Chlamydia pneumoniae infects and multiplies in lymphocytes in vitro

Abstract

The obligate intracellular pathogen Chlamydia (Chlamydophila) pneumoniae is known to be associated with some chronic inflammatory diseases, such as atherosclerosis. Interaction between C. pneumoniae and immune cells is important in the development of such diseases. However, susceptibility of immune cells, particularly lymphocytes, to C. pneumoniae infection has not been reported, even though lymphocytes play a pivotal role in the development of the diseases caused by this bacterium. In this regard, we examined the susceptibility of lymphocytes to C. pneumoniae infection in vitro. The results demonstrated that human peripheral blood lymphocytes as well as mouse spleen lymphocytes could be infected with C. pneumoniae. Furthermore, purified T lymphocytes as well as established T-lymphocyte cell line cells showed an obvious susceptibility to C. pneumoniae infection, indicating that T cells could be one of the host cells for this bacterial infection. These findings reveal a new infection site for C. pneumoniae, i.e., lymphocytes.

FIG. 1

Fluorescence stain micrographs of C. pneumoniae-infected lymphocytes. (A and B) Human peripheral blood lymphocytes were infected with C. pneumoniae by gentle shaking and then incubated for 3 days at 37°C in 5% CO₂. The infected cells were fixed on a glass slide by Cytospin and stained with FITC-conjugated anti-Chlamydia monoclonal antibody. Chlamydia inclusion bodies (arrow) were demonstrated in lymphocytes. Magnification, ×1,000. (C and D) Immunofluoresence micrographs of C. pneumoniae-infected mouse spleen lymphocytes. The mouse spleen lymphocytes were infected with C. pneumoniae by gentle shaking, incubated for 3 days, and then stained with PE-conjugated anti-mouse IgG CD3 antibody and FITC-conjugated anti-Chlamydia antibody after treatment with the permeabilization kit. Red color indicates surface molecules of lymphocytes. Green to yellow color indicates C. pneumoniae. Magnification, ×1,000. (E and F) Fluorescence micrographs of mouse T-cell-enriched lymphocyte cultures infected with C. pneumoniae. The purity of T cells in the cultures was more than 95% as determined by FACS analysis with anti-CD3 antibody. The T-cell cultures showed a few chlamydiae at 0 h (E). At 3 days after infection, there were many Chlamydia inclusion bodies (yellow spots) stained with FITC-conjugated anti-Chlamydia antibody in the culture (F). Magnification, ×200.

FIG. 2

Transmission electron micrographs of C. pneumoniae-infected human lymphocytes. The analysis of C. pneumoniae-infected human lymphocytes (A and B, time zero after infection; C and D, 3 days after infection) by electron microscopy showed the attachment of chlamydiae to the surface of a lymphocyte (A), internalization of chlamydiae in a lymphocyte (B), and many Chlamydia particles of various sizes in a cell (C and D). Arrows indicate Chlamydia particles.

FIG. 3

Relative number of Chlamydia organisms determined by ELISA in lymphocyte cultures. The amount of Chlamydia LPS antigen in mouse lymphocyte cultures infected with C. pneumoniae was measured by ELISA and converted to a relative number of Chlamydia organisms from the standard curve. The lymphocytes (10⁶ cells) at time zero and 72 h after infection were collected, and Chlamydia LPS in lymphocytes was extracted (1.0 ml). The amount of LPS in extracts (200 μl) was measured by ELISA. The data represent means ± standard deviation (SD) for three experiments. ∗, P < 0.05 compared to time zero culture, analyzed by Student's t test.

FIG. 4

C. pneumoniae DNA in lymphocyte cultures. The amount of C. pneumoniae DNA in mouse lymphocyte cultures was semiquantified by Amplifluor-PCR with primers specific for C. pneumoniae omp1. DNA (50 μl) was isolated from the infected lymphocytes (10⁶ cells) at the indicated time points, and 2 μl of DNA extracts was subjected to PCR. The data presented are representative of three experiments.

FIG. 5

Fluorescence micrographs and relative number of Chlamydia organisms in T-lymphocyte cell line Molt 3 cultures infected with C. pneumoniae. See the legend to Fig. 3 for details. The cultures (10⁶ cells) at time zero and 72 h after infection were collected and stained with FITC-conjugated anti-Chlamydia antibody, or Chlamydia LPS in cultures was extracted (1.0 ml). Fluorescence micrographs show the culture at time zero and 72 h after infection with viable C. pneumoniae. Relative number of Chlamydia organisms was calculated from the LPS concentrations determined by ELISA. The data shown as the relative bacterial number represent means ± SD for three experiments. Open bars, infected with viable bacteria; solid bars, infected with UV-killed bacteria. ∗, P < 0.05 compared to time zero culture, analyzed by Student's t test. Magnification, ×1,000.