| Chlamydia pneumoniae infection, inflammation and heat shock protein 60 immunity in asthma and coronary heart disease | ||
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The results of this study show an association of serum IgA antibodies to the Hsp60 protein of C. pneumoniae with asthma and thus support the hypothesis of an association between C. pneumoniae infection and asthma (Hahn 1999). The analysis of C. pneumoniae Hsp60 IgA antibodies in sputum samples showed the levels to be somewhat, though not statistically significantly, higher among the asthma patients than among the controls. IgA antibodies to the Hsp60 protein of C. pneumoniae were associated only with asthma, while IgA antibodies against the whole C. pneumoniae bacterium were associated with both asthma and acute bronchitis. No significant association was found between the corresponding IgG antibodies and asthma. The lack of association between IgG antibodies to the same recombinant C. pneumoniae Hsp60 protein and asthma was also observed in another study in Finland (von Hertzen et al., submitted).
In addition, serum IgA antibodies to C. pneumoniae Hsp60 were inversely correlated with pulmonary function, as measured by FEV1, suggesting an association with the severity of pulmonary obstruction. The association of C. pneumoniae Hsp60 IgA antibodies with both asthma and the severity of pulmonary obstruction support the possibility that the Hsp60 protein may play a role in the pathogenesis of bronchial hyperreactivity and/or pulmonary obstruction. Miyashita et al. (1996) have shown marked anti-Hsp60 seroreactivity in an exacerbation of culture-proven persistent C. pneumoniae lung infection and suggested that an allergic reaction to Hsp could produce pulmonary symptoms.
Although the highly conserved nature of the Hsp proteins raises the possibility of molecular mimicry and generation of an autoimmune response as an additional factor in immunopathogenesis, no association was found between human Hsp60 antibodies and asthma. Nor was any correlation found between human and chlamydial Hsp60 IgG antibodies. Our results agree with the study of Henriksen et al. (2000). Yet, there was a correlation between human and chlamydial Hsp60 IgA antibodies. This correlation may be related to a role of Hsp60 molecular mimicry in respiratory illnesses. It seems that IgG and IgA antibodies are formed against partly different antigenic epitopes, of which some are cross-reactive.
Asthma is associated with predominant Th2 and relatively deficient Th1 phenotype. A similar Th2/Th1 profile has been demonstrated in persons with severe trachomatous scarring. In these patients, C. trachomatis Hsp60 increased the numbers of peripheral blood mononuclear cells, producing interleukin-4 (IL-4), whereas C. trachomatis Hsp60 stimulated increased secretion of interferon-gamma (INF-) in controls (Holland et al. 1996). No comparable studies are available for C. pneumoniae-associated diseases. It is known, however, that chlamydial Hsp60 regulates macrophage tumour necrosis factor-alpha (TNF-) and matrix metalloproteinase expression (Kol et al. 1998). An imbalance in metalloproteinase expression has also recently been implicated in the pathogenesis of airflow obstruction in asthma and chronic bronchitis (Vignola et al. 1998).
It is possible that the Hsp60 responses simply represent a marker for exposure to chlamydial infection. On the other hand, there are two counter-arguments in favour of a role for Hsp60 protein in the pathogenesis. Firstly, despite the weak but significant correlation between IgA antibodies to C. pneumoniae antigens and to C. pneumoniae Hsp60, the results indicate that there is, nevertheless, an independent association between C. pneumoniae Hsp60 IgA antibodies and asthma, even after controlling for the effects of C. pneumoniae IgA antibodies. Another argument is the differential IgA antibody responses in controls with acute bronchitis. These findings support the need for further investigations on the role of C. pneumoniae Hsp60 in the pathogenesis of asthma.
In this study, we also showed, for the first time, that low-grade systemic inflammation, demonstrated as a slightly elevated CRP level, was present in stable asthma, despite therapy with inhaled corticosteroids. Furthermore, the CRP levels were highest among the patients with moderate asthma. The asthma patients were more obese than the controls, but after adjustment for BMI, the difference in CRP levels remained statistically significant between the healthy controls and the patients with moderate asthma. CRP might thus help to identify asthma patients with ongoing inflammation and a risk for airway remodelling. Theoretically, CRP could be an additional tool in asthma control. A follow-up study is needed to evaluate the role of CRP as an inflammatory marker in predicting the outcome of asthma both for monitoring the sufficiency of anti-inflammatory medication and as part of poor outcome risk assessment. Elevated CRP levels may also reflect longstanding tissue damage and airway remodelling in chronic airway inflammation. Furthermore, it was found that serum IgA antibodies against the Hsp60 protein of C. pneumoniae and the whole C. pneumoniae bacterium were both associated with an elevated CRP level, suggesting that low-grade inflammation in some asthma cases may be associated with chronic infection and immune reactions caused by C. pneumoniae. The corresponding IgG antibodies were not associated with elevated CRP.
Most of the studies demonstrating an association between C. pneumoniae and asthma, especially in adults, have been serological, and evidence of a causal relationship or even of the presence of C. pneumoniae in lung tissue is thus still lacking.
In this study, we found an association between human Hsp60 IgA antibodies and the coronary risk. An elevated level of these antibodies was shown to predict the occurrence of MI or coronary death several years later. We further showed that only the Hsp60 antibodies that were persistently elevated in the sera were associated with an increased risk. Wick and his collaborators have studied the role of microbial Hsp60 in the development of atherosclerosis, and they have found immunity to mycobacterial Hsp65 to be associated with the pathogenesis of both carotid and coronary atherosclerosis (Xu et al. 1993, Hoppichler et al. 1996). Hsp65 antibodies were found more often in patients with atherosclerotic lesions than in persons without such lesions, and they also predicted carotid atherosclerosis (Xu et al. 1999, Kiechl et al. 2001). They have also shown that these antibodies cross-react with E. coli Hsp60, chlamydial Hsp60 and human Hsp60 antibodies and are cytotoxic to endothelial cells (Mayr et al. 1999). In this study, we confirmed their findings and extended them to apply to coronary events: autoimmunity, as indicated by an elevated level of IgA antibodies against human Hsp60 protein, was shown to predict the occurrence of MI or coronary death several years later.
Even though there was a good correlation between the antibodies to human Hsp60 and C. pneumoniae Hsp60, only IgA antibodies to human Hsp60 were associated with the coronary risk. The association found between human Hsp60 and C. pneumoniae IgA antibodies suggests that the antibodies to human Hsp60 may have been induced, at least partly, by C. pneumoniae Hsp60 during chronic and repeated C. pneumoniae infections, but the risk for coronary events is present only when autoimmunity to human Hsp60 has developed. Human, chlamydial and other bacterial Hsps share a high sequence homology, 75% at the amino acid level, and it is thus naturally possible that other bacterial agents, especially H. pylori and dental bacteria, which have been associated with CHD, have also contributed to the development of autoimmunity to human Hsp60 (Birnie et al. 1998). However, no association was found between H. pylori infection and coronary risk (Roivainen et al. 2000). Furthermore, our results showed that only the antibodies that were persistently elevated in the sera of these middle-aged hypercholesterolemic men were associated with an increased risk, suggesting that chronic C. pneumoniae infection and persistent production of autoantibodies to Hsp60 may have played a role in the development of cardiac events.
Our results showed that the coronary risks associated with elevated levels of IgA antibodies to human Hsp60 increased considerably in the presence of an elevated CRP level, a marker of systemic inflammation. The same applied to C. pneumoniae IgA antibodies, as already shown by Roivainen et al. (2000), where no risk was associated with a high level of C. pneumoniae IgA antibodies, unless they were present together with a high CRP level. In this study, the risk was increased by an increasing number of persistently elevated risk factors, being highest when all the three factors representing chronic C. pneumoniae infection, inflammation and autoimmunity to human Hsp60 were elevated. Interestingly, only one participant with all these three markers present was found among the 138 controls compared to 17 among the same number of cases. Thus, sensitivity was 12.3% and specificity over 99%.
Our findings are in agreement with the findings of Kiechl et al. (2001), which showed that antibodies to mycobacterial Hsp65 predicted carotid atherosclerosis, and the association between chronic (respiratory, urinary tract, dental and other) infections and the risk of atherosclerosis increased clearly in the presence of an elevated ( > 1 mg/l) CRP level. Another study by Gattone et al. (2001) showed that seropositivity to both C. pneumoniae and cytomegalovirus infections was associated with increased CRP and a risk of MI. Indeed, atherosclerosis is nowadays accepted as an inflammatory disease (Ross 1999), and several prospective studies have shown CRP to be a strong independent predictor of future coronary events (Rifai & Ridker 2001, Ridker et al. 2002). Given that the coronary risk associated with elevated C. pneumoniae IC/IgA and human Hsp60 IgA antibody levels increased in the presence of an elevated CRP level, the risk associated with elevated CRP also increased clearly when elevated antibody levels were involved.
We found no multiplicative joint effect between human Hsp60 antibody levels and herpes simplex type 1 virus antibody levels, which, in the study of Roivainen et al. (2000), were a significant risk factor for coronary events (data not shown). This suggests that a Hsp60 response is essential for C. pneumoniae infection in the process leading to CHD, whereas in viral infections, an elevated Hsp60 antibody level is a coexisting phenomenon. This is in agreement with the study of Mayr et al. (2000), in which IgA antibodies to C. pneumoniae were associated with carotid and femoral atherosclerosis and correlated with antibodies to mycobacterial Hsp65, whereas no association was found between antibodies to cytomegalovirus and to Hsp65. Our results showed that the associations between C. pneumoniae antibodies, human Hsp60 antibodies and CRP were strong, indicating that these factors might contribute to the process leading to coronary events.
We also studied the possible modifying effect of smoking on the risk associated with elevated levels of human Hsp60 IgA antibodies. The joint effect was not negligible, but no synergistic effect was seen. Thus, our results indicate that these two factors may, at least partly, act independently. Similar finding were reported by Xu et al. (1999) and Zhu et al. (2001b). C. pneumoniae infection is more common in smokers than in nonsmokers, suggesting that smoking may predispose to the development of chronic C. pneumoniae infection (Hahn & Golubjatnikov 1992, Karvonen et al. 1994, Laurila et al. 1997b, Mayr et al. 2000). In identical twins, C. pneumoniae -specific IgA levels have been shown to be higher and cell-mediated immunity (measured as a lymphoproliferative response) lower in the smoking twins than in their nonsmoking co-twins, indicating that chronic C. pneumoniae infections in smokers are linked to lowered cell-mediated protective immunity (von Hertzen et al. 1998). In the Helsinki Heart Study, smoking significantly increased the risk associated with markers of chronic C. pneumoniae infection, and its effect with high CRP level was more than multiplicative, showing a high degree of synergy (Roivainen et al. 2000).
In this study, we found no risk to be associated with any of the IgG antibodies, suggesting that the presence of IgG antibodies is not a good marker for either chronic C. pneumoniae infection or autoimmunity to human Hsp60 protein in prediciting coronary risk. In the studies of Burian et al. (2001) and Zhu et al. (2001b), IgG antibodies to human Hsp60 were associated with the presence of CAD, whereas IgA antibodies were not measured. In the study of Mahdi et al. (2002), too, where it was found that antibodies to chlamydial Hsp60 but not to human and bacterial homologs were associated with CAD, only IgG antibodies were measured. In the study of Xu et al. (1999), which showed that mycobacterial Hsp65 antibody titres predicted 5-year mortality, only total antibodies (all Ig classes together) to Hsp65 were measured, and it is thus not known if IgA antibodies, rather than IgG antibodies, were an important risk factor in that study, too. The serological association between C. pneumoniae antibodies and atherosclerosis has recently been disputed, mostly due to studies where the lack of association was observed when studying only IgG antibodies from baseline samples (reviewed by Danesh et al. 2000). Several studies support our finding that an elevated level of IgA antibodies seems to be a better marker for chronic C. pneumoniae infection and to be more strongly associated with CHD and an increased risk of future coronary events (Saikku et al. 1992, Miettinen et al. 1996, Mayr et al. 2000, Kiechl et al. 2001, Danesh et al. 2002). The presence of IgA antibodies in serum samples collected a few years apart is an even better marker for chronic infection than the presence of IgA antibodies in a single serum sample (Saikku et al. 1992, Laurila et al. 1997a, Laurila et al. 1997b). However, the testing of paired sera at such a long interval may not be feasible in clinical settings.
If chronic C. pneumoniae infection is involved in atherogenesis by, for example, inducing inflammation and the development of autoimmunity to Hsp60, as suggested by this study, it would be possible to modify the disease process by antibiotic treatment. Unfortunately, C. pneumoniae antibodies are very common in middle-aged and elderly populations, and it is thus impossible to treat all antibody-positive persons by way of primary prevention. The results of this study suggest that a combination of elevated C. pneumoniae IgA antibodies and slightly elevated CRP values and/or elevated IgA antibodies to human Hsp60 would be helpful when screening persons who are at a high risk for a coronary event and would possibly benefit from prophylactic treatment.