Chlamydia pneumoniae antigens, rather than viable bacteria, persist in atherosclerotic lesions

Aims-To evaluate the nature of the presence of Chlamydia pneumoniae or of o ther members of the order Chlamydiales in atherosclerotic lesions. Methods-Consecutive sections of 13 carotid artery specimens obtained at nec ropsy and of C pneumoniae infected HEp2 cells were analysed using: (1) immu nocytochemistry (ICC) to detect C pneumoniae membrane protein; (2) in situ hybridisation (ISH) using a polymerase chain reaction (PCR) fragment of the omp1 gene to detect C pneumoniae specific DNA; (3) ISH using an oligonucle otide probe to detect Chlamydiales specific 16S rRNA; (4) PCR to detect C p neumoniae 16S rDNA; and (5) in situ DNA nick end labelling (TUNEL) to detec t fragmented DNA. Results-Staining by ICC and ISH of infected HEp2 cells showed characteristi c inclusions. Chlamydia pneumoniae membrane protein was demonstrated in mac rophages in advanced atherosclerotic lesions (six of six), but not in fatty streaks (none of two), or normal arteries (none of five). ISH assays using both probes and PCR were all negative, indicating the absence of both spec ific C pneumoniae DNA and Chlamydiales specific 16S rRNA. Only after treatm ent with DNAse I were uniformly sized dots demonstrated by the TUNEL assay in inclusions of infected HEp2 cells. The TUNEL assay showed a similar stai ning pattern in macrophages in five carotid artery specimens, of which four were also positive for C pneumoniae membrane protein. Both macrophage popu lations were morphologically similar and were similarly distributed. Conclusions-No evidence was obtained for the involvement of other members o f the order Chlamydiales in atherosclerosis. The presence of C pneumoniae a ntigen in the absence of DNA and 16S rRNA suggests that antigens, rather th an viable bacteria, persist in atherosclerotic lesions.