Chlamydia pneumoniae infection in human monocytes

Abstract

Chlamydia pneumoniae infection has been associated with cardiovascular diseases in seroepidemiological studies and by demonstration of the pathogen in atherosclerotic lesions. It has the capacity to infect several cell types, including monocyte-derived macrophages, which play an essential role in the development of atherosclerosis. However, the persistence of C. pneumoniae in mononuclear cells is poorly understood. To study the morphology and biological characteristics of the infection, human peripheral blood monocytes were infected with C. pneumoniae. Freshly isolated monocytes resisted the development of infectious progeny, and confocal and transmission electron microscopy showed that the morphology of the inclusions and chlamydial particles was abnormal. Addition of tryptophan or antibodies against gamma interferon did not diminish the inhibition of C. pneumoniae, suggesting that other factors are involved in the chlamydiostatic activity of the monocytes. Chlamydial mRNA was expressed at least 3 days after infection, however, and a capability for infected monocytes to induce a positive lymphocyte proliferative response was detected for up to 7 days, indicating that C. pneumoniae remains metabolically active in the monocytes in vitro. These results are in accordance with the hypothesis that C. pneumoniae may participate in the maintenance of local immunological response and inflammation via infected monocytes and thus enhance atherosclerosis.

FIG. 1

A confocal micrograph of C. pneumoniae-infected human monocytes stained with FITC-conjugated anti-Chlamydia antibody. The monocyte contains several inclusions that are variable in size and staining intensity.

FIG. 2

Transmission electron micrographs of C. pneumoniae-infected HL cells and monocytes 72 h postinfection. (a) Large inclusions filled with C. pneumoniae particles are found in the HL cells. Mature EBs are small, with an electrodense core (thick arrow), while RBs are large, with a coarse inner structure (thin arrow). (b) Monocytes can carry very small multiple inclusions (arrows), some containing only one C. pneumoniae particle. A thin membrane always covers these inclusions. (c) The inclusions in monocytes contain far fewer C. pneumoniae particles than do those in HL cells. Only a few mature-looking EBs can be seen (thick arrow), and the RBs are clearly transformed (thin arrows). Magnification, ×10,400.

FIG. 2

Transmission electron micrographs of C. pneumoniae-infected HL cells and monocytes 72 h postinfection. (a) Large inclusions filled with C. pneumoniae particles are found in the HL cells. Mature EBs are small, with an electrodense core (thick arrow), while RBs are large, with a coarse inner structure (thin arrow). (b) Monocytes can carry very small multiple inclusions (arrows), some containing only one C. pneumoniae particle. A thin membrane always covers these inclusions. (c) The inclusions in monocytes contain far fewer C. pneumoniae particles than do those in HL cells. Only a few mature-looking EBs can be seen (thick arrow), and the RBs are clearly transformed (thin arrows). Magnification, ×10,400.

FIG. 2

Transmission electron micrographs of C. pneumoniae-infected HL cells and monocytes 72 h postinfection. (a) Large inclusions filled with C. pneumoniae particles are found in the HL cells. Mature EBs are small, with an electrodense core (thick arrow), while RBs are large, with a coarse inner structure (thin arrow). (b) Monocytes can carry very small multiple inclusions (arrows), some containing only one C. pneumoniae particle. A thin membrane always covers these inclusions. (c) The inclusions in monocytes contain far fewer C. pneumoniae particles than do those in HL cells. Only a few mature-looking EBs can be seen (thick arrow), and the RBs are clearly transformed (thin arrows). Magnification, ×10,400.

FIG. 3

Viability of C. pneumoniae in monocytes, studied by RT-PCR. (A to C) The mRNA expression of gene transcripts for 16S rRNA (463 bp); (A) and for Crp60 (252 bp); (B) was positive up to 3 days after infection, and that for HSP60 (507 bp); (C) was positive up to 7 days after infection. (D) The chromosomal β-actin gene of monocytes was expressed at all the time points. IFN-γ treatment of the infected monocytes seemed not to be able to inhibit gene expression. The arrows indicate the amplification products. Lanes: 1 and 14, molecular weight markers; 2 to 5, infected monocytes; 6 to 9, infected, IFN-γ treated cells; 10 to 13, uninfected cells. For each set of lanes, the samples were obtained right after infection and on days 1, 3, and 7 after infection, respectively.

FIG. 4

Ability of C. pneumoniae-infected monocytes to induce lymphocyte activation. Purified T cells were added to the infected monocyte cultures at different time points and incubated for 6 days, and lymphocyte proliferation responses were measured. Clear specific T-cell activation is seen up to 7 days. Values represent means and standard deviations of triplicate measurements. Cpn 1, C. pneumoniae (8 × 10³ IFU/well); Cpn 2, C. pneumoniae (8 × 10² IFU/well). PPD, purified protein derivative.