ABSTRACT
Strong epidemiological and pathological evidence supports a role for Chlamydia pneumoniae infection in atherosclerosis and human coronary heart disease. Animal models have shown that C. pneumoniae disseminates hematogenously in infected monocytes and macrophages, while in vitro data suggest that infected macrophages can transmit C. pneumoniae infection directly to endothelial cells. Endothelial cells may be key in vivo targets for C. pneumoniae infection; given that these cells are important in regulating the dynamics of the vessel wall, we used cDNA microarrays to study the transcriptional response of endothelial cells to infection with C. pneumoniae. cDNA arrays were used to characterize the mRNA expression profiles for 268 human genes following infection with C. pneumoniae, which were compared to mRNA profiles of uninfected cells. Selected genes of interest were further investigated by reverse transcription-PCR throughout a 24-h period of infection. C. pneumoniaeinfection upregulated mRNA expression for approximately 20 (8%) of the genes studied. Genes coding for cytokines (interleukin-1), chemokines (monocyte chemotactic protein 1 and interleukin-8), and cellular growth factors (heparin-binding epidermal-like growth factor, basic fibroblast growth factor, and platelet-derived growth factor B chain) were the most prominently upregulated. In addition to these families of genes, increases in mRNA levels for intracellular kinases and cell surface receptors with signal transduction activities were observed. Time course experiments showed that mRNA levels were upregulated within 2 h following infection. These results expand our knowledge of the response of endothelial cells to C. pneumoniae by further defining the repertoire of C. pneumoniae-inducible genes and provide new insight into potential mechanisms of atherogenesis. In addition, the use of cDNA microarrays may prove useful for the study of host cell responses to C. pneumoniae infection during latent and replicative stages of infection and related pathology.
Chlamydia pneumoniae is an obligate intracellular pathogen of humans and causes acute respiratory illnesses such as pneumonia, sinusitis, bronchitis, and pharyngitis (15). An association of this organism with chronic diseases such as atherosclerosis and coronary heart disease has been established based on several seroepidemiological and pathological studies. Pathological studies have identified the organism in diseased atherosclerotic tissue by a variety of techniques including PCR, immunocytochemistry, electron microscopy, and culture (recently reviewed in references 14, 19, and 27). Chronic infection of cells with C. pneumoniae may be facilitated by the ability of this organism to persist within host cells in an aberrant, nondividing morphological form (1). Furthermore, infected cells shedding chlamydial envelope antigens have been shown to promote a sustained inflammatory response in vitro (43). Given that atherosclerosis is a chronic inflammatory response at the vessel wall (37, 38), interaction of C. pneumoniae with host cells and the subsequent host cell response to infection may be important in the pathogenesis of atherosclerosis (13, 16).
Studies attempting to identify mechanisms by which C. pneumoniae may alter the hemodynamic properties of the vessel wall are ongoing. Data emerging from these in vitro experiments focus on the host cell response to infection and have identified several important pathways that are activated in atherogenesis. For example,C. pneumoniae lipopolysaccharide has been shown in vitro to enhance foam cell formation in macrophages exposed to oxidized low-density lipoprotein (LDL) (21). Another C. pneumoniae component, heat shock protein −60, has been shown to promote the oxidation of LDL to its proatherogenic form (22) and to stimulate the synthesis of matrix metalloproteinases in macrophages (25).
One of the hallmark features of atherosclerosis is the migration and proliferation of medial smooth muscle cells (SMC) into the arterial intima (32, 37, 39). Studies from our laboratory have shown that infection of human umbilical vein endothelial cells (HUVEC) resulted in the production of a endothelial cell-derived soluble factor(s) that stimulated DNA synthesis in SMC and increased SMC proliferation (3). Cellular proliferation and induction of various genes are tightly controlled by intercellular cytokine, chemokine, and growth factor networks, which may be affected by C. pneumoniae infection. Evidence for this is suggested by the in vitro finding that C. pneumoniae activates several host cell signaling pathways whose downstream effector proteins are transcription factors capable of transactivating several genes with important immunological and regulatory functions. A recent report shows that signal transduction cascades involving several host cell protein tyrosine kinases are induced within 5 min of C. pneumoniae binding to host endothelial cells (26), and activation of the transcription factor NF-κB has been shown to translocate to the nucleus of C. pneumoniae-infected endothelial cells within 15 min following infection (26), potentially affecting the transcriptional regulation of various host cell genes.
Transcriptional activity of endothelial cells following infection withC. pneumoniae has been reported. Induction of various molecules with immunological and procoagulant activity, including monocyte chemotactic protein 1 (MCP-1) and interleukin 8 (IL-8), has been observed (24, 31). These findings are consistent with a role of C. pneumoniae in the pathogenesis of atherosclerosis. These reports, however, focus only on a small number of genes encoding immunoregulatory proteins that may represent a small subset of inducible genes that are activated in endothelial cells following infection with C. pneumoniae.
Microarray technology is now readily available and allows characterization of the mRNA levels for a large number of genes simultaneously, thus providing a useful tool to identify broad spectrum changes in gene expression in cells in response to a given stimulus (5, 7, 41). cDNA arrays have been used to analyze transcription in host cells in response to several intracellular pathogens including Salmonella (8) andStaphlococus aureus (42), yet this approach has not been applied toChlamydia-infected cells. In an effort to expand the repertoire of human host cell genes that are upregulated by C. pneumoniae, we have used cDNA microarrays to analyze mRNA expression for a large number of genes in human microvascular endothelial cell line HMEC-1 following infection with C. pneumoniae.
MATERIALS AND METHODS
Cell culture.HEp-2 cells (ATCC CCL-23) were grown in 75-cm2 culture flasks with minimal essential medium (Gibco BRL, Gaithersburg, Md.) containing Earle's salts and supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) and 2 mMl-glutamine. HEp-2 cells were subcultured into 25-cm2 flasks or shell vials containing glass coverslips prior to infection with C. pneumoniae. HMEC-1 cells, obtained from E. Ades (Centers for Disease Control and Prevention, Atlanta, Ga.), were cultured in MCDB-131 medium (Gibco BRL) supplemented with 10% heat-inactivated FBS, epidermal growth factor (10 ng/ml; Sigma, St. Louis, Mo.), and hydrocortisone (1 μg/ml; Sigma) at 37°C and 5% CO2. Prior to infection, cells were seeded into 25-cm2 flasks (for cDNA array experiments) or into six-well plates (for reverse transcription-PCR [RT-PCR] experiments) at a density of 1.7 × 105cells/cm2 without supplements and allowed to adhere for 24 h. HUVEC (ATCC 1730-CRL) were maintained in Ham's F12K medium (Gibco BRL) supplemented with 10% FBS, 30 μg of endothelial cell growth supplements (Sigma) per ml, and 10 U of heparin (Sigma) per ml. Cells were maintained at 37°C and 5% CO2 in gelatin-coated culture flasks. Prior to infection, cells were seeded into gelatin-coated six-well plates and allowed to adhere for 24 h in the absence of supplements.
C. pneumoniae propagation. C. pneumoniae VR-1310 (ATCC 1310-VR) was propagated in HEp-2 cells as described by Roblin et al. (35), with slight modifications. C. pneumoniae was inoculated onto confluent monolayers of HEp-2 cells, centrifuged at 1,000 × gfor 60 min at 25°C, and then incubated at 37°C for 1 h. The inoculum was removed and replaced with growth medium consisting of minimal essential medium containing cycloheximide (1 μg/ml) and incubated for 72 h at 37°C and 5% CO2. C. pneumoniae was harvested by disruption of HEp-2 cells with glass beads followed by sonication and centrifugation at 500 ×g to remove cellular debris. Supernatants containingC. pneumoniae were centrifuged at 30,000 ×g for 30 min at 4°C to pellet C. pneumoniaeelementary bodies (EBs). EB pellets were suspended in sucrose-phosphate-glutamate buffer, aliquoted, and stored at −70°C.C. pneumoniae titrations were performed on frozen stocks using immunofluorescent staining with a genus-specific fluorescein isothiocyanate-labeled monoclonal antibody (Kallestad, Chaska, Minn.).C. pneumoniae titers were expressed as inclusion-forming units per mililiter.
HMEC-1 infection protocol.HMEC-1 cells were infected as described above at a multiplicity of infection of 1. Following centrifugation at 1,000 × g and incubation at 37°C for 1 h, the inoculum was removed, and cells were washed twice with Hanks balanced salt solution and cultured in MCDB-131 medium containing 0.1% FBS but lacking growth supplements and cycloheximide. Host cell RNA was isolated at various times as indicated in the figure legends. Intracellular inclusions could be seen in C. pneumoniae-infected cultures under these growth conditions after 48 to 72 h, but the viability of the bacterial progeny was not examined.
Analysis of mRNA expression using cDNA arrays.Infected and uninfected HMEC-1 cells were trypsinized and collected by centrifugation. Total cellular RNA was isolated by lysis of cells in 4.0 M guanidinium thiocyanate followed by a series of phenol-chloroform extractions. The final aqueous phase containing total RNA was treated with RNase-free DNase to remove genomic DNA and reextracted with phenol-chloroform-isoamyl alcohol, and RNA was isolated by precipitation with 2.5 volumes of absolute ethanol and 0.1 volume of 2 M sodium acetate (pH 4.5). Total RNA was collected by centrifugation and washed with ice-cold 75% ethanol. The integrity of RNA transcripts was verified by electrophoresis through denaturing agarose-formaldehyde gels followed by ethidium bromide staining according to standard protocols (40). Subsequently, poly(A)+ RNA was purified from total RNA using Oligotex polystyrene-latex resin (Qiagen, Mississauga, Ontario, Canada) according to the manufacturer's instructions. mRNA expression was analyzed by hybridization of radioactively labeled cDNA to membrane-bound cDNAs corresponding to various genes. The array used in this study was the Atlas cytokine/receptor cDNA expression microarray from Clontech Laboratories (Palo Alto, Calif.). Preparation of radiolabeled cDNAs and hybridizations were performed as outlined by the manufacturer. Briefly, 1 μg of poly(A)+ RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase in the presence of 35 μCi of [α-32P]dATP and 268 gene-specific primers. cDNA was purified by passage through a CHROMA SPIN-200 column (Clontech), and column fractions were analyzed by scintillation counting for incorporation of radioactive label. Each cDNA probe pool was adjusted to 106 cpm/ml and hybridized to separate nylon Atlas arrays at 68°C overnight in ExpressHyb hybridization solution (Clontech). Membranes were washed three times in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% sodium dodecyl sulfate for 30 min at 68°C and twice in 0.1× SSC–0.5% sodium dodecyl sulfate for 30 min at 68°C. Membranes were exposed to X-ray film with intensifying screens at −70°C for 1 to 3 days, and mRNA expression levels were analyzed by scanning densitometry of autoradiographs using Image Master VDS version 2.0 (Amersham Pharmacia Biotech). Analysis of differential patterns of gene expression was assessed by preparing cDNA probe pools from both uninfected HMEC-1 controls and C. pneumoniae-infected HMEC-1 cells and hybridizing these cDNAs in parallel to pairs of identical cDNA arrays. Array data are expressed as relative changes in mRNA expression following normalization of gene signals (based on optical density [OD]) to levels of β-actin mRNA to ensure analysis of equivalent amounts of RNA.
Analysis of mRNA expression using RT-PCR.Total RNA was isolated from uninfected and C. pneumoniae-infected HMEC-1 cells at various times after infection using RNeasy columns (Qiagen) according to the manufacturer's instructions. Total RNA was treated with RNase-free DNase and further purified by salted alcohol precipitation as described above. For cDNA preparation, 1.5 μg of total RNA was reverse transcribed with moloney murine leukemia virus reverse transcriptase in the presence of 0.5 μg of oligo(dT)12–18. Primer sequences for RT-PCR were as follows: MCP-1 (forward) 5′-CAAACTGAAGCTCGCACTCTCGCC-3′, MCP-1 (reverse) 5′-ATTCTTGGGTTGTGGAGTGAGTGTTCA-3′ (28), IL-8 (forward) 5′-ATGACTTCCAAGCTGGCCGTCGCT-3′, IL-8 (reverse) 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′ (9), β-actin (forward) 5′-CCAACCGCGAGAAGATGACC-3′, and β-actin (reverse) 5′-GATCTTCATGAGGTAGTCAGT-3′ (20). Sequences for PCR primers specific for other individual genes of interest were obtained from Clontech, and primers were synthesized by the Central Facility of the Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada; 2 μl of cDNA was used as the template for individual PCRs with pairs of gene-specific primers. Each PCR mixture on contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate, 0.5 μM each primer, and 1.5 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer). Thermal cycling programs consisted of 10 min of denaturation at 95°C, followed by 1 min at 94°C, 1 min at 56°C, and 2 min at 72°C for 23 cycles and a final extension of 5 min at 72°C. PCR products were analyzed by electrophoresis through 2% agarose gels and visualized by ethidium bromide staining. RT-PCR data were analyzed by scanning densitometry of gel bands and normalized to β-actin signals obtained from the same time point. The normalized data were expressed as relative changes in mRNA levels betweenC. pneumoniae-infected HMEC-1 cells and uninfected controls. The numerical data were analyzed using a two-tailed Studentt test. A P value of <0.05 was considered significant.
RESULTS
Analysis of endothelial cell mRNA expression by cDNA microarrays in response to C. pneumoniae infection.To study changes in mRNA expression in endothelial cells in response to infection with C. pneumoniae, we employed a cDNA microarray approach using the Clontech Atlas microarray. This array represents 268 different human genes, including those encoding cytokines and other immunological regulatory proteins such as chemokines, growth factors, and cellular receptors. Each gene is represented on the array as duplicate spots containing immobilized cDNA fragments, to which experimentally prepared cDNAs are hybridized. mRNA was isolated from uninfected control and C. pneumoniae-infected HMEC-1 cells at 18 h postinfection and converted to radioactively labeled cDNAs by reverse transcription using a single gene-specific primer for each gene represented on the array. cDNA pools from uninfected cells and C. pneumoniae-infected cells were hybridized in parallel to identical pairs of cDNA array membranes under identical hybridization conditions. Subsequent wash steps, generation of autoradiographs, and densitometric analysis of data were performed in parallel. This approach facilitates the direct comparison of mRNA levels between infected and uninfected cells.
mRNAs for approximately 84 out of 268 arrayed genes (31%) were expressed in uninfected HMEC-1 cells. In C. pneumoniae-infected cells, 99 out of 268 genes (37%) were expressed (Fig. 1). Upregulation of 20 genes (7.5%) of the total arrayed genes was identified using the cDNA microarrays, where 15 genes were induced from undetectable levels and 5 genes were upregulated. Therefore, the majority of genes expressed by HMEC-1 cells under these experimental conditions were not affected byC. pneumoniae infection, indicating that the endothelial cell mRNA responses to C. pneumoniae infection was relatively narrow and specific. Table 1identifies the C. pneumoniae-induced genes in HMEC-1 cells at 18 h after infection.
cDNA microarray analysis of gene expression in response to C. pneumoniae infection. Radioactively labeled cDNA probes generated from poly(A)+ mRNA from uninfected HMEC-1 cells (A) and HMEC-1 cells infected with C. pneumoniae for 18 h (B) were hybridized in parallel to pairs of identical cDNA arrays. Hybridization patterns were assessed by autoradiography for 24 to 72 h and analyzed by scanning densitometry. Relative expression levels for specific genes were normalized using housekeeping gene controls (boxed area). Arrows indicate the locations of several representative genes whose expression levels increased in response to C. pneumoniae infection.
Identification of C. pneumoniae-induced genes in HMEC-1 cells by cDNA arraya
Analysis of mRNA expression using RT-PCR.To confirm the data obtained using the cDNA arrays and to further characterize the mRNA expression profiles for selected genes of interest, we chose a panel of genes representing various chemokines and cellular growth factors whose expression levels were increased in infected HMEC-1 cells. We also included one gene (encoding CD40) that was not expressed by control HMEC-1 cells or cells infected with C. pneumoniaeto verify the specificity of the cDNA array for unexpressed transcripts. The mRNA expression levels of these selected genes were then analyzed by RT-PCR at various time points after C. pneumoniae infection ranging from 0 to 24 h. RNAs from infected and uninfected HMEC-1 cells were harvested and processed in parallel under identical conditions for each time point. In some cases, primer sequences for selected genes of interest were obtained from Clontech. RT-PCR was performed for a minimum number of cycles (18 to 23 cycles) previously determined to be within the linear range of amplification (data not shown). As shown in Fig.2 for the genes chosen for further analysis, RT-PCR confirmed most of the data obtained using the cDNA arrays. As summarized in Table 2, of seven genes chosen for further analysis whose mRNA levels were increased in the cDNA array, RT-PCR confirmed upregulation for five. IL-8, MCP-1, heparin-binding epidermal-like growth factor (HBEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor B chain (PDGF-B) genes were all upregulated at least twofold. Table 2 lists the maximum fold induction of these genes in C. pneumoniae-infected HMEC-1 cells compared to uninfected control cells, which in most cases occurred between 2 to 4 h postinfection. Also listed in Table 2 are the expression levels of these genes at 12 h post infection, demonstrating that expression levels were lower as the time of infection increased. Insulin-like growth factor (IGF)-binding protein 4 (IGFBP4) and thrombin receptor, however, showed no differences in the mRNA expression levels using RT-PCR. Figure3 shows the levels of mRNAs for the seven genes selected for further study at various times after infection. Five genes (encoding IL-8, MCP-1, PDGF-B, bFGF, and HBEGF) were upregulated as early as 2 h postinfection. Levels of mRNA for these genes declined from 2 to 4 h, reaching basal levels by 12 to 24 h in most cases. Using the more sensitive technique of RT-PCR, the finding that CD40 was not expressed by HMEC-1 cells under the conditions used in this study confirmed the cDNA array result and verified the ability of the cDNA array to provide a true negative result for a nonexpressed gene.
RT-PCR analysis of mRNA expression for various genes in uninfected HMEC-1 cells and HMEC-1 cells infected with C. pneumoniae. Total RNA was harvested at 0, 2, 4, 8, 12, and 24 h, and levels of specific mRNAs were determined by RT-PCR as described in Materials and Methods. RT-PCR products were analyzed by scanning densitometry for relative changes in mRNA expression. Lane −, no RNA control. Sizes of amplified cDNAs are shown at the right.
RT-PCR analysis of mRNA expression in HMEC-1 cells following C. pneumoniae infection
RT-PCR analysis of mRNA expression in C. pneumoniae-infected HMEC-1 cells. Levels of specific mRNAs following infection of HMEC-1 cells with C. pneumoniaewere assessed by RT-PCR at various time points as described in Materials and Methods. RT-PCR products were first measured by plotting the densitometric [gene]/[β-actin] RT-PCR product ratios for both infected and uninfected HMEC-1. Normalized data for infected cells were then converted to fold induction by expressing the densitometric data as a ratio of uninfected controls (OD ratioinfected cells/OD ratiouninfected controls). Data points represent the means ± standard errors from two separate experiments. ∗, P < 0.03 .
Confirmation of mRNA responses in another endothelial cell culture model.To extend the results obtained for HMEC-1 cells, we used the well-characterized HUVEC as a secondary cell culture model system to study the mRNA responses for the two genes with the highest increases in mRNA expression as measured by RT-PCR. HUVEC were infected with C. pneumoniae as outlined for HMEC-1 cells, and mRNAs for IL-8 and MCP-1 were assessed by RT-PCR at 2 h after infection. As shown in Fig.4, MCP-1 and IL-8 mRNAs were upregulated in HUVEC 11- and 21-fold, respectively. This was similar to the 8.3- and 17.4-fold induction levels seen in HMEC-1 cells (Table 2).
Analysis of gene expression in HUVEC by RT-PCR. HUVEC were infected with C. pneumoniae or left uninfected for 2 h and then processed for RT-PCR as described in Materials and Methods for IL-8, MCP-1, and β-actin. (A) RT-PCR products resolved by agarose gel electrophoresis. (B) Numerical representation of the RT-PCR data as determined by densitometric analysis of gel bands. Fold induction is calculated as the maximum OD of C. pneumoniae-infected samples/OD of uninfected control samples, normalized to β-actin. NC, negative control; U, uninfected HMEC-1 cells; I, C. pneumoniae-infected HMEC-1 cells.
DISCUSSION
C. pneumoniae infection has been associated with atherosclerosis in many seroepidemiological studies and has been demonstrated in coronary, carotid, or popliteal arteries in over 40 studies using a variety of techniques, including PCR, immunohistochemistry, and culture (recently reviewed in reference14). C. pneumoniae may enter preformed or forming atheromas from infected peripheral blood mononuclear cells or via endothelial cells that become infected indirectly from infected mononuclear cells. Studies aimed at characterizing the host response toC. pneumoniae infection are necessary in order to discern how infection with this organism contributes to the pathophysiology of atherosclerosis. In the present study, we have shown that approximately 20 genes are upregulated in human vascular endothelial cells in response to C. pneumoniae infection. These genes include those encoding cytokines, chemokines, growth factors, and cellular receptors, all of which are involved in inflammation and may include a pathophysiological role for C. pneumoniae in atherogenesis.
In this study, we used cDNA microarray technology to characterize gene expression in human endothelial cells infected with C. pneumoniae. Since atherosclerosis is a chronic inflammatory process involving several cell types within the vessel wall (viz., endothelial cells, SMC, and macrophages), we chose an array containing 268 cDNA probes representing known human genes whose products included cytokines, cellular receptors, and other secreted growth-regulatory molecules (Clontech Atlas cytokine/receptor cDNA microarray). These arrays are composed of gene-specific cDNA probes immobilized on a solid-phase nylon membrane. mRNA pools from cultured cells are detected by their ability to hybridize to a given cDNA probe on the array. Upregulation of specific genes with cDNA microarrays was confirmed using a semiquantitative RT-PCR to measure fold increases in mRNA between C. pneumoniae-infected HMEC-1 cells and uninfected control cells.
Previous studies from our laboratory have shown that infection of human endothelial cells with C. pneumoniae leads to the production of soluble factors with mitogenic and proliferative activity towards SMC (3). SMC proliferation in the neointima is a hallmark feature of atherosclerosis and is controlled, in large part, by paracrine growth factors secreted by neighboring cells (2, 37). Communication between arterial cells mediated by soluble molecules and cellular receptors likely plays an important role in the progression of the chronic inflammatory atherosclerotic lesion since these molecules control tightly regulated cellular and molecular events. It has been suggested that perturbation of these networks due to intracellular infection with C. pneumoniae may contribute to the cellular dysfunction associated with atherogenesis (J. B. Mahony and B. K. Coombes, submitted for publication).
Our approach using a cDNA microarray demonstrated that infection of HMEC-1 cells with C. pneumoniae induced expression of relatively few genes (20 out of 268) and suggested that the endothelial mRNA response to infection is not a generalized response. Of these responses, some of the findings confirmed previous studies whereas others are novel. MCP-1 and IL-8 were detected in the supernatants ofC. pneumoniae-infected endothelial cell cultures by Molestina et al., suggesting transcriptional induction of mRNA for these proteins (30). Of interest was the novel finding that mRNAs coding for several growth factors including bFGF, PDGF-B, and HBEGF were induced by C. pneumoniae infection of endothelial cells. Recently, Rödel et al. (36) reported the accumulation of bFGF mRNA in SMC infected withC. pneumoniae, indicating a possible common induction cascade in endothelial cells and SMC.
The production of growth factors by endothelial cells that may induce proliferation of SMC supports our previous finding of SMC proliferation in response to culture supernatants from C. pneumoniae-infected endothelial cells (3) and could represent a significant new mechanism of C. pneumoniaeinvolvement in atherogenesis. For example, HBEGF has been shown to be a potent mitogen with apparent specificity for SMC (17) and has been implicated in a variety of pathological processes, including SMC hyperplasia and atherosclerosis (34). Similarly, homodimers of PDGF-B with SMC growth-promoting activity have also been associated with neointimal proliferation of SMC (39). PDGF-associated protein, a growth factor accessory molecule that modulates the activity of specific growth factors, was also upregulated by C. pneumoniae. Together, HBEGF and PDGF-BB could initiate or regulate the migration and proliferation of medial-derived SMC in the intima of a progressing atherosclerotic lesion. However, we do not yet know whether one or more of these specific growth factors was responsible for SMC proliferation in our previous study.
The finding of increased mRNA expression for activin A or erythroid differentiation protein was also a novel finding in our study. Activin A is a member of the transforming growth factor β (TGF-β) superfamily and is functionally composed of a homodimer of βA chains of the inhibin/activin group (44). This molecule has recently been shown to modulate monocyte/macrophage functions including immunological activation of monocytes (11) and induction of matrix metalloprotease 2 (33). Association of activin A during atherogenesis has also been reported by others. Using cDNA microarrays, de Waard et al. reported induction of activin A mRNA from human endothelial cells exposed to conditioned medium from monocytes exposed to oxidized LDL (6). Upregulation of activin A may be important since this molecule has been demonstrated in atherosclerotic lesions of humans (M. A. Engelse et al., unpublished data) and in animal models (18). Its role in lesion progression may be to influence phenotypic changes in SMC (10) or act as a paracrine or autocrine mediator of macrophage activation as described above.
Time course analysis of gene expression in C. pneumoniae-infected endothelial cells by RT-PCR revealed a tightly controlled temporal regulation of gene induction. For the genes studied in C. pneumoniae-infected cells, mRNA was maximal between 2 to 6 h postinfection and declined thereafter, reaching a steady state at 24 h in most cases. This was a consistent finding for all genes studied. These findings, at least for MCP-1, differ somewhat from those reported by Molestina et al. (31). Although an early MCP-1 mRNA response was noted following C. pneumoniae infection in their study, this response was not maximal until 12 h post infection and remained significantly elevated at 24 h postinfection. These differences may be due to cell-specific variations, as the endothelial cells used in their study were derived from human umbilical vein, while HMEC-1 cells are derived from the human microvasculature.
However, our data on early activation of mRNA responses are consistent with early activated signal transduction pathways in endothelial cells following chlamydial infection (12, 26). A recent report shows that signal transduction cascades involving several host cell protein tyrosine kinases including p42/p44 mitogen-activated protein kinase (MAPK) are induced within 5 min ofC. pneumoniae binding to host endothelial cells (26). In addition, the ubiquitous transcription factor NF-κB, which controls inducible transcriptional activation of several immunological genes, has been shown by several investigators to be activated and nucleus associated within 10 to 15 min following C. pneumoniae interaction with host cells (4, 26, 31). This early transcription factor activation is reduced to basal levels by 24 h postinfection, indicating that the transcriptional response of cells to C. pneumoniae infection is elicited at an early time point after infection. An early transcriptional response in less than 2 h would be consistent with signal transduction events following contact of EBs with specific membrane molecules, leading to cytosol activation and nuclear relocation of transcription factors such as NF-κB. Consistent with this chronology is the finding that maximal increases of E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 mRNAs in endothelial cells occur at 2 h after infection with C. pneumoniae, with a return to basal levels by 24 h postinfection (26). These genes all contain consensus NF-κB-binding sequences within their promoter regions and are known to be inducible following activation of NF-κB (29).
In addition to these signal transduction pathways, our data suggest the activation of other signal transduction cascades in C. pneumoniae-infected endothelial cells. The upregulation of mRNA corresponding to TKT tyrosine kinase, a member of a cell adhesion kinase receptor family (23), was observed, along with IGF receptor 1, which is a transmembrane tyrosine kinase linked to the Ras-Raf MAPK cascade. Other intracellular gene products whose mRNAs were upregulated included the gamma interferon (IFN-γ-responsive transcription factor IFN regulatory factor 1. This transcription factor binds to regulatory DNA-binding sequences upstream from IFN-inducible genes and controls their transactivation. This finding further suggests that endothelial cells may play an important role in controlling initial immunological responses to C. pneumoniae infection at the vessel wall and may play an important role in the production of inflammatory mediators in the atherosclerotic plaque.
Despite these early initial responses of endothelial cells to infection with C. pneumoniae in vitro, sustained activation of these molecules may occur in vivo, during different stages of infection. For example, the infection of various cells by C. pneumoniae would not be synchronized, so it may be possible that specific gene products accumulate to high levels in tissues as new cells become infected during an ongoing chlamydial infection. Alternatively, the apparent ability of chlamydiaeto enter into a persistent stage of infection where the organism aborts its normal developmental cycle and appears to reside in viable, nonreplicating form may provide a sustained antigenic stimulation of both immune and nonimmune cells which contributes to a chronic activated state of cells present in atheromatous lesions. This idea of chronic cell activation is supported by the in vitro demonstration of sustained activation of endothelial cells in response to persistentChlamydia envelope antigens following antibiotic treatment of infected cell cultures (43).
Technical issues relating to the use of cDNA arrays for the study of differential gene expression following a given stimulus were noted in our study. For example, in some cases for genes whose expression levels were found to be upregulated by the cDNA arrays, induction could not be confirmed by RT-PCR analysis. This was the case for two genes out of seven chosen for further study, the IGFB4 and thrombin receptor genes. In these cases, the confirmatory approach revealed mRNA expression for these genes, yet their levels were not significantly higher in infected cells than in uninfected controls. This issue underscores the importance of confirmatory testing of cDNA microarray results using a second technology. A similar conclusion has been reached in other studies using oligonucleotide arrays for analysis of differential gene expression in cells (8). It is likely that the discrepancies noted above relate to the different sensitivities between the cDNA arrays and, in this case, RT-PCR. Where RT-PCR can provide exquisitely better sensitivity owing to amplification of starting products, thereby improving the detection of mRNA in low abundance, the cDNA microarray may not reach this level of sensitivity. In this case, small increases in mRNA abundance for a given gene in response to infection may become visible on the array, but the low-level expression from the uninfected samples may be below the detection threshold. During densitometric analysis of these signals, changes may be overestimated, since a weak signal from the infected array is being compared to an absent signal from the uninfected array. In these cases, RT-PCR may be a better indicator of actual differences in the mRNA populations between the two samples.
The use of cDNA microarrays for the study of host-pathogen interactions has proved to be a valuable tool for extending and characterizing the repertoire of host cellular responses toC. pneumoniae infection. An understanding of these responses at a molecular level will be necessary to evaluate the biological role infection may play in the development or progression of certain diseases.
ACKNOWLEDGMENTS
We are grateful to R. Austin for kindly providing the densitometry equipment used throughout this study.
Brian K. Coombes was the recipient of a scholarship from the Father Sean O'Sullivan Research Centre, St. Joseph's Hospital, while completing a portion of this work and is now supported by a doctoral training award from the Canadian Institutes for Health Research and the Heart and Stroke Foundation of Canada.
Notes
Editor: E. I. Tuomanen
FOOTNOTES
- Received 21 September 2000.
- Returned for modification 13 November 2000.
- Accepted 27 November 2000.
- Copyright © 2001 American Society for Microbiology
