Oxidative stress-induced antibodies to carbonyl-modified protein correlate with severity of chronic obstructive pulmonary disease

Abstract

Rationale: There is increasing evidence for the presence of autoantibodies in chronic obstructive pulmonary disease (COPD). Chronic oxidative stress is an essential component in COPD pathogenesis and can lead to increased levels of highly reactive carbonyls in the lung, which could result in the formation of highly immunogenic carbonyl adducts on "self" proteins.

Objectives: To determine the presence of autoantibodies to carbonyl-modified protein in patients with COPD and in a murine model of chronic ozone exposure. To assess the extent of activated immune responses toward carbonyl-modified proteins.

Methods: Blood and peripheral lung were taken from patients with COPD, age-matched smokers, and nonsmokers with normal lung function, as well as patients with severe persistent asthma. Mice were exposed to ambient air or ozone for 6 weeks. Antibody titers were measured by ELISA, activated compliment deposition by immunohistochemistry, and cellular activation by ELISA and fluorescence-activated cell sorter.

Measurements and main results: Antibody titer against carbonyl-modified self-protein was significantly increased in patients with Global Initiative for Chronic Obstructive Lung Disease stage III COPD compared with control subjects. Antibody levels inversely correlated with disease severity and showed a prevalence toward an IgG1 isotype. Deposition of activated complement in the vessels of COPD lung as well as autoantibodies against endothelial cells were also observed. Ozone-exposed mice similarly exhibited increased antibody titers to carbonyl-modified protein, as well as activated antigen-presenting cells in lung tissue and splenocytes sensitized to activation by carbonyl-modified protein.

Conclusions: Carbonyl-modified proteins, arising as a result of oxidative stress, promote antibody production, providing a link by which oxidative stress could drive an autoimmune response in COPD.

Figure 1.

Antibodies to carbonyl-modified protein in human serum from patients with chronic obstructive pulmonary disease (COPD), smokers, and nonsmokers. Human serum was screened for immunoreactivity toward carbonyl-modified human serum albumin by ELISA and titers determined as detailed in Methods. Antibody titers against human serum albumin that had been left (a) unmodified or had been modified by (b) acrolein, (c) malonyldialdehyde (MDA), (d) 4-hydroxynonenal, or (e) cigarette smoke extract are shown.(f) The cumulative titers against all the different carbonyl-modified HSAs tested for each patient group are shown. Results are expressed as a box and whiskers plot and displaying the mean for each patient group. Sera from 12 patients with severe persistent asthma (SA) are used as a disease control. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparison post test analysis. *P < 0.05, **P < 0.01, ***P < 0.001 compared with nonsmokers (NS). ^#P < 0.05, compared with smokers.

Figure 2.

Carbonyl-modified proteins are present in parenchymal lung tissue of patients with chronic obstructive pulmonary disease (COPD). Solubilized parenchymal lung tissue from three patients with COPD was analyzed by (A) Western blotting for carbonyl modified proteins after dinitrophenylhydrazine (DNPH) derivatization of carbonyl epitopes, and (B) Coomassie staining for total protein. In lanes 1, 3, and 5, protein samples were derivatized with DNPH before sodium dodecyl sulfate polyacrylamide gel electrophoresis; lanes 2, 4, and 6 were left underivatized. Further details are available in the online supplement.

Figure 3.

Antibodies to carbonyl-modified self-protein in mice chronically exposed to ozone. Murine serum was screened for immunoreactivity toward carbonyl-modified murine serum albumin by ELISA and titers determined by ELISA as detailed in Methods. Murine serum from mice either acutely exposed (1 d) or chronically exposed (6 wk) to air or ozone was screened on ELISA plates coated with murine serum albumin (MSA) that had been carbonyl-modified with malonyldialdehyde (MDA). Chronic ozone exposure results in a significant increase in antibody titer against carbonyl-modified protein. Results are expressed as the mean ± SEM for the titer determination from 6 to 8 mice in each treatment group. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparison post test analysis. *P < 0.05, compared with control mice exposed to air.

Figure 4.

Complement (C4d) activation in peripheral lungs of chronic obstructive pulmonary disease (COPD) and control subjects. Photomicrographs showing immunostaining for activated compliment C4d with a polyclonal rabbit IgG antibody on peripheral lung from (A) a nonsmoker, (B) a healthy smoker with normal lung function, (C) a patient with mild/moderate COPD, and (D) the negative control wherein the primary antibody is substituted for a nonspecific polyclonal rabbit IgG antibody on COPD lung. C4d+ cells are identified by a brown immunostain. Results are representative of those from 14 nonsmokers, 20 smokers with normal lung function, 16 with mild/moderate COPD. A significant increase in staining between nonsmokers versus healthy smokers and COPD groups was observed (P < 0.05 as determined using the Kruskal-Wallace test). Further methodological details are described in the online supplement.

Figure 5.

Autoantibodies to endothelial cells in human serum from patients with chronic obstructive pulmonary disease (COPD), smokers, and nonsmokers. Human serum was screened for immunoreactivity toward human endothelial cells by ELISA and titers determined as detailed in Methods. Plates were coated with live human umbilical vein endothelial cells, then treated with serum from COPD, smokers, or nonsmokers before detecting bound antibody. Results are expressed as the mean ± SEM for immunoreactivity in each patient group. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparison post test analysis. *P < 0.05 compared with control nonsmokers.