Background—Recent evidence has implicated Chlamydia pneumoniae in the aggravation of atherosclerosis. However, the mechanisms by which this agent affects atherogenesis remain poorly understood. Chlamydiae produce large amounts of heat shock protein 60 (HSP 60) during chronic, persistent infections, and C pneumoniae localizes predominantly within plaque macrophages. Several studies have furnished evidence that endogenous (human) HSP 60 may play a role in atherogenesis. We tested here the hypothesis that atheroma contains chlamydial HSP 60 and that this bacterial product might stimulate macrophage functions considered relevant to atherosclerosis and its complications, such as production of proinflammatory cytokines as tissue necrosis factor-α (TNF-α) and matrix-degrading metalloproteinases (MMPs).

Methods and Results—Surgical specimens of human carotid atherosclerotic arteries (n=19) and normal arterial wall samples (n=7, 2 carotid arteries and 5 aortas) were tested immunohistochemically for the presence of chlamydial HSP 60 and human HSP 60. Macrophage localization of these antigens was assessed by double immunostaining. Murine peritoneal macrophages, maintained in serum-free conditions for 48 hours after harvesting, were incubated with C pneumoniae, chlamydial HSP 60, human HSP 60, or Escherichia coli lipopolysaccharide (LPS). Culture supernatants, collected at 24 hours for concentration-dependence experiments and at up to 72 hours for time-dependence experiments, were analyzed for TNF-α by ELISA and for MMP by gelatin zymography. Atherosclerotic lesions showed immunoreactive chlamydial HSP 60 in 47% (9 of 19) of the cases and human HSP 60 in 89% (17 of 19) of the cases. Chlamydial HSP 60 colocalized with human HSP 60 within plaque macrophages in 77% (7 of 9) of the cases. Nonatherosclerotic samples contained neither HSP. Both C pneumoniae and recombinant chlamydial HSP 60 induced TNF-α production by mouse macrophages in a concentration- and time-dependent fashion. E coli LPS and human HSP 60 produced similar effects. Similarly, C pneumoniae and HSPs induced MMPs in a concentration- and time-dependent manner. Heat treatment abolished the effect of C pneumoniae and HSPs on both TNF-α and MMP production, but it did not alter the ability of E coli LPS to induce these functions.

Conclusions—Chlamydial HSP 60 frequently colocalizes with human HSP 60 in plaque macrophages in human atherosclerotic lesions. Chlamydial and human HSP 60 induce TNF-α and MMP production by macrophages. Chlamydial HSP 60 might mediate the induction of these effects by C pneumoniae. Induction of such macrophage functions provides potential mechanisms by which chlamydial infections may promote atherogenesis and precipitate acute ischemic events.