Abstract
Rationale: In patients with chronic inflammatory lung disease, pulmonary proteases can generate neoantigens from elastin and collagen with the potential to fuel autoreactive immune responses. Antielastin peptide antibodies have been implicated in the pathogenesis of tobacco-smoke–induced emphysema. Collagen-derived peptides may also play a role.
Objectives: To determine whether autoantibodies directed against elastin- and collagen-derived peptides are present in plasma from three groups of patients with chronic inflammatory lung disease compared with a nonsmoking healthy control group and to identify whether autoimmune responses to these peptides may be an important component of the disease process in these patients.
Methods: A total of 124 patients or healthy control subjects were recruited for the study (Z-A1AT deficiency, n = 20; cystic fibrosis, n = 40; chronic obstructive pulmonary disease, n = 31; healthy control, n = 33). C-reactive protein, IL-32, and antinuclear antibodies were quantified. Antielastin and anti–N-acetylated-proline-glycine-proline autoantibodies were measured by reverse ELISA.
Measurements and Main Results: All patients were deemed stable and noninfective on the basis of the absence of clinical or radiographic evidence of recent infection. There were no significant differences in the levels of autoantibodies or IL-32 in the patients groups compared with the healthy control subjects.
Conclusions: Antielastin or anti–N-acetylated proline-glycine-proline autoantibodies are not evident in chronic inflammatory lung disease.
Autoimmune responses to elastin- and collagen peptides may have a role in the pathogenesis of tobacco-smoke-induced emphysema.
We demonstrate no evidence for systemic autoantibodies directed against elastin peptides or N-acetylated-proline-glycine-proline in chronic inflammatory lung disease.
Recently, Lee and colleagues demonstrated the presence of antielastin antibodies in the peripheral blood of individuals with tobacco-smoke–induced COPD and were the first to describe COPD as an autoimmune disease (10). Conversely, Cottin and colleagues (11) failed to detect significant differences in antielastin autoantibody levels in patients with combined pulmonary fibrosis and tobacco-smoke–induced emphysema versus control subjects. Calabrese and colleagues demonstrated elevated IL-32 expression in lung tissue of patients with COPD (12). IL-32 is a relatively recently described proinflammatory cytokine (13) reported to have a role in rheumatoid arthritis and inflammatory bowel disease (14, 15).
Z-A1AT deficiency is a genetic disease that predisposes to emphysema. Similar to smoking-induced emphysema, Z-A1AT deficiency is associated with higher-than-normal levels of pulmonary proteases. Although CF is pathologically distinct from COPD and Z-A1AT deficiency, it is characterized by excessive lung proteases. In this study, we sought to detect and quantify the presence of antielastin and anti-PGP autoantibodies in plasma from Z-A1AT-deficient patients with CF and COPD and to compare these levels with healthy control groups. We also quantified IL-32 levels in each sample to identify whether systemic IL-32 may be an important component of the disease process in these patients. Some of the results of these studies have been previously reported in the form of an abstract (16).
A total of 124 patients were included in this study. All patients were diagnosed by standard criteria: Adult patients with CF were genotyped for cystic fibrosis transmembrane conductance regulator mutations and had positive sweat testing of chloride greater than 60 mmol/L, adult patients with Z-A1AT deficiency were homozygous for the Z allele and had serum A1AT ≤11 μM, and adult patients with COPD had obstructive lung disease and a history of smoking. Patients and control subjects were excluded on the basis of recent or current (within 6 weeks) respiratory tract infections, known autoimmune diseases (e.g., connective tissue disorder, Graves Disease), age less than 18 years, or refusal to give consent. All participants gave written informed consent to participate in the study, which was approved by Beaumont Hospital Ethics Committee. None of our patient cohorts was on systemic corticosteroids. Regarding inhaled corticosteroids, 17 of 20 (85%) Z-A1AT-deficient patients, 29 of 31 (94%) patients with COPD, and 35 of 40 (87.5%) patients with CF were on inhaled corticosteroids. The daily dose of inhaled corticosteroid is less than 1,000 μg/d. The control subjects were recruited from a nonpaid group of patients. Smoking and second-hand smoke exposure were excluded from the history alone.
Blood was taken from patients into Sarstedt Monovette tubes containing Li-Heparin. The blood was centrifuged and plasma was processed into aliquots.
Total IL-32 and IL-32α (BioLegend, San Diego, CA) levels in concentrated, neat, and diluted plasma were quantified by ELISA. Assays were performed in duplicate. C-reactive protein (CRP) levels were measured by Olympus System CRP latex reagent (Olympus, Southend on Sea, UK) according to the manufacturer's instructions. Antinuclear antibodies were detected by using indirect immunofluorescence using Hep-2 (Binding Site, Birmingham, UK) as the substrate. Bound IgG was detected using polyclonal rabbit antihuman IgG/FITC Rabbit F(ab')2 (Dako, Glostrup, Denmark). Titers ≥ 1 in 80 were considered positive.
Antielastin IgG antibodies were quantified by using a modified ELISA as described by Lee and colleagues (10) with the following changes. After sample addition, signals were detected using peroxidase-conjugated goat antihuman IgG (1:15,000) (Sigma-Aldrich, Dublin, Ireland) and ABTS (Zymed, San Francisco, CA). Optical density (OD) of the individual wells was determined at 405 nm using a standard microplate spectrophotometer. One sample was selected for inclusion on each ELISA plate to control for interassay variability. Assays were performed in duplicate. Anti-IgM, anti-IgA, anti-IgD, and anti-IgE antibodies were detected in a similar manner using appropriate secondary antibodies.
Anti–N-Ac-PGP antibodies were quantified by indirect ELISA as follows. High–binding, flat-bottom polystyrene microtiter plates (Immunlon; Thermo Scientific, Waltham, MA) were coated with 25 μg/ml N-Ac-PGP (Custom Synthesis; GeneScript, Piscataway, NJ) in Voller's buffer overnight at 4°C, washed with PBS-Tween (0.05%), and blocked with 0.2% I-Block (Tropix). Initially, a selection of diluted human plasma samples (1:10–1:1,280) were incubated for 2 hours at 37°C, washed, and incubated with peroxidase-conjugated goat antihuman IgG (1:15,000 Sigma-Aldrich). After a final wash, ABTS (Zymed) was added, and the OD of the individual wells was determined at 405 nm using a standard microplate spectrophotometer. The sensitivity of the assay extended past 1:1,280 dilution of plasma with good linearity evident over the range of 1:10 to 1:1,280 dilutions. Immunoreactivity of plasma samples was inhibited by pretreatment with N-Ac-PGP. All samples were subsequently tested at 1:80 dilution. One sample was selected for inclusion on each ELISA plate to control for interassay variability. Assays were performed in duplicate.
Data are expressed as mean +SD or ± SEM as indicated. Test of normality was performed by the Kolmogorov-Smirnov test. All data were of nonparametric distribution. Differences between two individual groups were assessed by Mann-Whitney U test. Statistical tests were performed using SPSS 15.0 and Prism 3.0 software. P values 0.05 or less were considered to be significant.
A total of 124 individuals were included in this study (Z-A1AT deficiency, n = 20; CF, n = 40; COPD, n = 31; healthy control, n = 33). Table 1 provides details of their baseline characteristics.
A1AT | CF | COPD | Control | |||||
|---|---|---|---|---|---|---|---|---|
| No. of subjects | 20 | 40 | 31 | 33 | ||||
| Age, years (± SD) | 52.8 ± 7.0 | 23.5 ± 5.5 | 66.9 ± 9.0 | 34.7 ± 11.1 | ||||
| Sex, % male/female | 70/30 | 70/30 | 55/45 | 42/58 | ||||
| Smoking status, % | ||||||||
| Smokers | 15 | 13 | 26 | – | ||||
| Nonsmokers | 20 | 87 | – | 100 | ||||
| Ex-smokers | 65 | – | 74 | – | ||||
| BMI, kg/m2 (± SD) | 26 ± 4 | 22 ± 3 | 26 ± 5 | 24 +/- 4 | ||||
| FEV1, % predicted (± SD) | 50.2 ± 18.7 | 63.1 ± 26.3 | 49.7 ± 19.0 | – | ||||
| FVC, % predicted (± SD) | 88.6 ± 15.5 | 75.0 ± 22.4 | 71.8 ± 20.9 | – | ||||
| Emphysema | ||||||||
| CT evidence, % | 85 | – | 68 | – | ||||
| No CT evidence, % | 10 | – | 19 | – | ||||
| CT unavailable, % | 5 | – | 13 | – | ||||
All patients with chronic inflammatory lung disease recruited for this study were deemed stable and noninfective on the basis of the absence of clinical symptoms (absence of increasing cough, sputum production, dyspnea, and fever) or the absence of radiographic evidence of recent infection. Circulating levels of CRP were also quantified. The majority of patients' CRP levels were within the normal limits. The mean CRP for Z-A1AT deficiency is 8.9 ± 4.8 SEM mg/L, for CF is 13.8 ± 2.9 SEM mg/L, for COPD is 10.8 ± 3.0 SEM, and for healthy control subjects is 1.7 ± 0.3 SEM mg/L.
We quantified antielastin autoantibody levels in individuals with Z-A1AT deficiency and in patients with CF and COPD and compared these with the healthy control subjects (Figure 1). Although variable levels of antielastin IgG antibodies were detectable in each cohort (Figure 1A), there were no significant differences between the patient groups when compared with the healthy control subjects. Mean antielastin antibody levels were lower in the COPD group versus the control group. There were no differences in total antielastin autoantibody levels in the patient groups versus the control group (Figure 1B). ANA are present in the normal population at a prevalence of 13.3% at 1:80 dilution (17), and their frequency increases in older individuals. Of the Z-A1AT-deficient patients, 7 of 20 (35%) had ANA assay performed, and all were negative for ANA. Nineteen percent (6/31) of patients with COPD had ANA assay performed; two of six were ANA negative, and four of six were ANA positive. In the CF group, 9 of 40 of patients (23%) had ANA assay performed, of which seven were ANA negative and two were ANA positive. Samples from individuals with positive ANA assays in our COPD and CF cohorts showed a trend toward lower antielastin antibodies levels, but the numbers are too small to make any significant interpretation.
Figure 1. Antielastin autoantibody levels. (A) IgG antielastin antibody levels and (B) total antielastin antibody levels (optical density [OD] 405 nm) were quantified in plasma from Z-A1AT deficiency (Z; n = 20), cystic fibrosis (CF; n = 40), chronic obstructive pulmonary disease (COPD; n = 31), or healthy control subjects (C; n = 33) by reverse ELISA. Median is depicted by the black bar; squares = Z; triangles = CF; diamonds = COPD; circles = C.
[More] [Minimize]N-Ac-PGP is a collagen-derived peptide known to be present in the lung in CF and COPD, and this could potentially fuel autoreactive immune responses. We developed an anti–N-Ac-PGP reverse ELISA to quantify anti–N-Ac-PGP IgG autoantibody levels in each of our patient groups. Figure 2 shows the levels of detectable anti–N-Ac-PGP antibodies in each sample. There were no significant differences between the patient groups and the healthy control subjects.
Figure 2. Anti–N-Ac-PGP autoantibody levels. Levels of anti–N-Ac-PGP IgG antibodies (OD 405 nm) were quantified in plasma from Z-A1AT deficiency (Z; n = 20), CF (n = 40), COPD (n = 31), or healthy control subjects (C; n = 33) by reverse ELISA. Median is depicted by the black bar; squares = Z; triangles = CF; diamonds = COPD; circles = C.
[More] [Minimize]IL-32 is a relatively recently identified proinflammatory cytokine that has been shown to have a role in rheumatoid arthritis and inflammatory bowel disease (13–15). A number of IL-32 isoforms exist, with non-α isoforms β, γ, and δ reported to be abundant in lung tissue of patients with COPD (12). We quantified total IL-32 and IL-32α levels in plasma of each of our patients and control subjects (Figure 3). The Z-A1AT–deficient, CF, and COPD plasma samples had lower-than-normal levels of total IL-32 (Figure 3A). There was no significant increase in IL-32α levels in the patient versus healthy control cohorts (Figure 3B). Mean IL-32α levels in all groups were less than 2 pg/ml. These results showed that IL-32, a proinflammatory cytokine previously demonstrated in situ in COPD (12), was not detectable systemically in Z-A1AT–deficient patients or in patients with CF and COPD in our study.
Figure 3. IL-32 levels. Levels of (A) total IL-32 (OD 405 nm) and (B) IL-32α (pg/ml) were quantified by sandwich ELISA in plasma from Z-A1AT deficiency (Z; n = 20), CF (n = 40), COPD (n = 31), or healthy control subjects (C; n = 33). Median is depicted by the black bar; squares = Z; triangles = CF; diamonds = COPD; circles = C.
[More] [Minimize]A number of studies have suggested a role for autoimmunity in the pathogenesis of COPD (10, 18). Recently autoantibodies directed against elastin-derived peptides have been implicated as having a key role in tobacco smoke–induced emphysema (10). Here we report no evidence of abnormally high antielastin antibody concentrations in the plasma of patients with COPD. We did not detect a relationship between plasma anti-elastin antibody levels and Z-A1AT deficiency or CF. Our analysis of anti–collagen-derived peptide autoantibodies and plasma IL-32 levels demonstrated no marked increase in our patient versus control cohorts and together point to no role for systemic antielastin or anti–N-Ac-PGP autoantibodies or systemic IL-32 in COPD, Z-A1AT deficiency, or CF.
In addition to higher-than-normal intrapulmonary levels of neutrophil elastase during neutrophil-dominated airway inflammation, there is evidence to suggest that MMP-8, MMP-9, and prolyl endopeptidase are elevated, for example in the CF lung, and that together these enzymes can degrade collagen in vivo to generate the tripeptide PGP, which is detectable as PGP or N-Ac-PGP in CF sputum (2, 4) or in experimental animals with emphysema (19). Because NE can activate MMPs, a similar phenomenon may occur in Z-A1AT deficiency because it too is a neutrophil-dominated disease of the airways. PGP is chemotactic for human neutrophils and has been linked to neutrophil superoxide production, alveolar enlargement, and right ventricular hypertrophy, which contribute to the pulmonary inflammatory manifestations of CF. To address whether protease-derived PGP peptides function as autoantigens in Z-A1AT deficiency and CF, we investigated whether anti-PGP antibodies were raised to this neoantigen in vivo in patients' plasma. Lee and colleagues reported that they failed to detect anticollagen autoantibodies in their COPD cohort (10); we considered it important to repeat these experiments using N-Ac-PGP rather than collagen as the target and to include CF and Z-A1AT-deficient cohorts to provide an unequivocal answer to this question. Although we detected anti-PGP antibodies in plasma from all the cohorts, there was no significant difference between the patient groups and healthy control subjects. The individuals with detectable anti-PGP antibodies did not correlate with those who had antielastin antibodies.
IL-32 is a relatively recently described cytokine (13). Previously termed “NK transcript 4,” IL-32 is produced by mitogen-activated lymphocytes, IFN-γ–activated epithelial cells and, IL-12– and IL-18–activated NK cells. It induces the expression of TNF-α, IL-1β, IL-6, and C-X-C chemokine family members and has been shown to have a role in rheumatoid arthritis (14), inflammatory bowel disease (15), lung cancer (20), and pancreatic cancer (21). Recently, Calabrese and colleagues (12) demonstrated increased expression of IL-32, particularly its non-α isoforms, in lung tissue of patients with COPD, where it was colocalized with TNF-α and correlated with the degree of airflow obstruction. These data suggest that IL-32 is implicated in the characteristic immune response of COPD, with a possible impact on disease progression. However, IL-32 production is neither unique nor specific for autoimmunity because there is production of IL-32 in cell culture systems devoid of autoantibodies or self-reactive T cells. There have been no reports linking IL-32 to CF or Z-A1AT deficiency. We failed to detect elevated plasma levels of IL-32 or the IL-32α isoform in our cohorts of patients with CF or COPD or in Z-A1AT-deficient patients, suggesting that IL-32 expression in these patients may be tissue specific.
Our findings show that neither antielastin nor anti–N-Ac-PGP autoantibodies are increased in patient plasma. That does not exclude the potential for other autoantibodies, particularly at specific tissue sites. Other evidence supports a role for autoantibodies in the pathogenesis of CF, COPD, and Z-A1AT deficiency. For example, a number of studies have detected circulating autoantibodies directed against bactericidal permeability increasing protein (BPI-ANCA) in CF, suggesting that BPI-ANCA levels are associated with P. aeruginosa colonization and may be predictive for lung damage (22, 23). Others have detected anti-GAD65, but not β-cell autoantibodies, in CF-related diabetes (24, 25) and observed an increase in anti-hsp60 autoantibody levels before the development of glucose intolerance in this population (26). Z-A1AT deficiency has also been linked to autoimmune disease, in particular due to the presence of antineutrophil cytoplasmic antibodies (ANCA) and antiendothelial cell antibodies (AECA) in some Z-A1AT-deficient individuals. ANCA are autoantibodies directed against neutrophil cell antigens, mainly proteinase 3 (PR3) and myeloperoxidase but also lactoferrin, NE, and BPI. The observation that PR3-ANCA is associated with Z-A1AT deficiency has been previously demonstrated (27, 28). Audrian and colleagues (29) also demonstrated a high incidence of antibodies directed against NE in Z-A1AT deficiency, concluding that ANCA not directed against classical antigens (myeloperoxidase and PR3) may be found in these patients. Notwithstanding the conflicting reports regarding antielastin antibodies in emphysema, there is evidence that autoantibodies with avidity for pulmonary epithelium are present in COPD plasma (18). Antielastin antibodies have been detected in normal healthy subjects as reported by Baydanoff and colleagues (30). They demonstrated the presence of antielastin antibodies (IgG, IgM, IgA, and IgD) in normal healthy subjects between 1 and 75 years of age. Antielastin antibodies are also present in connective tissue diseases, such as scleroderma and systemic lupus erythematous (31), and in Type I diabetes mellitus (32, 33).
Possible reasons for differences in our findings and those of Lee and colleagues include the differences in the populations' ethnicity and that Lee's control group may have had very low circulating antielastin autoantibodies (30). Our healthy control subjects were nonsmokers and were younger compared with the other groups (at least the COPD cohort). This could have biased this study toward finding more antibody in the COPD cohort because autoantibody titers increase with age and among smokers. However, antielastin autoantibodies are lower in COPD compared with healthy control subjects in our study. The converse is true for the greater proportion of women among healthy control subjects in our study, which could bias the results toward decreasing intergroup difference because women typically have more autoantibodies than men. In summary, we failed to detect systemic autoantibodies directed against elastin or collagen peptides in COPD, CF, and Z-A1AT deficiency, all of which are associated with a high pulmonary protease burden. Despite the fact that elastin, PGP, and/or IL-32 are evident in the lung in these diseases, the effects appear to be more important in the lung than throughout the circulation.
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