In this study, we have demonstrated that serum anti-CK8, anti-CK18, and anti-CK19 autoantibodies are present in certain populations of bronchial asthma patients. The prevalences of anti-CK18 and anti-CK19 autoantibodies were significantly higher in patients with ASA-intolerant asthma than in healthy controls. While some of the ASA-tolerant asthmatics had anti-CK18 and anti-CK19 antibodies, the prevalences did not differ from those of normal controls. Furthermore, a proportion of the asthma patients, regardless of ASA sensitivity, had laboratory markers of autoimmunity, including ANA, CIC, and IgG antibody to TGase, although the prevalences of these markers were too low to be statistically significant.
The mechanism of induction of autoantibodies in asthma remains unknown. Disruption of bronchial epithelial cells and subsequent exposure of autoantigens or ineffective antigen elimination during the inflammatory process may cause chronic immune stimulation and autoantibody production. In asthma, the bronchial epithelium is characteristically damaged, with shedding of the columnar cells into the airway lumen. Recently, it has been demonstrated that high doses of acetaminophen reduce the levels of glutathione in lung tissues (13
), and that the asthmatic bronchial epithelium is more susceptible to oxidant-induced apoptosis (14
). During this early apoptosis, activated caspases cleave a variety of structural proteins. Therefore, it may be postulated that disruption of the cytoskeleton leads to the loss of apoptotic cells from the epithelium and that the altered epithelium becomes an important source of autacoid mediators, chemokines, and growth factors, which contribute to ongoing inflammation (15
Cytokeratin is a cytoskeletal structure that is expressed only in epithelial cells. Pairs of keratins seem to be consistently co-expressed in different types of epithelial cells. Thus, CK8, CK18, and CK19, which were used in this study, have been found only in simple epithelia, including both bronchial and lung alveolar epithelial cells (17
), which are the major target tissues of asthma. Previously, CK18 has been identified as a bronchial epithelial autoantigen that is associated with non-allergic asthma (12
). In isocyanate-induced asthma, CK18 has been identified as a major diisocyanate-binding protein (18
), and significantly higher levels of serum IgG to CK19 have been detected (19
). CK8 and CK18 contain the caspase cleavage site and have been reported to undergo marked re-organization during apoptosis (16
). These findings raise the possibility that fragments of CKs and intracytoplasmic materials are released to the blood vessels and may play a role in the formation of circulating autoantibodies, including ANA and IgG to CKs and TGase.
Recent in vitro studies have indicated that the opsonization of extracellular keratin aggregates by IgG-anti-CK autoantibodies plays an important role in promoting the phagocytosis of cytokeratin aggregates (21
). This may relate to our results, which show that asthma patients with anti-CK18 and anti-CK19 antibodies have higher prevalences of CIC as well as more severe airway hyperresponsiveness to methacholine. These results suggest that persistent airway inflammation in some patients with bronchial asthma results from a non-IgE-mediated reaction to endogenous or exogenous antigen, possibly an autoantigen, or to a chronic viral infection (22
). These possibilities are supported by a number of studies, which have shown that some patients with ASA-intolerant asthma have elevated markers of autoimmunity with rheumatic symptoms. Enhanced IgG4
synthesis in association with viral infection and a positive association with the HLA gene marker have been noted (10
). Our previous study has demonstrated that HLA-DPB1*0301 is a valuable gene marker for ASA-intolerant asthma (10
). However, in the present study, no direct relationship was found between this HLA gene marker and the prevalences of IgG antibodies to the three CKs, ANA, IgG antibody to TGase, and CIC. Moreover, none of subjects with these autoantibodies complained of rheumatic symptoms.
Tissue transglutaminase (TGase I) is a member of the Ca2+
-dependent enzymes that catalyze the cross-linking of proteins. TGase I is expressed in tissues that contain simple epithelia, such as bronchial epithelia, skin epidermis, liver, gastrointestinal tract, kidney, and endothelial cells (23
). The induction and activation of TGase is part of the apoptotic cascade and plays an effector role in the stabilization of apoptotic bodies to limit the leakage of intracellular components into the extracellular space (24
). Recently, anti-TGase antibodies have been found not only in various autoimmune diseases, such as celiac disease, diabetes mellitus type I, and SLE, but also in toluene diisocyanate-induced asthma (19
). Based on these findings, we examined the impact of anti-TGase antibodies on persistent airway inflammation in patients with ASA-intolerant asthma, to evaluate the potential of TGase as an endogenous antigen. However, the prevalence of IgG to TGase in patients with ASA-intolerant asthma was <5%, which was similar to that in ASA-tolerant patients, and it had no effect on asthma phenotype.
Considering the results of the present study, an autoantibody-mediated mechanism for ASA-intolerant asthma appears to be unlikely. We cannot rule out the possibility that the existence of anti-CK and anti-TGase autoantibodies is a non-specific consequence of epithelial injury, since anti-CK18 and anti-CK19 antibodies have been reported in patients with various airway diseases, which included non-atopic (13
) or TDI-induced asthma (19
), pulmonary fibrosis (25
), adenocarcinoma (26
), and non-airway diseases like an autoimmune hepatitis (27
). Besides, the positivity levels of these autoantibodies were neither high enough nor related to ASA-sensitivity, atopy, asthma duration or baseline FEV1
. However, long-term prospective studies are needed to address whether autoantibodies cause clinical symptoms of autoimmunity several years later, have a pathogenic role, or merely reflect the epiphenomenon of polyclonal immune stimulation.
A mechanism by which IgG antibody to CK could be involved in the pathogenicity of asthma has been suggested in the case of hexamethylene diisocyanate-induced asthma. Inhaled hexamethylene diisocyanate can bind to the CK of epithelial cells, which may induce T cell and cytokine production (18
). Considering the clinical features of the patients with ASA-intolerant asthma enrolled in our study, with most of the patients having moderate to severe persistent asthma, we hypothesize that self-antigen or possibly CK-driven immune responses contribute to the development of persistent airway inflammation in these patients. Patients who are more susceptible to epithelial damage following airway inflammation, or who have impaired clearance of apoptotic bodies, may develop serum IgG antibodies to CK. It is clear from this study that asthmatics with circulating anti-CK18 and anti-CK19 autoantibodies suffer from more severe airway hyperresponsiveness and have more complement-binding immute complexes.
The prevalence of IgG to CK18 in non-atopic asthma was a little lower than that seen in the previous investigation using immunoblot analysis (13
). This difference may be due to the different antigen and antibody detection methods used; the ELISA system using recombinant CK was applied in this study to screen a larger population of patients with ASA-intolerant asthma.
In conclusion, we have confirmed the existence of several autoantibody types, with the highest prevalence of IgG to CK18, IgG to CK19, and CIC in patients with bronchial asthma, irrespective of ASA sensitivity or atopy. In addition, the correlation with airway hyperresponsiveness suggests that anti-CK18 and anti-CK19 autoantibodies are involved in the persistent airway inflammation of bronchial asthma. To confirm the role of anti-CK autoantibodies, further studies are needed to show the presence of autoreactive helper T cells specific for CKs and the deposition of activated complement components in patients with bronchial asthma.