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. 2000 Dec;53(12):911–916. doi: 10.1136/jcp.53.12.911

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

A Meijer 1, P Roholl 1, S Gielis-Proper 1, J Ossewaarde 1
PMCID: PMC1731134  PMID: 11265175

Abstract

Aims—To evaluate the nature of the presence of Chlamydia pneumoniae or of other members of the order Chlamydiales in atherosclerotic lesions.

Methods—Consecutive sections of 13 carotid artery specimens obtained at necropsy and of C pneumoniae infected HEp2 cells were analysed using: (1) immunocytochemistry (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 oligonucleotide probe to detect Chlamydiales specific 16S rRNA; (4) PCR to detect C pneumoniae 16S rDNA; and (5) in situ DNA nick end labelling (TUNEL) to detect fragmented DNA.

Results—Staining by ICC and ISH of infected HEp2 cells showed characteristic inclusions. Chlamydia pneumoniae membrane protein was demonstrated in macrophages 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 specific C pneumoniae DNA and Chlamydiales specific 16S rRNA. Only after treatment with DNAse I were uniformly sized dots demonstrated by the TUNEL assay in inclusions of infected HEp2 cells. The TUNEL assay showed a similar staining pattern in macrophages in five carotid artery specimens, of which four were also positive for C pneumoniae membrane protein. Both macrophage populations were morphologically similar and were similarly distributed.

Conclusions—No evidence was obtained for the involvement of other members of the order Chlamydiales in atherosclerosis. The presence of C pneumoniae antigen in the absence of DNA and 16S rRNA suggests that antigens, rather than viable bacteria, persist in atherosclerotic lesions.

Key Words: Chlamydia pneumoniae • Chlamydiales • immunocytochemistry • in situ hybridisation • polymerase chain reaction • DNA fragmentation • atherosclerosis

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Figure 1 Representative photomicrographs of an area with atherosclerotic changes in consecutive sections of the carotid artery specimen of case 11 (A–D, and F–H), and of HEp2 cells infected with Chlamydia pneumoniae (E). Several cells in this atherosclerotic area were immunoreactive for C pneumoniae membrane protein (A; some positive cells indicated with an arrow). These cells are part of the population of cells that immunostained for macrophage marker CD68 (B). In situ hybridisation reactivity for eukaryotic 28S rRNA (C) demonstrated the suitability of the specimen for hybridisation experiments. However, C pneumoniae DNA was not detected by in situ hybridisation (D). Fragmented DNA of C pneumoniae in a section of HEp2 cells infected with C pneumoniae pretreated with DNAse I and then processed in the in situ DNA nick end labelling (TUNEL) assay was visible as uniformly sized dots in clearly recognisable inclusions (E; arrows) next to TUNEL stained nuclei of the HEp2 cells. Uniformly sized dots stained using the TUNEL assay were also found in the carotid artery specimen (F and G; arrows) in a cell type in which C pneumoniae membrane protein was also observed. An enlargement of the area enclosed by the arrowheads in (F) is shown in (G). Note that cells similar in morphology to TUNEL reactive cells were also not reactive in the TUNEL assay (G; arrowheads). These vacuolated cells are part of the population of cells that immunostained for macrophage marker CD68 (B). No staining was observed without transferase in the TUNEL reaction mixture (H). Counterstaining, panels A–D, nuclear fast red; panels E–H, Mayer's haematoxylin. Original magnifications, x31 (A, B, and F), x62 (C), x125 (D, G, and H), and x250 (E).

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