Novel autoantigens immunogenic in COPD patients
- Petra Leidinger1_734,
- Andreas Keller2_734,
- Sabrina Heisel1_734,
- Nicole Ludwig1_734,
- Stefanie Rheinheimer1_734,
- Veronika Klein1_734,
- Claudia Andres2_734,
- Jürg Hamacher3_734,
- Hanno Huwer4_734,
- Bernhard Stephan5_734,
- Ingo Stehle6_734,
- Hans-Peter Lenhof2_734Email author and
- Eckart Meese1_734Email author
© Leidinger et al. 2009
Received: 16 October 2008
Accepted: 12 March 2009
Published: 12 March 2009
Chronic obstructive pulmonary disease (COPD) is a respiratory inflammatory condition with autoimmune features including IgG autoantibodies. In this study we analyze the complexity of the autoantibody response and reveal the nature of the antigens that are recognized by autoantibodies in COPD patients.
An array of 1827 gridded immunogenic peptide clones was established and screened with 17 sera of COPD patients and 60 healthy controls. Protein arrays were evaluated both by visual inspection and a recently developed computer aided image analysis technique. By this computer aided image analysis technique we computed the intensity values for each peptide clone and each serum and calculated the area under the receiver operator characteristics curve (AUC) for each clone and the separation COPD sera versus control sera.
By visual evaluation we detected 381 peptide clones that reacted with autoantibodies of COPD patients including 17 clones that reacted with more than 60% of the COPD sera and seven clones that reacted with more than 90% of the COPD sera. The comparison of COPD sera and controls by the automated image analysis system identified 212 peptide clones with informative AUC values. By in silico sequence analysis we found an enrichment of sequence motives previously associated with immunogenicity.
The identification of a rather complex humoral immune response in COPD patients supports the idea of COPD as a disease with strong autoimmune features. The identification of novel immunogenic antigens is a first step towards a better understanding of the autoimmune component of COPD.
Chronic obstructive pulmonary disease (COPD) is a common pulmonary affection, which is characterized by a range of pathophysiological changes including airflow limitation based on an obstructive bronchiolitis and persistent inflammation with neutrophils, macrophages, B and T lymphocytes and dendritic cells, a mucociliary dysfunction, apoptosis, and on structural changes in the airways causing emphysema, and by extrapulmonary systemic effects [1–3]. The overall global prevalence of COPD in adults that are 40 years or older is approximately 10% . While COPD is currently the fourth leading cause of death worldwide it is expected to be the third leading cause of death in 2020 . Besides, COPD is a major factor for disease-related loss of quality of life, health expenditure and loss of productivity.
Various host and environmental risk factors are supposed to contribute to the development of COPD. Host factors include airway hyperresponsiveness and aberrant lung growth. Several candidate genes are found to contribute to the individual risk. Environmental risk factors include smoking as the first cause followed by air pollution and occupational dust or chemicals .
COPD shares many clinical and pathophysiological features with autoimmune diseases . There is strong evidence for an active adaptive T-cell response in COPD patients . Antielastin antibody and T-helper type 1 responses characterize emphysema as an autoimmune disease . The most recent evidence for a prevalence of IgG autoantibodies in COPD patients further supports the idea of a strong autoimmune component in COPD . However, there is rather limited information on the nature of antigens reacting with autoantibodies in COPD patients. In this study we identified a large number of different antigens immunogenic in COPD patients and determined how many antigens reacted with each COPD serum. Likewise, we determined how many COPD sera reacted with each antigen. Additionally, we compared the reactivity of each antigen between COPD sera and control sera of healthy individuals. The identification of novel immunogenic antigens that allow differentiation of COPD patients from controls is a first step towards the development of new diagnostics and therapies as recently suggested .
Information on COPD patients
former smoker, 100 py
former smoker, py unknown
former smoker, 30 py
former smoker, py unknown
former smoker, py unknown
former smoker, 50 py
former smoker, py unknown
former smoker, 100 py
former smoker, 20 py
former smoker, 20–25 py
former smoker, 120 py
former smoker, 30 py
former smoker, 20 py
former smoker, 30 py
former smoker, 30 py
Gender and age of patients and controls
Protein macroarray screening
We assembled 1827 different immunogenic clones from high-density protein macroarrays consisting of 38,016 E. coli expressed proteins from the hex1 library  by carrying out a pre-screening with sera of patients with different human diseases. These 1827 E. coli expressed proteins were screened in duplicates with 17 COPD sera and 60 normal control sera. In brief, macroarrays were washed twice with TBSTT (TBS, 0.05% Tween 20, 0.5% Triton X-100) and 4 times with TBS. After blocking with 3% non-fat dry milk powder in TBST (TBS, 0.05% Tween 20), macroarrays were incubated over night with sera 1:1000 diluted in blocking solution (3% non-fat dry milk powder in TBST). After the incubation, sera were stored for the second incubation round. Three washing steps with TBST were followed by incubation with stripping solution at 70°C. Macroarrays were washed twice with TBST and 4 times with TBS. Incubation with blocking solution was followed by a second round of serum incubation over night. Macroarrays were washed three times with TBST, and incubated with secondary antibody (rabbit anti-human IgG, IgA, IgM-Cy5 (H+L)) 1:1000 diluted in blocking solution. Macroarrays were washed four times with TBST, twice with TBS and scanned by the GE Healthcare Typhoon 9410 scanner. The evaluation of the scanned protein arrays was carried out by the image analyzing software tool AIDA version 4.15 and by a newly developed computer-aided analysis procedure.
Computational analysis of seroreactivity patterns
Visual analysis of protein macroarrays
Next, we asked how frequent does any of the peptide clones react with sera of COPD patients. We detected 17 clones that reacted with more than 60% of all tested COPD sera and seven clones that reacted with more than 90% of all tested COPD sera.
Computational comparison between COPD sera and controls
In silico analysis of the in frame peptide clones
Sequence motives like coiled coil, ELR, RGD, and granzyme B cleavage sites can be characteristic for autoantigens [16–18]. We analyzed the 58 different genes with the statistical gene analysis tool "GeneTrail" . Our analysis revealed that 20.7% of the 58 sequences are containing coiled coil motives, whereas only 11.3% of all human gene sequences show such motives. We found approximately 10% more sequences with ELR motives in our test set, i.e. the 58 genes, as compared to the training set. Predicted granzyme B cleavage sites were found in 43.1% of the sequences in our test set and in 40.4% of the sequences in the reference set. In contrast, RGD motives were found in only 5.2% of the sequences in our test set but in 7.5% of the sequences in the reference set.
Out of the 58 genes, eight genes namely RPL37, RPL35, RPS27A, RPL23A, RPL6, RPS2, RPS6, and LOC388720are involved in the KEGG (Kyoto Encyclopaedia of Genes and Genomes) ribosome pathway. RPS6 is also involved in the insulin signalling and the mTOR signalling pathway. MCM3 and VIM are involved in the pathways cell cycle and cell communication, respectively. GTF2B, also known as TFIIB, is a member of the pathway basal transcription factors. HSPD1 is involved in the two pathways Prion disease and Type I diabetes mellitus.
The concept of COPD as an autoimmune disease is supported by the recent findings of frequent IgG autoreactive antibodies in COPD patients [7–10]. In our study, we identified 381 antigens that are immunogenic in COPD patients by screening protein macroarrays with patients' sera followed by a visual evaluation. We identified seven clones that were reactive in more than 90% of all COPD sera. To overcome a bias possibly caused by visual evaluation of the protein macroarrays, we additionally evaluated the macroarrays with a computer aided image analysis procedure that ensures a standardized evaluation of the arrays. Here, we identified 212 peptide clones with informative AUC values < 0.3 and > 0.7 by comparing seroreactivities of COPD patients and healthy controls. Clones with informative AUC values offer themselves as future biomakers for COPD. The identification of biomarkers is however not the focus of our study. Biomarker evaluation requires a study design that tests antigens with informative AUC values for their reactivity against a large number of patients' sera and control sera in a prospective manner. The identified peptide clones provide the basis for such a prospective study. The clones with AUC < 0.3 and > 0.7 included 67 in frame clones, representing 58 different genes and 145 out of frame clones that are termed mimotopes. Mimotopes are defined as peptides capable of binding to the antibody but unrelated in sequence to the natural protein that the antibody recognizes . Since some of the mimotopes yielded rather informative AUC values as for example clone FAM36A, it will be worthwhile to reveal the nature of the natural protein that react in vivo with autoantibodies of COPD patients.
As indicated above a larger number of clones identified in this study were previously associated with an immune response in cancer or with autoimmune diseases. In addition, the two genes TRAF4 and NME2 have been associated with non-cancer lung diseases. TRAF4 deficiency leads to tracheal malformation resulting in airflow limitations in TRAF4-deficient mice . NME2 negatively regulates Rho activity through interactions with other proteins involved in the Rho pathway . In embryonic mouse lungs, the inhibition of the Rho pathway leads in vitro to reduced lung bud formation after 48 hours . Since none of the clones identified in our study has previously been associated with the development or progression of COPD, the newly identified antigens may give leads to as of yet not analyzed cellular processes underlying COPD. Several of the identified antigens are currently discussed as biomarkers including HSP1, MAZ, and RPS2 that are up-regulated in colorectal cancer, acute myeloid leukaemia, and astrocytoma, respectively [24–26]. In addition, future biomarkers may be identified among the antigens NME2, CDC42BPB, RPS2, PTBP1, SON, MCM3, CD320, VIM, CENPB, PDE4DIP, CCNL2, HMG-14, HSPD1, MAZ, RPL6, STUB1, and MBTPS1 all of which have been associated with different human cancers. However, none of the identified antigens has been introduced into clinical practice.
It remains to elucidate what mechanisms contribute to the immunogenicity of each antigen in COPD patients. One hypothesis is that the humoral immune response against disease associated antigens results from overexpression. Although there is some evidence for an association of overexpression and immunogenicity of antigens [27, 28], conclusive experimental proof for a causative role of overexpression has still to be provided. Posttranslational modifications including altered protein folding and processing may also cause a humoral immune response against disease-associated antigens. In addition, mutations are discussed as cause for the humoral immune response against immunogenic antigens . However, we analyzed the reactivity of COPD sera using a cDNA library expressed in E. coli that lack the post-translational modification machinery. In addition, the sequences are highly unlikely to contain specific COPD mutations. Several sequence motives including coiled coil, RGD, ELR and granzyme B cleavage sites have previously been associated with the immunogenicity of antigens [16–18]. We found a slightly elevated percentage of coiled coil motives, ELR motives, and granzyme B cleavage sites in COPD associated antigens. Single analysis of each of these antigens will be required to elucidate the contribution of these motives and other factors to the immunogenicity of the antigens.
Our study provides clear evidence that COPD shows strong autoimmune features. In contrast to previous studies, we not only demonstrated the prevalence of IgG autoantibodies but determined the nature of antigens reacting with autoantibodies in COPD patients. The identification of novel immunogenic antigens that allow differentiation of COPD patients from healthy controls will help to contribute to improved diagnostics and therapies.
This work was funded by Homburger Forschungsförderungsprogramm (HOMFOR) and by Globus Baumarkt.
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