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Growth in Vascular Cells and Cytokine Production by Chlamydia pneumoniae Charlotte A. Gaydos
Division of Infectious Diseases, Johns Hopkins University, Baltimore, Maryland
The proposed pathogenesis of Chlamydia pneumoniae in atherosclerosis is supported by the finding that C. pneumoniae can initiate and sustain growth in human vascular cells. In vitro growth of C. pneumoniae is found in macrophages, peripheral blood monocyte (PBMC)–derived macrophages, endothelial cells, and aortic artery smooth muscle cells. U937 macrophages infected with C. pneumoniae are capable of transmitting the infection to human coronary artery endothelial cells (CAEC) with direct cellular contact. Production of cytokines by cells infected with C. pneumoniae indicates that the organism can stimulate the immune system. CAEC infected with C. pneumoniae produce more interleukin-8 than cells sham inoculated with negative control cells. When interferon-g is used to stimulate HEp-2 cells, U-937 cells, and PBMC (before infection with C. pneumoniae), inhibition of a productive growth cycle occurs in a dose-related response. Studies are needed to learn the relationship between productive infection and persistence, the ability of C. pneumoniae to affect the immune response, and the potential for C. pneumoniae to influence atheromatous lesions.
The pathogenesis of atherosclerosis involves an immunologically mediated inflammatory response by the host [1, 2]. The site of intimal rupture of coronary atherosclerotic plaques is always marked by inflammation. This may play a role in destabilizing fibrous tissue and enhancing the risk of coronary thrombosis [3]. The proposed pathogenesis of Chlamydia pneumoniae with coronary heart disease has been suggested by seroepidemiology and detection of the organism in atheromas by culture, polymerase chain reaction (PCR), immunohistochemistry, and in situ hybridization [4–10]. The presence of C. pneumoniae in macrophages and within the intima of atherosclerotic plaques [9–11] supports an association of an infectious process in the initiation of atherosclerosis or in plaque instability. Animal models demonstrate that after respiratory inoculation, C. pneumoniae is capable of inducing the formation of fatty streaks and atheromas [12–18]. Persistence of C. pneumoniae in the host may contribute to continual immune stimulation. The ability of the organism to persist in a primate model for up to a year with three respiratory inoculations has been documented [19]. Because C. pneumoniae appears to have a specificity for cardiovascular tissue and is detected infrequently in granulomatous tissue and noncardiovascular tissues, it appears not to be an innocent bystander [20]. Researchers have demonstrated the ability of C. pneumoniae to grow in human vascular cells and to stimulate these cells to produce cytokines, chemokines, and adhesion molecules [21–25].
Reprints or correspondence: Dr. Charlotte A. Gaydos, 1159 Ross Research Bldg., 720 Rutland Ave., Johns Hopkins University, Baltimore, MD 21205 (
[email protected]). The Journal of Infectious Diseases 2000; 181(Suppl 3):S473–8 q 2000 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2000/18106S-0021$02.00
Infection of Vascular Cells C. pneumoniae can grow in vascular cells of various types and can be passaged in them [21]. The in vitro growth of C. pneumoniae has been studied in macrophages, endothelial cells, and smooth muscle cells during investigation of its ability to directly infect, disseminate, and proliferate in atherosclerotic lesions and aortic artery smooth muscle cells. Five C. pneumoniae strains were capable of at least three of three passage attempts in human U-937 macrophages and in murine RAW 246.7 macrophages. Growth titers were suppressed in both macrophage cell lines with each passage as compared with growth in HEp-2 cells [21]. Both human bronchoalveolar lavage macrophages and peripheral blood monocyte (PBMC)–derived macrophages could inhibit C. pneumoniae after incubation for 96 h compared with growth in permissive HEp-2 cells [21]. Growth of 11 C. pneumoniae strains was also supported by several endothelial cell lines (human umbilical vein cells, pulmonary artery endothelial cells, and aortic artery endothelial cells). Infection in human aortic artery smooth muscle cells was also established for 13 strains of C. pneumoniae [21]. The in vitro ability of C. pneumoniae to infect human macrophages, vascular endothelial cells, and aortic smooth muscle cells in vitro provides support for the hypothesis that C. pneumoniae can infect such cells in vivo.
Transmission of Infection by Macrophages Infected with C. pneumoniae to Coronary Artery Endothelial Cells (CAEC) In order to determine whether C. pneumoniae can grow in human CAEC in addition to endothelial cells from the aorta, cadaver-derived cells (Clonetics, San Diego) were inoculated with a vascular isolate (A-03) [26] of C. pneumoniae. The results
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given reflect the average of titers counted from three different titer wells after being stained with fluorescein-conjugated antilipopolysaccharide (LPS) monoclonal antibody (MAb; Kallestad, Chaska, MN). The isolate could infect and replicate in CAEC with or without artificial laboratory culture enhancements (e.g., centrifugation or use of cycloheximide). Although infectious titer counts were higher when cycloheximide was used (8.2 3 10 6 inclusion-forming units [ifu]/mL vs. 3.0 3 10 5 ifu/ mL), the organism could infect and grow without the usual laboratory enhancement of the addition of cycloheximide [25]. Figure 1 shows the growth of C. pneumoniae inclusions in CAEC. Also studied was whether U-937 macrophages that were infected with C. pneumoniae were capable of transmitting the infection to endothelial cells when they were brought into contact with the CAEC in culture [25]. U-937 human macrophages infected with three different doses (105, 106, and 107 ifu/mL) of C. pneumoniae were brought into contact with CAEC in three ways: centrifugation, rocking overnight, and direct layering overnight. All three methods of cell-to-cell contact between macrophages and CAEC produced infection in CAEC resulting in inclusions of C. pneumoniae when infected U-937 macro-
Figure 1.
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phages were used as the infecting dose. The counts are mean of three replicate wells. The most adequate dose for infecting the U-937 cell for use in transmission to the CAEC was 106 ifu/mL, resulting in average counts of 1, 21, and 19 inclusions per well for cell-to-cell contact by centrifugation, rocking, and direct layering, respectively [25]. Figure 2A and 2B show two different representative fields with chlamydial inclusions in CAEC stained with fluorescein-conjugated anti-LPS MAb (Kallestad) after infection with infected macrophages. Quality control staining with phycoerythrin-conjugated anti-CD14 (Becton Dickinson, Sparks, MD) demonstrated that there were also adherent macrophages on the CAEC cell layers. Macrophages Infected with C. pneumoniae Are Stimulated to Produce Cytokines C. pneumoniae can induce various cytokine responses when macrophages are infected with the organism in vitro. This demonstrates a potential role for infection to stimulate an immune response in vivo [27]. It has been postulated that alveolar macrophages play an important role in the initial pulmonary infectious process with C. pneumoniae. By becoming vascular
C. pneumoniae strain A-03 growing in human coronary artery endothelial cells. Inoculum was 105 inclusion-forming units/mL.
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Figure 2. C. pneumoniae strain A-03. A and B, Separate experiments with strain growing in human coronary artery endothelial cells infected by U-937 macrophages at 106 inclusion-forming units (ifu)/mL. C, Stain growing in peripheral blood mononuclear cell–derived macrophages. Inoculum was 104 ifu/mL.
monocytes, these cells may play a critical role in dissemination of the organism. Animal model studies support this hypothesis since C. pneumoniae has been recovered from circulating PBMC [12, 19, 28–30]. To study the cytokine response of U-937 cells, this macrophage cell line was infected in vitro with C. pneumoniae, strain 2023. U-937 cells (4 mL) were infected with 1 mL of 3.5 3 104 ifu/mL C. pneumoniae by gentle agitation every 20 min for 6 h before the addition of 10 mL of growth media without cycloheximide [25]. Flasks were sampled by removing and freezing 1 mL of the infected cells at 24, 48, and 96 h and at 7 days. A control flask was treated in an identical manner, except that uninfected growth medium was used in place of the inoculum.
Growth of C. pneumoniae in U-937 was monitored for each sample time point from the infected flask of U-937 cells by performing growth titers in triplicate in HEp-2 cells. Levels of cytokines were determined in triplicate from the samples from both the infected and noninfected flasks for interleukin (IL)1b, interferon (IFN)-g, and tumor necrosis factor (TNF)-a. The assays were performed by sandwich EIA (Endogen, Woburn, MA; R&D Systems, Minneapolis). The results of the viability or growth of C. pneumoniae in the cells yielded titers ranging from 103 ifu/mL at 24 h to 104 ifu/mL at 72 h; however, at 7 days there were no viable organisms [25]. Cytokine levels (pg/mL) for IL-1b were similar for infected and uninfected cells for up to 72 h, but by 96 h
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and 7 days, levels for the infected cells were 5.3–9.3 times greater than for uninfected cells. For IFN-g, results were similar up to 48 h, but at 72 and 96 h, levels were 15.2 and 48.1 times higher for the infected cells than for uninfected cells, respectively. Likewise, for TNF-a, levels remained similar for 48 h, but at 72 and 96 h and at 7 days, the respective levels were 1.9, 1.2, and 2.1 times higher than in uninfected cells [25]. These results indicate that C. pneumoniae infection in U-937 macrophages stimulates increased production of IL-1b, IFNg, and TNF-a. Mechanisms of action of these cytokines may include, but are not limited to, induction of adhesion molecule expression on endothelial and epithelial cells (IL-1b); enhancement of intracellular killing, TNF-a cytotoxicity, and NK cell activity (IFN-g); and enhanced macrophage killing and cytotoxic T cell differentiation (TNF-a). Other investigators have also demonstrated that C. pneumoniae increases production of various inflammatory cytokines [31]. Further study will enhance our understanding of the role of these and other cytokines in the pathogenesis of vascular infections with C. pneumoniae infection.
IL-8 Response of Human CAEC Infected with C. pneumoniae C. pneumoniae A-03 can elicit an IL-8 response in cultured CAEC [25, 32]. IL-8 levels were determined in triplicate at various times from culture supernatants (BioSource, Camarillo, CA). More IL-8 was produced by cells that were inoculated with C. pneumoniae A-03 (inoculum of 2.1 3 104/mL) than by sham-inoculated cells at 24, 48, and 72 h. Levels peaked at 72 h for cells infected with viable C. pneumoniae compared with sham-inoculated negative control cells (928.5 vs. 604.6 pg/mL, respectively; figure 3). Others have also reported that C. pneumoniae can stimulate umbilical vein endothelial cells to produce
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increased levels of IL-8 [33]. Thus, infected endothelial cells can be stimulated to release chemokines in response to infection, and these molecules may play a role in the recruitment of inflammatory cells to the site of inflammation.
IFN-g–Restricted Growth of C. pneumoniae in Cells We previously reported on the role of IFN in restricting the growth of C. pneumoniae in HEp-2 cells and U-937 macrophages [25]. Persistent infection in macrophages could mediate the transport of C. pneumoniae to vascular lesions. IFN-g restricts the growth of other chlamydiae (e.g., C. trachomatis) [34, 35]. The role of IFN-g on intracellular growth of C. pneumoniae–infected PBMC-derived macrophages was investigated. Figure 2C shows C. pneumoniae A-03 growing in human PBMC without any additives (e.g., IFN-g) with an inoculum of 2.1 3 10 4/mL. Human recombinant IFN-g was added to PBMC in culture in replicate concentrations of 0.05–10 ng/mL (1.25–250 U/mL). IFN-g was added 24 h before inoculation with C. pneumoniae. After 24, 48, 72, 96, and 120 h of incubation, cells from each IFN-g concentration were scraped, and titers of infectious progeny were determined in triplicate in HEp-2 cells. Decreased titers of infectious progeny were observed in a dose-response fashion for increasing amounts of IFN-g (figure 4). Prior studies showed that when IFN-g is removed by washing cell cultures infected with C. pneumoniae or with C. trachomatis and the cells are fed new growth media, the cells again can produce infectious progeny [25, 34]. These data may indicate that IFN-g can suppress the intracellular replication of C. pneumoniae in these cells and that this phenomenon may lead to persistently infected tissue cells or blood PBMC, although no in vivo data support this. There is evidence that C. pneumoniae may remain metabolically active in monocytes in vitro for up to 7 days by the production of a lympho-
Figure 3. Interleukin-8 levels from C. pneumoniae–infected coronary artery endothelial cells (CAEC) compared with mock-infected control CAEC over time. Inoculum was 2.1 3 104/mL. Lines with bars are mean levels of triplicate measurements 5 SD.
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Figure 4. Suppression of interferon (IFN)-g on productive infection of C. pneumoniae in peripheral blood mononuclear cell–derived macrophages. Inoculum was 2.1 3 104 ifu/mL. Cells were stimulated for 24 h by IFN-g before inoculation of C. pneumoniae. Viable progeny were counted in HEp-2 cells. Lines with bars are mean levels of triplicate measurements 5 SD.
proliferative response and up to 3 days by the production of mRNA, despite the decreased production of infectious progeny [36]. In addition, Boman et al. [11] found that PCR can show C. pneumoniae–infected PBMC in patients with coronary heart disease. These data demonstrate that chronic infection by C. pneumoniae in macrophage and nonmacrophage cell types may perpetuate an inflammatory response and could contribute to the inflammatory process that is the hallmark of atherosclerosis [37]. Kalayoglu and Byrne [38] demonstrated that C. pneumoniae induce foam cell formation by human monocyte–derived macrophages, with a marked increase in the number of foam cells and an accumulation of cholesterol esters upon exposure of the macrophages to the organism. They also showed that the C. pneumoniae component that induces macrophage foam cell formation is chlamydial LPS [39]. In summary, all of the in vitro studies of C. pneumoniae infection and data from animal models potentially support, but do not confirm, a biologic role for the involvement of this organism in the complex and multifactorial processes of the initiation and progression of atheromatous lesions. Further studies that encompass many scientific disciplines will better elucidate the role of infection in the complicated disease of atherosclerosis.
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