Differences in Cell Activation by Chlamydophila pneumoniae and Chlamydia trachomatis Infection in Human Endothelial Cells

Abstract

Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH₂ kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.

Chlamydophila pneumoniae, a gram-negative obligate intracellular bacterium, is a widespread respiratory pathogen (15). Chronic infection may be an important risk factor for chronic obstructive lung disease or adult-onset asthma (10) and has been suggested as a trigger/promoter of inflammatory reactions and development of vascular lesions (16, 24). The field is troubled by the “chicken versus egg” problem, and causative proof is difficult because, besides different antichlamydial isotypes of antibodies, there are no good markers to differentiate among new versus old (immunoglobulin M [IgM] versus IgG) as well as acute versus chronic persistent (IgM versus IgA) C. pneumoniae infection.

Airway-derived organisms may be able to spread systemically in at least two different ways: (i) carried within monocytes (or possibly lymphocytes) following pulmonary infection and (ii) by direct access to the bloodstream, causing a short interval of chlamydial bacteriemia (18, 21).

The role of C. pneumoniae in atheroma formation has not been studied in detail. Chronic infection of susceptible target cells (monocytes, macrophages, endothelial cells, fibroblasts, and smooth muscle cells) as well as direct activation of endothelial cells may initiate and perpetuate a local inflammation by inducing the release of cytokines (e.g., interleukin-6 [IL-6], IL-8), increased expression of adhesion molecules (P- and E-selectins, ICAM-1, VCAM-1), and subsequent adhesiveness of the endothelium for leukocytes or platelets, key pathomechanisms during atherogenesis (5, 8, 12-14, 16, 23). Although chlamydiae may reside and replicate in different cell types and induce a chronic immune activation, little is known about the mechanisms of C. pneumoniae-induced target cell alteration. In addition, it is still unclear if these pathogen-mediated effects on endothelial cell activation are specific for C. pneumoniae or if other chlamydial species such as Chlamydia trachomatis are also able to invade and activate endothelial cells.

There is limited knowledge of the mechanisms of chlamydiae entry into host cells. The chlamydial growth cycle is initiated when an infectious elementary body attaches to a susceptible target cell, promoting entry into a host cell-derived phagocytic vesicle. Elementary bodies develop into reticular bodies, a process which could be detected metabolically within 15 min and microscopically 12 to 15 h after addition of chlamydiae to HEp-2 and HeLa-229 cells (22, 28). The length of the complete developmental cycle, as studied in cell culture models, is 48 to 72 h (1,19, 27).

The mitogen-activated protein kinase (MAPK) family members are ubiquitously expressed protein kinases activated in response to a variety of extracellular stimuli (3). They play important roles in cell activation (p38 kinase), stress response (c-Jun-NH₂ kinase [JNK]), differentiation and cell growth (extracellular receptor kinase [ERK]). p38-MAPK, in particular, has been demonstrated to be a key player in the development of a proinflammatory and prothrombotic phenotype in target cells (7, 17, 26). At least four distinctly regulated groups of MAPK are controlled through three upstream kinases cascades composed of a MAPK, MAPK kinase (MAPKK, MKK, or MEK) and a MAPKK kinase (MEK kinase). Cross talk among the different MAPK results in direct modulation of downstream signal transduction pathways. Subsequent activation or translocation of transcription factors is an essential prerequisite for altered gene expression in stimulated target cells (3).

We have recently demonstrated that infection of human endothelial cells with C. pneumoniae activates different signal transduction pathways such as phosphorylation of serine/threonine and tyrosine kinases and translocation of nuclear factor-κB (NF-κB [5, 14]). The first objective of the present study, therefore, was to clearly assess the ability of C. pneumoniae to activate different endothelial MAPK pathways and to analyze the effects of activated ERK, p38, and JNK on the expression of different proinflammatory mediators. We could demonstrate that MAPK cascade-mediated induction of a proinflammatory phenotype in C. pneumoniae-infected human endothelial cells required a viable pathogen. Endothelial pretreatment with a specific p38-MAPK inhibitor reduced C. pneumoniae-mediated IL-8 secretion markedly. The second objective of our study was to elaborate the specificity of endothelial cell activation for C. pneumoniae. We therefore infected human endothelial cells with C. pneumoniae strain TW183 and C. trachomatis serovar K to compare infectivity and target cell activation with respect to IL-8 release. In addition, we ruled out the importance of a paracrine release of endothelial cell-derived mediators such as platelet-activating factor (PAF), NO, prostaglandins, or leukotrienes for chlamydia-mediated target cell activation.

(Parts of this work are included in the M.D. theses of J. Kramp, T. Petrov, and C. Walter.)

MATERIALS AND METHODS

Materials.

Tissue culture plasticware was obtained from Becton Dickinson (Heidelberg, Germany). MCDB131 medium, phosphate-buffered salt solution (PBS), trypsin-EDTA solution, HEPES, and fetal calf serum (FCS) were from GIBCO (Karlsruhe, Germany). Collagenase type II was purchased from Worthington Biochemical Corp. (Freehold, N.J.), U0126 and SB202190 were from Calbiochem (San Diego, Calif.), an anti-C. pneumoniae major outer membrane protein-specific monoclonal antibody was from Dako (Hamburg, Germany), and all other reagents were from Sigma (Munich, Germany).

HUVEC and chlamydial strain.

Human umbilical cord vein endothelial cells (HUVEC) were isolated from human umbilical cord veins and identified as described previously (13). Briefly, cells obtained from collagenase digestion were washed, resuspended in MCDB131-5% FCS, and seeded into 6- or 24-well plates with 1.2 × 10⁶ or 2.5 × 10⁵ cells/well, respectively.

C. pneumoniae strain TW183 (ATCC VR2282) was cultured and purified as described by Maass et al. (16). Briefly TW183 was grown to high titers in cycloheximide-treated HEp-2 cells. Infected monolayers were harvested from culture flasks and sonicated for 30 s. Cellular debris was removed by centrifugation at 500 × g for 10 min at 4°C. Aliquots diluted with an equal volume of sucrose-phosphate-glutamate (SPG) buffer supplemented with 10% FCS were stored at −75°C until use. Titer determination in cycloheximide-treated HEp-2 cells was performed with a thawed aliquot (in triplicate). Infection was performed as described previously (14), except that the cells were not centrifuged. HUVEC monolayers were inoculated with C. pneumoniae using a multiplicity of infection (MOI) of 5 and, after incubation at 37°C, processed for further experiments at the times indicated. C. trachomatis serovar K was a kind gift of J. G. Kuipers (Department of Rheumatology, Medical School Hannover, Hanover, Germany) and was first isolated as described in reference 25.

Confocal laser-scanning microscopy (CLSM) analysis of endothelial cell infection by C. pneumoniae and C. trachomatis.

After infection of HUVEC grown on Thermanox slides (Falcon Culture Slide; Becton Dickinson, Rutherford, N.J.) with C. pneumoniae strain TW183 or C. trachomatis serovar K (CTK) at an MOI of 5 for 24 h, cells were fixed with 3% paraformaldehyde for 20 min and permeabilized with 1% Triton X-100 for 15 min. To reduce nonspecific background staining, the cells were treated with 5% goat serum for 30 min in PBS before the addition of antibodies. For chlamydia staining, the primary antibody (a genus-specific monoclonal antibody; “chlamydia culture conformation system,” Sanofi Diagnostics Pasteur, Freiburg, Germany) was incubated overnight at 4°C. Bound antibodies were detected with ALEXA-488-conjugated goat anti-rabbit monoclonal antibody at 4°C overnight; (Molecular Probes, Eugene, Oreg.). Endothelial F-actin was counterstained using ALEXA-546-conjugated phalloidin (Molecular Probes). After each step, the cells were washed three times with sterile PBS. Coverslips were sealed, and cells were analyzed with the use of a Pascal 5 confocal laser- scanning microscope (CLSM; Zeiss, Jena Germany).

Western blot analysis.

For determination of MAPK phosphorylation, HUVEC were processed as described previously (11). Briefly, the cells were starved for 12 h in serum-free medium, stimulated as indicated, and washed twice in ice-cold HEPES buffer (pH 7.4) containing 100 mM sodium fluoride, 2 mM sodium vanadate, and 15 mM sodium pyrophosphate. They were then harvested on ice by scraping with lysis buffer containing 1 mM EDTA, 1% Triton X-100, 1 mM, phenylmethylsulfonyl fluoride, and 2 μg each of leupeptin, pepstatin and antipain per ml. After removal of the cell debris by centrifugation, cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide) and blotted onto Hybond-ECL membranes (Amersham Biosciences, Freiburg, Germany). Each lane contains 80 μg of protein (ascertained by the Bradford assay). Immunodetection of phosphorylated p38-MAPK, ERK1/2, or JNK/SAPK was carried out with phospho-specific MAPK antibodies (Cell Signaling, Frankfurt, Germany). Their nonphosphorylated isoforms (Santa Cruz Biotechnologies, Heidelberg, Germany) were detected simultaneously to confirm equal protein loading. Proteins were visualized by incubation with secondary IRDye 800- or Cy5.5-labeled antibodies respectively, using an Odyssey infrared imaging system (LICOR Inc., Bad Homburg, Germany).

Immune complex kinase assays.

For MAPK immune complex kinase assays, stimulated HUVEC monolayers were lysed and equal amounts of protein lysates were incubated with 25 μl of protein A-agarose (Boehringer GmbH, Mannheim, Germany) or 1 μl of rabbit antiserum against ERK, JNK, or p38 kinase (Santa Cruz Biotechnology) per ml for 2 h at 4°C (11). After being washed, the samples were incubated with either myelin basic protein as substrate for ERK, glutathione S-transferase (GST)-c-Jun as substrate for JNK, or 3pK/MAPKAP-K3 as a substrate for p38 MAPK in the presence of 100 μM unlabeled ATP, 5 μCi of [γ-³²P]ATP, and kinase buffer as previously described (20). Samples were subsequently subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and visualized by autoradiography. Western blot analysis was performed to confirm equal loading of each MAPK.

IL-8 ELISA and cell surface ELISA for ICAM-1 expression.

HUVEC were grown to confluence in 24-well plates and stimulated as indicated above (HUVEC and chlamydial strain). After incubation, expression of ICAM-1 on endothelial monolayer (cell surface enzyme-linked immunosorbent assay [ELISA]) and IL-8 in supernatants (ELISA) was determined as described previously (11, 14).

Statistical methods.

A one-way analysis of variance was used for data in Fig. 3 through 5. P < 0.01 was considered significant.

FIG. 3.

C. pneumoniae-induced activation of MAPK. HUVEC were incubated with C. pneumoniae, strain TW183 (MOI, 5) for the periods indicated or mock infected (control [C]) 15 min. for ERK (A), p38-MAPK (B), and JNK (C) activity was assessed by an immune complex kinase assay. Equal gel loading was confirmed by ERK, p38 kinase, and JNK immunoblotting. Representative gels (of four for all figures) are demonstrated. Data presented from semiquantitative analysis (expressed as relative kinase activity) are the mean and standard error of the mean of four separate experiments for all panels. *, P < 0.01 versus control for 5 min.

FIG. 5.

C. pneumoniae-mediated target cell activation is independent of paracrine endothelial mediators. HUVEC were pretreated for 30 min with 10 μM l-NMMA (inhibition of nitric oxide synthase), 0.5 mM acetylsalicylic acid (inhibition of cyclooxygenases), 10 μM MK-886 (inhibition of lipoxygenase), or 1 μM BN 50727 (PAF receptor antagonist) and subsequently infected with strain TW183 (MOI, 5). The level of IL-8 in the supernatant was determined by ELISA as described in Materials and Methods. Note that endothelial cell preincubation with each inhibitor did not significantly modify C. pneumoniae-mediated IL-8 expression. Data presented are mean and standard error of the mean for three separate experiments.

RESULTS

Infection of HUVEC by C. pneumoniae strain TW183 or CTK.

Infection of endothelial cells by C. pneumoniae or C. trachomatis was demonstrated by CLSM of HUVEC 24 h after the addition of bacteria (Fig. 1). ALEXA-546-phalloidin was used to counterstain endothelial F-actin; Fig. 1A represents uninfected endothelial cells. CTK (Fig. 1B) and TW183 (Fig. 1C) were inoculated onto HUVEC monolayer without centrifugation, using an MOI of 5. This resulted in a bacterial adhesion after the first minutes of cell contact and in subsequent internalization. After 24 h of exposure, only a few bacteria were adherent to the cell surface; instead, perinuclear formation of internalized bacteria was observed. Heat-inactivated bacteria adhered to the surface of endothelial cells, but only very few were subsequently internalized (Fig. 1D). Intracellular location of those was not associated with the perinuclear region. At 72 h postinfection there were no detectable inclusion-forming units in the cytoplasm compared to those in cells infected with viable C. pneumoniae (data not shown).

FIG. 1.

Infection of HUVEC by C. pneumoniae strain TW183 or CTK. Infection was demonstrated by CLSM. HUVEC were grown on glass slides and exposed to chlamydiae at a MOI of 5. At 24 h postinfection, the cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and labeled with phalloidin as indicated in Materials and Methods. (A) Uninfected control cells. (B and C) Z-stack analysis confirmed that in infected HUVEC, all visualized chlamydiae were intracellular (CTK [B] and TW183 [C]). Note that internalized bacteria have started to decondense and form typical perinuclear inclusion body-like structures. (D) Only very few of the heat-inactivated TW183 were phagocytosed and were located diffusely in the cytoplasm. Representative pictures (from three independent experiments) are presented. Magnification, ×598.

C. pneumoniae-mediated phosphorylation of MAPK.

Activation of MAPK is an early step in intracellular signaling after target cell activation. Western blot analysis demonstrated that preincubation of HUVEC with C. pneumoniae strain TW183 (MOI, 5) increased the phosphorylation of MAPK ERK1/2 (Fig. 2A), p38 MAPK (Fig. 2B), and SAPK/JNK (Fig. 2C) in a time-dependent fashion. Chlamydiae were added to the HUVEC medium and inoculated on the endothelial monolayer without centrifugation. Phosphorylation peaked at 15 min (ERK1/2 and p38) to 30 min (SAPK/JNK) and decreased again to almost baseline after 60 min for ERK1/2 and p38; SAPK/JNK phosphorylation remained elevated for up to 60 min and decreased after 120 min. Cell incubation without bacteria for more than 120 min did not have any effect on MAPK phosphorylation. Neither chlamydial UV treatment or heat inactivation (60 min at 90°C) nor TW183 preincubation with polymyxin B in order to block chlamydial lipopolysaccharide (LPS) reduced MAPK phosphorylation markedly (data not shown). Endothelial cell preincubation with different concentration of C. trachomatis (MOI, 0.05 to 5) for 15 min did not induce phosphorylation of p38-MAPK (Fig. 2D), ERK1/2, or SAPK/JNK in endothelial cells. Tumor necrosis factor alpha (10 ng/ml for 15 min) was used as a positive control.

FIG. 2.

C. pneumoniae-induced phosphorylation of ERK1/2, p38, and JNK MAPK in human endothelial cells. (A to C) Western blot analysis demonstrates the time course of ERK1/2 (A), p38 (B), and JNK (C) phosphorylation in HUVEC. Cells were incubated with C. pneumoniae strain TW183 (MOI, 5) for 2, 5, 10, 15, 30, or 60 min or mock infected for 15 min (control [C]). Proteins were separated using a 10 % polyacrylamide gel, and equal gel loading was confirmed by immunoblotting of nonphosphorylated MAPK. Representative gels (of four for all figures) are demonstrated. (D) Endothelial preincubation for 15 min with CTK (MOI, 0.05 to 5) did not induce p38 MAPK phosphorylation. at TNF-α 10 ng/ml was used as a positive control (15-min treatment). Representative gels (of three) are demonstrated.

C. pneumoniae-mediated activation of MAPK.

Next, MAPK activity after stimulation with C. pneumoniae strain TW183 was studied. Again, chlamydiae were added to the HUVEC medium and inoculated onto the endothelial monolayer without centrifugation. C. pneumoniae (MOI, 5) induced a time-dependent increase of ERK1/2 (Fig. 3A), p38 MAPK (Fig. 3B), and SAPK/JNK (Fig. 3C) activity in HUVEC 15 to 30 min after endothelial cell contact with TW183, as shown by phosphorylation of the target proteins MBP (ERK1/2), pK/MAPKAP-K3 (p38 MAPK), and GST-c-Jun (SAPK/JNK). No MAPK activity was detectable after more than 120 min of TW183-HUVEC preincubation (data not shown). Time points of maximal kinase activity are congruent with maximal phosphorylation of MAPK (Fig. 2). Again, neither chlamydial UV treatment or heat inactivation (60 min at 90°C) nor TW183 preincubation with polymyxin B in order to block chlamydial LPS reduced MAPK activation markedly (data not shown).

C. pneumoniae-mediated protein expression.

ELISA (IL-8) and cell surface ELISA (ICAM-1) were performed 24 h postinfection to assess whether infection and activation of endothelial MAPK by C. pneumoniae was followed by IL-8 and ICAM-1 expression. Chlamydiae were added to the HUVEC medium and inoculated onto the endothelial monolayer without centrifugation. CLSM analysis confirmed a marked infection of endothelial cells 24 h postinfection (Fig. 1). Preincubation of HUVEC with 10⁻⁶ M U0126 (ERK1/2 inhibitor) for 15 min or 10⁻⁶ M SB202190 (p38-MAPK inhibitor) for 15 min reduced viable TW183-mediated endothelial cell IL-8 secretion by 40 and 66%, respectively (Fig. 4A). Coincubation with both inhibitors had an almost additive effect (84%) but did not result in complete abolition of IL-8 synthesis. Inhibition of ERK or p38 kinase had only a slight but significant effect (30% for U0126 and 18% for SB202190) on C. pneumoniae-induced ICAM-1 expression (Fig. 4B). Although phosphorylating and activating MAPK, heat-inactivated bacteria did not induce IL-8 and ICAM-1 protein expression (Fig. 4). Infection of endothelial cells with CTK (MOI, 5) for 24 h did not induce the expression of either IL-8 or ICAM-1.

FIG. 4.

Chlamydia-induced release and expression of endothelial IL-8 and ICAM-1. IL-8 (A) and ICAM-1 (B) expression was determined 24 h postinfection by ELISA and cell surface ELISA, respectively. HUVEC were pretreated with the ERK (U0126, 10⁻⁶ M) or p38 kinase (SB202190, 10⁻⁶ M) inhibitor for 30 min before being stimulated with 6.5 × 10⁴ IFU of strain TW183 per ml (MOI, 5). Infection of endothelial cells with CTK (MOI, 5) did not induce the expression of either IL-8 (A) or ICAM-1 (B). Data presented are the mean and standard error of the mean of four separate experiments. (A) #, P < 0.01 versus none; *, P < 0.01 versus TW183. Data for heated chlamydiae (“TW heated”) were not significantly different from those for mock-infected control cells (“none”).

Importance of endothelial cell-derived mediators for C. pneumoniae-mediated target cell activation.

Several recent studies have demonstrated the importance of paracrine target cell activation by endothelial cell-derived mediators on stimulation with different stimuli (2, 6). To rule out the importance of major endothelial products, HUVEC were pretreated for 30 min with 10 μM l-N-monomethyl orginine (l-NMMA) (inhibition of inducible nitric oxide synthase), 0.5 mM acetylsalicylic acid 1 μM (inhibition of cyclooxygenases), 10 μM MK-886 (inhibition of lipoxygenases), or BN 50727 (PAF receptor antagonist) and subsequently infected with strain TW-183 (MOI, 5). The level of IL-8 in the supernatant was determined by ELISA as described in Materials and Methods. Note that endothelial cell preincubation with each inhibitor did not significantly modify C. pneumoniae-mediated IL-8 expression (Fig. 5).

DISCUSSION

The study presented here demonstrates the following. (i) C. pneumoniae strain TW183 induced the phosphorylation and activation of the MAPK ERK1/2, p38, and JNK in human endothelial cells. (ii) p38-MAPK and—to a minor degree—ERK1/2 activity contributed substantially to C. pneumoniae-induced secretion of IL-8. TW183-associated ICAM-1 expression was only partly dependent on chlamydia-mediated p38- kinase and ERK1/2 activation. (iii) While C. trachomatis was also able to infect endothelial cells, it did not induce the phosphorylation of p38-MAPK or the expression of endothelial IL-8 or ICAM-1. (iv) These effects were specific for direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators. These observations have unearthed important new properties to this bacterium, namely, its capacity and specificity to initiate different early signal transduction events leading to endothelial cell activation. Endothelial cell infection in vivo is considered to occur via cell-to-cell spread from infected adherent mononuclear phagocytes (21). Moreover, mice intranasally infected with C. pneumoniae showed evidence of systemic chlamydial dissemination via macrophages (18). However little is known about the mechanisms of C. pneumoniae-induced target cell alteration.

Members of the MAPK family are ubiquitously expressed and activated in response to a variety of stimuli. We were recently able to identify the importance of ERK1/2 in C. pneumoniae-mediated endothelial cell activation (14). In addition, Coombes and Mahony could demonstrate that activation of the MEK-ERK1/2 pathway is important for C. pneumoniae invasion of epithelial cells (4). Our studies aimed to identify early intracellular signaling steps and demonstrated that phosphorylation and activation of all three MAPK pathways (ERK1/2, p38, and JNK) occurred within 10 to 15 min of chlamydial contact with endothelial cells. This immediate cell activation suggests that chlamydial attachment is sufficient to initiate an endothelial cell response and that bacterial uptake may not be required. Moreover, chlamydial heat treatment or UV inactivation did not reduce MAPK phosphorylation markedly, suggesting that viable bacteria may only in part be required for initial cell activation. Heat- or UV-killed chlamydiae were not able to infect endothelial cells, as demonstrated by CLSM. A profound and prolonged endothelial cell activation therefore could be initiated only by viable chlamydiae infecting the endothelial cells, suggesting that MAPK activation—initiated also by heat-resistant chlamydial membrane compounds—is insufficient, by itself, for this response. Endothelial cell activation by TW183 was followed by enhanced expression of IL-8 and ICAM-1. Inhibition of the MAPK pathway by specific inhibitors demonstrated that chlamydial stimulation of p38-MAPK and to a minor degree ERK1/2, but not JNK, appeared to be of particular importance for IL-8 secretion. ICAM-1 expression, however, could be only slightly reduced by p38-MAPK or ERK1/2 inhibition, suggesting that additional signal transduction pathways distal to MAPKs or two parallel signaling pathways are operative in C. pneumoniae-infected HUVEC.

MAPK phosphorylation and activation was already initiated by attachment of C. pneumoniae or chlamydial membrane compounds to the endothelial cell surface. Heat-resistant chlamydial membrane compounds such as LPS, chlamydial heat shock proteins (hsp60), outer membrane proteins (OMP-1 and OMP-2), and other not yet identified virulence factors are possible candidates for initiation of early signal transduction events in target cells. Preliminary experiments using a commercially available antibody against chlamydial OMP-1 suggested that this membrane component may indeed have some effects during target cell activation. TW183 preincubation with polymyxin B in order to inactivate chlamydial LPS had no effects on endothelial MAPK phosphorylation or subsequent cytokine expression (data not shown). Additional studies involving purified OMP-1, LPS, or groEL-1/hsp60 from C. pneumoniae as well as monoclonal antibodies against chlamydial LPS will help to clarify the role of these virulence factors during target cell activation.

Effects (MAPK phosphorylation and activation, expression of proinflammatory marker) were specific for C. pneumoniae since infection of endothelial cells with C. trachomatis did not induce phosphorylation of MAPK, release of endothelial IL-8, or upregulation of ICAM-1. Infection of HUVEC with C. trachomatis leads to the development of many small atypical serovar K inclusions. This “suboptimal” infection procedure could affect the results and may occur because endothelial cells are not primary target cells and so serovar K—although inducing a productive infection—might not grow very well in HUVEC. However, preliminary data demonstrated that C. trachomatis serovars E and L2 induced similar results (infection but no activation [data not shown]).

After initial attachment, chlamydiae are internalized; they dissociate themselves from the endocytotic pathway by actively modifying the vacuole to become fusogenic with exocytic vesicles. Interaction with this secretory pathway appears to provide a pathogenic mechanism that allows chlamydiae to establish themselves in a site that is not destined to fuse with lysosomes (9). Further studies are required to determine the relationship between distinct steps of this chlamydial development cycle, the importance of different chlamydial virulence factors, and the initiation of host cell signaling pathways.

Overall, the data presented suggest that C. pneumoniae triggers a cascade of early signal transduction events that could lead to endothelial damage and inflammation, which in turn may result in or may promote atherosclerosis. Development of a proinflammatory phenotype in endothelial cells is dependent on the existence of viable C. pneumoniae; C. trachomatis was without effect on HUVEC activation in our system.