersburg, MD) diluted 1:800 in PBS-1% BSA for 1 h at 37°C. After five more washes with PBS1% BSA, 3,39,5,59-tetramethylbenzidine (KPL Protein Research Products) was added as a substrate and incubated at room temperature. After 10 min, the enzyme reaction was stopped by the addition of 1-M H2SO4, and optical density was measured at 450 nm using an ELISA reader (BioTek; Winooski, VT). The results were expressed as an ELISA ratio (ER) calculated as (S 2 A)/(B 2 A), where S is the optical density of the sample tested, and A and B are the optical densities of the negative and positive controls, respectively. All assays included at least one positive and one negative control sample on each plate. As a positive control, a highly positive serum sample from a patient with
The characteristics of the 116 patients with COPD are shown in Table 1. All patients, except one, had a smoking history, with a median of 51 pack-years, and 34 (29%) patients were currently smoking. All of the patients had FEV1/FVC ratios of , 70%, and the median predicted FEV1 was 62.4%. The patients were well distributed into the four GOLD stages (stage I, 32 [28%]; stage II, 40 [34%]; stage III, 24 [21%]; stage IV, 20 [17%]). With regard to the treatments for COPD, 42 (35%) patients were treated with single-agent therapy, and 24 were treated with combinations of two or three drugs. Although anticholinergic therapy was widely used for all stages, the use of a long-acting b2-agonist and inhaled corticosteroids increased with advancing disease severity. Ninety-six percent of the patients with COPD showed low attenuation areas on high-resolution CT scan, and most had moderate to severe emphysematous changes. In the reference population, 75 subjects had never smoked (non-COPD never smokers), and 82 had smoking statuses and pack-year histories similar to those of the patients with COPD but who had normal spirometry (non-COPD smokers). As shown in
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Table 1—Characteristics of Patients With COPD COPD
Reference Population
Characteristic
All Cases
Stage Ia
Stage IIa
Stage IIIa
Stage IVa
All Cases
Never Smokers
Smokers
Number Male sex, % Mean age, y Range BMI, kg/m2 Range Smoking status, % Current Former Never Pack-years Range PFT FEV1/FVC ratio IQR FEV1 % predicted IQR
116 91 73 56-88 21.0 13.3-31.0
32 91 73 60-88 22.0c 17.9-29.0
40 93 72 56-85 21.9c 13.3-26.1
24 96 73 56-87 20.4c 16.6-25.2
20 85 74 56-86 17.1 14.0-31.0
157 86 73 50-90 21.1 11.9-29.4
75 77b 72 51-85 20.5 11.9-28.5
82 94 74 50-90 21.3 14.2-29.4
29 70 1 51d 0-150
34 66 0 60 8-125
35 63 2 53 0-100
21 79 0 46 15-150
20 80 0 50 5-125
15 37 48 25 0-105
0.55e 0.42-0.64 62.4e 41.2-82.2
0.64 0.61-0.67 91.0 84.1-97.7
0.60 0.52-0.64 63.6 60.0-73.0
0.40 0.39-0.48 40.9 36.0-46.5
0.35 0.32-0.43 27.1 23.7-35.3
0.82 0.77-0.87 94.0 82.1-112
0 0 100 0
0.83 0.77-0.88 95.1 83.8-111
28 72 0 40 5-105 0.82 0.75-0.87 89.8 80.8-117
Values are expressed as number or median, unless otherwise indicated. IQR 5 interquartile range; PFT 5 pulmonary functional test. Stages according to the Global Initiative for Chronic Obstructive Lung Disease criteria. bP , .001 compared with patients with COPD or smokers in the reference population. cP , .01 compared with patients with COPD patients in stage IV. dP , .0001 for patients with COPD compared with never smokers or all cases in the reference population. eP , .0001 for patients with COPD compared with never smokers, smokers, or all cases in the reference population. a
Table 1, the reference population had similar clinical features of being elderly, a predominance of men, and a lower BMI. The patients with COPD had a significantly more-frequent smoking status (P , .001) and a larger number of pack-years (P , .001) than the reference population. As expected, the patients with COPD showed a significantly lower FEV1 (P , .001), percent-predicted FEV1 (P , .001), and FEV1/FVC ratio (P , .001) than the reference population. Even when compared with the non-COPD smokers, the patients with COPD showed a significantly lower FEV1 (P , .001), percent-predicted FEV1 (P , .001), and FEV1/FVC ratio (P , .001). Expression of AECA in Serum There were no significant correlations of the serum AECA values with age (r 5 20.11; P 5 .166), sex (P 5 .58), and pack-year history (r 5 20.10; P 5 .28) in all subjects. In the present study, the mean serum ER for AECA in the reference population was 0.346 6 0.106, and the cutoff value was determined to be 0.664 by the Rosenbaum method.12 Using this threshold, 35 (31%) of the 116 patients with COPD were considered to be positive for AECA. There were no significant differences in the clinical features, smoking habits, pulmonary function, or emphysema score on high-resolution CT scan between AECA-positive patients and AECA-negative patients with COPD (Table 2). The median ER of AECA was 0.566 (0.490-0.696) in the patients with www.chestpubs.org
COPD, which was significantly higher than that in the reference population (P , .0001) (Fig 1). The serum ERs of AECA in the patients with COPD were Table 2—Characteristics of Antiendothelial Cell Antibodies-Positive and Antiendothelial Cell Antibodies-Negative Patients With COPD Characteristic Patients, No. Sex Male Female Mean age (range), y Smoking status Current smoker Former smoker Never smoker Pack-years (range) PFT FEV1/FVC ratio FEV1 % predicted GOLD stagea I II III IV Emphysema score on CT scanb
AECA-Positive Patients
AECA-Negative Patients P Value
35
81
ns
31 (89) 4 (11) 73 (56-87)
75 (93) 6 (7) 73 (56-88)
ns
11 (31) 23 (66) 1 (3) 50 (0-150)
23 (28) 58 (72) 0 (0) 55 (5-129)
ns
0.55 (0.47-0.67) 63.0 (52.9-87.3) 10 (28) 15 (43) 3 (9) 7 (20) 14 (6-16.5)
0.53 (0.40-0.64) 60.6 (39.0-80.7) 22 (27) 25 (32) 21 (25) 13 (16) 10 (4-16)
ns
ns ns ns ns
ns
Values are expressed as No. (%) or median (IQR), unless otherwise indicated. AECA 5 antiendothelial cell antibodies; GOLD 5 Global Initiative for Chronic Obstructive Lung Disease; ns 5 not significant. See Table 1 legend for expansion of other abbreviations. aBased on GOLD criteria. bThe visual emphysema score based on high-resolution CT scan. CHEST / 138 / 6 / DECEMBER, 2010
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compared with the non-COPD smokers, the ERs for AECA were significantly increased in the patients with COPD (P , .001) (Fig 1).
Discussion
Figure 1. Expression of serum AECA in patients with COPD and the reference population. Horizontal lines, boxes, error bars, and circles represent the median, the 25th and 75th percentiles, the 10th and 90th percentiles, and data apart from the 10th or 90th percentiles, respectively. AECA 5 antiendothelial cell antibodies; ER 5 enzyme-linked immunosorbent assay ratio. *P , .0001 for patients with COPD compared with never smokers, smokers, or all cases in the reference population.
not correlated with age, BMI, smoking history, pulmonary function, emphysema score on high-resolution CT scan, or antinuclear autoantibody levels. Neither the prevalence nor the ERs of AECA differed among the GOLD stages (Fig 2). Even when the comparisons were restricted to the patients with positive AECA expression, the ERs were not correlated with the clinical findings and pulmonary functions (data not shown). We then divided the reference population into two groups according to their smoking habits (Table 1). Interestingly, the 78 non-COPD smokers showed similar median ER values to those in the nonCOPD never smokers group (0.369 [0.290-0.427] and 0.325 [0.258-0.401], respectively) (Fig 1). Even when
Figure 2. Expression of serum AECA in patients with COPD according to the GOLD criteria. GOLD 5 Global Initiative for Chronic Obstructive Lung Disease. See Figure 1 legend for specifics of the box plot and expansion of other abbreviations.
The present study examined the expression of AECA in patients with COPD. We found that patients with COPD had a significantly higher prevalence and level of serum AECA than the reference population. Onethird of the patients with COPD had elevated AECA, whereas none of the subjects in the non-COPD reference population had these antibodies. There were no significant relationships between the expression of AECA and pulmonary function or patient characteristics. In the reference population, smokers and never smokers had similar low AECA expression levels. Interestingly, there has been accumulating evidence that COPD is regarded as having the properties of an autoimmune disease.5,17-20 The expression of autoantibodies has been reported in patients with COPD.21,22 Antielastin antibodies are markedly increased in patients with COPD. Increases in interferon-g and IL-10 secretion after stimulation with elastin peptides also have been demonstrated.21 In this previous study, the authors proposed that cigarette smoking initiated T- and B-cell-mediated immunity against elastin in susceptible individuals. Feghali-Bostwick et al22 reported that the prevalence of circulating antipulmonary airway epithelial cell IgG antibodies was elevated among patients with COPD and that these antibodies had the potential to mediate cytotoxicity against pulmonary epithelial cells in vitro. They further evaluated the antipulmonary artery endothelial cell antibodies using indirect immunofluorescence assays, but the study was very small, targeting only 12 patients with COPD. Recently, Leidinger et al19 established protein macroarrays containing 1,827 immunogenic clones and screened serum samples from patients with COPD. They detected 381 peptide clones that reacted with the COPD serum samples. Surprisingly, 17 of these clones reacted with . 60% of the COPD sera, whereas seven clones reacted with . 90% of the sera. The findings that not all smokers developed clinically significant COPD5,7,8 and some smokers develop COPD even after smoking cessation5,17 suggest that additional determinants are involved in onset of the disease. In this context, it is interesting that we found that AECA were found only in the patients with COPD, whereas AECA expression in the non-COPD smokers, who had similar smoking habits to the patients with COPD but normal spirometry, was not elevated. Although the identities of the antigens that
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induce autoantibodies in patients with COPD remain unknown,21,22 COPD has an autoimmune profile, and antibodies, including AECA, may be associated with its pathogenesis. Cigarette smoke contains approximately 4,700 xenobiotic compounds, some of which are harmful and potentially immunogenic in some cases.5,26 These compounds can precipitate in the lungs and induce adaptive immune responses, including autoantibody production.27 Agustí et al17 proposed the possibility that cigarette smoke itself damages airway cells and creates autoantigens that drive immune and inflammatory responses. Because there is a loss of lung microvessels and significant reductions in capillary length density, COPD has a vascular disease component.20,28 Vascular endothelial growth factor (VEGF) is critical for vascular maintenance and endothelial cell survival.29 The gene and protein expressions of VEGF and VEGF receptor are decreased in lung tissues from patients with COPD,29,30 supporting the idea that the breakdown of a VEGF-dependent maintenance program leads to the death of alveolar endothelial cells.20,30 In in vivo studies, VEGF receptor blockade in adult rats causes lung endothelial cell apoptosis and airspace enlargement.18,31 Interestingly, intraperitoneal injection of human endothelial cells leads to the production of antibodies directed against VEGF receptor and endothelial cells and induces alveolar cell apoptosis and pathologic features similar to COPD.18,20,23 Furthermore, antibodies against human endothelial cells in rats induce apoptosis of endothelial cells in vitro and alveolar airspace enlargement in passively immunized mice. Taken together, these findings indicate that injury and damage to endothelial cells contribute to the pathologic changes in patients with COPD.30 In the present study, we investigated the presence of AECA in serum samples and found that only the patients with COPD had elevated AECA levels. Although further studies are required to clarify the vascular involvement in COPD both pathologically and immunologically, AECA may be involved in COPD development through vascular or endothelial cell injury caused by autoantibodies. There are some limitations to the present study. First, we could not determine the vascular involvement pathologically because no specimens were taken from the patients and reference population. Second, although AECA bind to endothelial cell membrane antigens and induce endothelial cell damage,9-11 their target antigen remains to be investigated. Because the issue of whether AECA actually cause vascular damage or are merely the result of vascular damage remains unclear in humans, further studies, including characterization of the antigens recognized by AECA, will elucidate the precise roles of AECA in the pathophysiology of COPD. www.chestpubs.org
In conclusion, the present study demonstrated that patients with COPD had a significantly higher prevalence and levels of AECA than a reference population, suggesting that an autoimmune component associated with endothelial cell injury is involved in the pathogenesis of COPD. Investigations from the viewpoint of autoimmune mechanisms, including AECA, may provide important information toward a better understanding of COPD and a new therapeutic strategy for this disease. Acknowledgments Author contributions: Dr. Karayama: contributed to the study design; data analysis and interpretation; and critical review, revision, and final approval of the manuscript. Dr Inui: contributed to the study design; data analysis and interpretation; and the drafting, critical review, revision, and final approval of the manuscript. Dr Suda: contributed to the data collection, analysis, and interpretation; and to the drafting, critical review, revision, and final approval of the manuscript. Dr Y. Nakamura: contributed to the data collection, analysis, and interpretation; and to the drafting, critical review, revision, and final approval of the manuscript. Dr H. Nakamura: contributed to the statistical analysis; data analysis and interpretation; and critical review, revision, and final approval of the manuscript. Dr Chida: contributed the study design; data analysis and interpretation; and critical review, revision, final approval of the manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.
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