Antibody Response to Chlamydial Heat Shock Protein 60 Is Strongly Associated With Acute Coronary Syndromes
Background— Heat shock proteins (HSPs) are a family of proteins with immunogenic and proinflammatory properties. Human and Chlamydia pneumoniae (Cp) HSP60 were found in patients with stable coronary disease.
Methods and Results— We measured the levels of anti–Cp-HSP60 and anti-Cp immunoglobulin G (IgG) in 179 patients with unstable angina, 40 with acute myocardial infarction, and 40 with stable angina (SA), as well as 100 control subjects. Forty-one patients with acute coronary syndromes (ACS) were also studied at follow-up. We also measured plasma levels of high-sensitivity C-reactive protein (hs-CRP) and troponin T (TnT). Seropositivity to Cp-HSP60 was found in 99% of ACS patients but in only 20% of SA patients and none of the control subjects. Seropositivity to Cp was detected in 67% of ACS patients, 60% of SA patients, and 30% of the control subjects. No differences in Cp-HSP60 IgG and in Cp IgG were observed between patients with myocardial infarction and patients with unstable angina. No correlation was found between Cp-HSP60 IgG, TnT, and hs-CRP or between IgG against Cp and hs-CRP. In ACS patients at follow-up, Cp-HSP60 IgG decreased from 0.88±0.25 to 0.45±0.14 arbitrary units (P<0.0001), becoming negative in 12 patients.
Conclusions— Seropositivity for Cp-HSP60 appears to be a very sensitive and specific marker of ACS, unrelated to Cp IgG antibody titers or hs-CRP and TnT levels. Its causal involvement in instability and its diagnostic role in ACS deserve further study.
Original received November 6, 2002; de novo received March 19, 2003; revision received May 6, 2003; accepted May 7, 2003.
Heat shock proteins (HSPs), a family of chaperone proteins with strong antigenic properties, may represent a plausible link between infection, inflammation, and acute coronary syndromes (ACS). Antibodies to mycobacterial HSP 65 correlate with carotid thickening,1 and human (hu) HSP60 was reported to be associated with ischemic heart disease.2–5 The HSP60 produced by Chlamydia pneumoniae (Cp-HSP60) is of particular interest because of its proinflammatory and immunogenic properties.6,7 In a recent methodological study, we have observed a strikingly high prevalence of elevated Cp-HSP60 immunoglobulin G (IgG) antibody titers in patients with ACS compared with zero prevalence in control subjects.8 Therefore, we investigated whether this high prevalence of Cp-HSP60 IgG antibody titers was related to the presence of coronary artery disease, myocardial necrosis, and levels of high-sensitivity C-reactive protein (hs-CRP) and whether it decreases during follow-up, which would suggest a specific link with the acute phase of instability.
We studied 219 patients admitted to the Coronary Care Unit: 179 with unstable angina (UA) Braunwald class IIIB and 40 with acute myocardial infarction (AMI). The clinical characteristics of the patients are reported in the Table. We also studied 40 patients with stable angina (SA), who had been free from angina for at least 2 weeks, and 100 control subjects, who were matched for age, sex, and risk factors. To investigate the time course of Cp-HSP60 and Cp IgG levels in ACS, we reassessed these parameters in 41 patients at an average of 350±260 days’ follow-up. Cp-HSP60 IgG was measured by an in-house ELISA, as described elsewhere.8 All sera were diluted 1:50, and seropositivity was defined as an ELISA reading of >0.40, corresponding to twice the maximum value of antigen-only–coated wells. This value also corresponds to the upper limit of the mean±2SD of the control subjects. Anti-Cp IgG antibody levels were measured by a commercially available microimmunofluorescence assay (Labsystems). IgG titers ≥1:32 were regarded as positive. hs-CRP was measured by latex-nephelometry (Dade-Behring) and troponin T (TnT) by Elecsis (Roche).
Percentages were analyzed by χ2 test. Because anti-Cp IgG levels were not normally distributed but IgG Cp-HSP60 levels were normally distributed, the Spearman rank correlation or the linear correlation and the Wilcoxon test or the paired t test were used, as appropriate. A 2-tailed probability value <0.05 was considered significant.
Seropositivity to Cp-HSP60 was detected in 217 (99%) of 219 patients with ACS but in only 8 (20%) of 40 patients with SA and in none of the control subjects (P=0.0001, ACS patients versus control subjects; P=0.003, ACS patients versus SA patients; and P=0.05, SA patients versus control subjects). Seropositivity to Cp was detected in 67%, 60%, and 30% of ACS patients, SA patients, and control subjects, respectively (P=0.001, ACS and SA patients versus control subjects). Elevated hs-CRP was found in 60% of ACS patients versus 25% of SA patients and 8% of control subjects (P<0.001, ACS patients versus SA patients and control subjects). Considering the cutoff level of 0.40, Cp-HSP60 IgG antibody levels had 99% specificity and 94% sensitivity for ACS. No differences in anti–Cp-HSP60 or in anti-Cp IgG antibody titers and prevalence of seropositivity were observed between AMI and UA patients (Table) or, in patients with UA, between those with (n=65) and those without (n=114) raised TnT levels. Anti–Cp-HSP60 and anti-Cp antibody titers correlated neither with each other nor with hs-CRP levels. In the 41 patients with ACS, from whom a second sample was obtained at a mean follow-up of 350±260 days, anti–Cp-HSP60 IgG antibody titers decreased from 0.88±0.25 to 0.45±0.14 (P<0.0001), becoming seronegative in 12 patients (P=0.002). In contrast, anti-Cp antibody titers decreased by only one dilution (from 128 [range 0 to 512] to 64 [range 0 to 512]) and no Cp-seropositive patient became seronegative. These data were confirmed by the determination of IgG antibody titers against the outer membrane protein 2 of Chlamydia pneumoniae.8
Our study demonstrates that anti–Cp-HSP60 IgG antibody titers are above the detection threshold in 99% of patients with ACS but in only 20% of SA patients and in none of the control subjects. In addition, they become weakly positive or negative after about 1 year from the acute event and therefore appear to be a strikingly sensitive and specific marker of ACS. Anti–Cp-HSP60 IgG clearly diverged from anti-Cp IgG, suggesting that they probably reflect an immune response not directly related to Chlamydia pneumoniae infection, possibly involving the patient’s host immune response. As described in the previous methodological study,8 recombinant proteins (in particular HSP60) of Chlamydia pneumoniae were generated to assess antibody responses in subjects with ACS. In the present study, we specifically addressed the question of whether elevated anti–Cp-HSP60 IgG is a specific finding of ACS or more generically a marker of coronary artery disease.
Several authors have assessed the relationship between hu- or Cp-HSP and atherosclerotic disease; however, patients with ACS seldom have been considered,1,3,4,8 and different antigenic preparations of HSP60 were used. We used a nondenatured protein capable of finely detecting antibodies against the native Cp-HSP60, compatible with a good conformational epitope preservation,4 and our data, consistent with previous reports, do not support a direct association between anti-HSP60 response and infection.3,5,6
Potential Mechanisms Involved in the Response to Cp-HSP60
The high degree of sequence similarity between Cp- and hu-HSP60 suggests the possibility that antibodies formally measured against a preparation of Cp-HSP60 were in fact generated against hu-HSP60, which is compatible with the lack of correlation between Cp-HSP60 and Cp seropositivity. Priming with Cp-HSP60 may lead in some patients to anti-self immune response and raised titers of hu-HSP60 antibodies.3,9,10 Alternatively, the seropositivity to Cp-HSP60 may result from an anti-self immune response related to release of hu-HSP60 caused by cellular stress.3 The progressive reduction seen in Cp-HSP60 IgG levels and the very low levels found in SA patients argue for a mechanism tightly linked to ACS. However, no differences were observed in Cp-HSP60 IgG antibody titers levels between UA and AMI patients or between TnT-positive and TnT-negative UA patients, suggesting that the process is not likely to be related to myocardial necrosis. Evidence of colocalization of hu-HSP60 and Cp-HSP60 in human atheromatous plaques11,12 suggests the possible production of these proteins at the plaque level, where they can regulate tumor necrosis factor-α and matrix metalloproteinase expression and activate endothelium and smooth muscle cells. Independently of its origin, Cp-HSP60 may exacerbate the inflammatory cascade by stimulating innate immunity.13 The absence of correlation between Cp-HSP60 with hs-CRP in our study is consistent with the possibility that anti–Cp-HSP60 antibodies may develop as a result of an anti-self immune response and may be explained by the variable increase in CRP resulting from such a process.14
Antibodies against Cp-HSP60 appear to be a strikingly sensitive and specific marker of ACS. Their levels are unrelated to anti-chlamydial antibodies, hs-CRP, or TnT levels, and markedly decrease after the unstable phase. The anti–Cp-HSP60 antibody response might be caused or enhanced by an anti-self response related to antigenic mimicry between Cp- and hu-HSP60. This immune component of ACS may account for the widespread coronary inflammation reported in UA.15 The pathogenetic role of Cp-HSP60 in ACS remains to be elucidated, and its possible significance as a diagnostic marker of instability awaits confirmation in larger studies.
This study was supported by Instituto Superiore di Sanitá Special Project “Role of Infectious Agents in Cardio and Cerebrovascular Diseases,” Fondazione Internazionale Ricerche Per il Cuore, onlus,” Rome, Italy, and by grant 2001067817_008 of the Italian Departement of Research (MIUR).
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