INFECTION AND IMMUNITY, Aug. 2004, p. 4900–4904 0019-9567/04/$08.00⫹0 DOI: 10.1128/IAI.72.8.4900–4904.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 8
Chlamydia pneumoniae Decreases Smooth Muscle Cell Proliferation through Induction of Prostaglandin E2 Synthesis Ju ¨rgen Ro ¨del,1* Dirk Prochnau,2 Katrin Prager,1 Ju ¨rgen Baumert,1 1 Karl-Hermann Schmidt, and Eberhard Straube1 Institute of Medical Microbiology1 and Clinic of Internal Medicine, Department of Cardiology,2 Friedrich Schiller University of Jena, D-07740 Jena, Germany Received 23 January 2004/Returned for modification 25 February 2004/Accepted 27 April 2004
Chlamydia pneumoniae may modulate the proliferation of smooth muscle cells (SMC) in atherosclerotic plaques. Conditioned medium from C. pneumoniae-infected SMC decreased the proliferation of uninfected SMC. Treatment of infected cells with the cyclooxygenase-2 inhibitor NS-398 {N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide} suppressed the up-regulation of prostaglandin E2 (PGE2) and abolished the antimitogenic effect of conditioned medium, suggesting that C. pneumoniae can decrease SMC proliferation via stimulation of PGE2 synthesis. Atherosclerotic diseases have been linked to Chlamydia pneumoniae infection (reviewed in reference 1). The organism has been found within endothelial cells, macrophages, and smooth muscle cells (SMC) in atherosclerotic plaques (25). C. pneumoniae may contribute to the pathogenesis of atherosclerosis by eliciting cellular responses associated with inflammation, cell proliferation, and tissue remodeling (13). During atherogenesis, SMC migrate from the media to the intima. Proliferation of neointimal SMC and production of extracellular matrix proteins by these cells result in the formation of a fibrous cap overlying the prothrombotic lipid-rich core of the atherosclerotic plaque (11). Growth factors, such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), are thought to be largely responsible for the accumulation of SMC in the intima (8, 10, 14). Coombes et al. reported that C. pneumoniae infection of endothelial cells increases the expression of PDGF-BB (PDGF containing two B chains), which may contribute to the intimal thickening of aortic tissue in Chlamydia-infected rabbits (4). Besides endothelial cells, SMC themselves are discussed as an important source of growth factors in atherosclerotic plaques (14). A previous paper reported that SMC produce increased amounts of bFGF in response to infection with C. pneumoniae (18). Therefore, the aim of this study was to investigate the effects of conditioned medium from Chlamydia-infected SMC on the proliferation of uninfected SMC. Human vascular SMC (C-12511; PromoCell, Heidelberg, Germany) were infected with C. pneumoniae TW-183 (obtained from the Institute of Ophthalmology, London, United Kingdom) at a multiplicity of infection (MOI) of 5 as previously described (17). For heat inactivation, chlamydial suspensions were held at 75°C for 10 min prior to inoculation onto cell monolayers. For UV inactivation, chlamydial suspensions were placed under a UV lamp (15 W at 30 cm) for 15 min. Mock-infected and infected SMC were incubated with SMC
basal medium (PromoCell) containing 1% fetal calf serum (FCS) but no antibiotics. In some experiments, mock-infected and infected SMC were treated with NS-398 {N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide; Qbiogene-Alexis, Gru ¨nberg, Germany}. Chlamydial inclusions in infected cultures were visualized by immunofluorescence staining as previously described (17). Conditioned media from infected and mock-infected cultures were collected at 2, 24, 48, and 72 h after infection and clarified by centrifugation at 20,000 ⫻ g for 30 min before inoculation onto uninfected SMC. DNA synthesis in SMC was assessed as described by Padro ´ et al. with modifications (15). SMC were seeded onto 96-well plates at a density of 103 cells per well and incubated with basal medium containing 10% FCS for 24 h. The cells were then maintained in basal medium containing 1% FCS for a further 72 h. After this period, cells were incubated with conditioned media from infected and mock-infected SMC (100 l per well). In some experiments, SMC were treated with medium containing 1% FCS and prostaglandin E2 (PGE2; Qbiogene-Alexis). Complete SMC growth medium consisting of basal medium, 5% FCS, 0.5 ng of human recombinant epidermal growth factor (PromoCell) per ml, 2 ng of human recombinant bFGF (PromoCell) per ml, and 5 g of bovine insulin (PromoCell) per ml was used as a positive control for SMC proliferation. The next day, 5-bromo-2-deoxyuridine (BrdU; 10 M; Amersham Biosciences, Freiburg, Germany) was added, and the cultures were maintained for a further 24 h. Incorporation of BrdU into the DNA of proliferating cells was measured by enzyme-linked immunosorbent assay (Amersham Biosciences) according to the manufacturer’s protocol. Results were expressed in percentages as the ratio of absorbance of conditioned medium to that of SMC growth medium. For counting cells, SMC were plated onto 12-well plates at 104 cells per well, incubated with medium containing 10% FCS for 24 h, and then maintained in medium with 1% FCS for 72 h. The next day, SMC were treated with conditioned medium (1 ml per well) for a period of 4 days. For some experiments, cells were overlaid with 1:1 mixtures of complete SMC growth medium and conditioned medium. SMC numbers were determined every other day. Cells were trypsinized, resus-
* Corresponding author. Mailing address: Institute of Medical Microbiology, Friedrich Schiller University of Jena, Semmelweisstr. 4, D-07740 Jena, Germany. Phone: 49-3641-933105. Fax: 49-3641933474. E-mail:
[email protected]. 4900
VOL. 72, 2004
NOTES
4901
FIG. 1. DNA synthesis in SMC in response to conditioned medium harvested from C. pneumoniae-infected SMC. (A) Treatment of uninfected SMC with conditioned media from mock-infected and infected cells. Media were collected at 2, 24, 48, and 72 h after infection. Values are means (with standard deviations) of results from eight experiments. (B) Comparison of the effects of conditioned media from SMC infected with viable chlamydiae and those exposed to UV- and heat-inactivated bacteria. Media were collected at 24 h after infection. Values are means (with standard deviations) of results from four experiments. *, P of ⱕ0.001 compared to conditioned medium from mock-infected SMC; **, P of ⱕ0.01 compared to conditioned medium from SMC exposed to inactivated bacteria (Welch t test).
pended in medium, and counted under the microscope in a Neubauer chamber. Levels of PGE2 in conditioned medium were measured by enzyme-linked immunosorbent assay (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol. To examine whether soluble factors produced by C. pneumoniae-infected SMC modulate SMC proliferation, conditioned media collected from mock-infected and infected SMC cultures were examined for the ability to stimulate DNA synthesis in SMC. When SMC were incubated with medium containing 1% FCS as used in the infection experiments, the incorporation of BrdU was decreased by 60% compared to that measured when SMC were incubated with complete SMC growth medium (Fig. 1A). We found no differences in levels of DNA synthesis between SMC cultured with fresh medium containing 1% FCS and SMC stimulated with conditioned medium from mock-infected cells. However, conditioned medium harvested from Chlamydia-infected cells at 24, 48, and 72 h after infection caused a significant reduction in BrdU uptake in SMC compared to that in SMC incubated with conditioned medium from mock-infected SMC (Fig. 1A). Conditioned medium collected at 2 h after infection had no inhibitory effect on BrdU incorporation (Fig. 1A). The findings suggest that infected SMC released factors inhibiting SMC proliferation. The induction of antimitogenic factors required the infection of SMC by viable chlamydiae because conditioned medium prepared after exposure of cells to heat- and UV-inactivated bacteria did not markedly decrease BrdU uptake in SMC (Fig.
1B). Antimitogenic activities of conditioned medium could be found following chlamydial infection of SMC at different MOIs ranging from 2 to 20 (data not shown). PGE2 has been described to function as a negative regulator of SMC proliferation (24). C. pneumoniae stimulated the production of PGE2 by SMC (Fig. 2A). The release of PGE2 was slightly increased at 2 h, and large amounts of PGE2 were produced at 24, 48, and 72 h after infection (Fig. 2A). Because cyclooxygenase-2 (COX-2) is one of the key enzymes catalyzing PGE2 synthesis, conditioned medium was prepared in the presence of NS-398, a selective inhibitor of COX-2 activity (7). Treatment of infected cells with NS-398 not only suppressed the production of PGE2 but also abolished the capacity of conditioned medium to inhibit DNA synthesis in SMC, indicating that the antiproliferative factor induced by C. pneumoniae was a prostanoid (Fig. 2B and C). The percentages of Chlamydia-positive cells in infected cultures did not significantly differ between SMC with NS-398 treatment and those without NS-398 treatment (9% ⫾ 2% versus 12% ⫾ 3% inclusion-positive cells following infection at an MOI of 5). In comparative experiments, exogenous PGE2 induced a significant decrease in BrdU uptake in SMC at a concentration of 3,000 pg per ml, which corresponds to the amounts of PGE2 found in conditioned medium harvested from Chlamydia-infected cells at 24, 48, and 72 h after infection (Fig. 2D). In contrast, a low PGE2 concentration of 300 pg per ml had no inhibitory effect on BrdU incorporation (Fig. 2D). Direct cell counting was performed to evaluate whether the
4902
NOTES
INFECT. IMMUN.
FIG. 2. Role of PGE2 in decreased SMC proliferation. (A) PGE2 concentrations in conditioned media collected from mock-infected and C. pneumoniae-infected SMC. (B) PGE2 synthesis by C. pneumoniae-infected SMC in the absence or presence of NS-398 (10 M). PGE2 concentrations were measured at 24 h after infection. (C) DNA synthesis in SMC in response to conditioned media collected from SMC after NS-398 treatment. Conditioned media were harvested at 24 h after infection. (D) Effect of exogenous PGE2 on DNA synthesis in SMC. (A to D) Values are means (with standard deviations) of results from four experiments. *, P of ⱕ0.01 compared to values for mock-infected SMC at 24 h and infected SMC at 2 h; **, P of ⱕ0.001 compared to infected SMC without NS-398 treatment; ***, P of ⱕ0.01 compared to conditioned medium from infected SMC without NS-398 treatment; ****, P of ⱕ0.01 compared to medium without PGE2 (Welch t test).
VOL. 72, 2004
NOTES
4903
FIG. 3. SMC numbers in cultures treated with conditioned medium from C. pneumoniae-infected SMC. (A) Replication of SMC in response to conditioned medium from mock-infected SMC (F), conditioned medium from C. pneumoniae-infected SMC (■), and complete SMC growth medium (Œ). (B) Numbers of SMC after incubation with 1:1 mixtures of complete SMC growth medium and conditioned medium from mock-infected cells (E), conditioned medium from infected cells (䊐), or fresh medium containing 1% FCS (‚). (A and B) Cells were counted in a Neubauer chamber. Values are means (with standard deviations) of results from five experiments. *, P of ⱕ0.01 compared to conditioned medium from mock-infected SMC; **, P of ⱕ0.001 compared to the mixture of SMC growth medium and conditioned medium from mock-infected SMC (Welch t test).
inhibition of DNA synthesis in SMC after exposure to conditioned medium from Chlamydia-infected cells was accompanied by decreased cell proliferation. Conditioned medium from mock-infected cells had only moderate proliferative effects on SMC compared to complete SMC growth medium (Fig. 3A). However, cell numbers after 4 days of incubation were significantly higher in cultures overlaid with conditioned medium from mock-infected cells than in cultures treated with conditioned medium from C. pneumoniae-infected SMC (Fig. 3A). In further experiments, we investigated whether conditioned medium from Chlamydia-infected SMC can decrease the proliferation of SMC in the presence of complete growth medium. When conditioned medium from infected cells was added to SMC growth medium at a ratio of 1:1, cell numbers after 4 days in culture were reduced by 50% compared to those in cultures treated with conditioned medium from mock-infected SMC or fresh medium containing 1% FCS (Fig. 3B). Arterial C. pneumoniae infection may contribute to the pathogenesis of atherosclerosis by modulating SMC proliferation. This study shows that the infection of SMC by C. pneumoniae induces antiproliferative effects on uninfected SMC via secretion of PGE2. C. pneumoniae infection of SMC stimulated the production of PGE2, which could be abolished by treating the cells with the COX-2 inhibitor NS-398. This observation corresponds to those from previous work in which the induction of COX-2 expression and PGE2 synthesis in epithelial cells upon chlamydial infection was demonstrated (9, 26). PGE2 is known to inhibit mitogenesis in SMC by elevating cyclic AMP levels (16, 24). The suppression of PGE2 synthesis in SMC by NS-398
treatment abolished the capacity of conditioned medium to decrease SMC proliferation. Furthermore, the stimulation of SMC with exogenous PGE2 at a concentration found in conditioned medium caused a significant reduction in DNA synthesis. PGE2 has been described as being more effective in inhibition of SMC growth than other prostaglandins (12, 23). However, it cannot be excluded that other COX-2 products may also contribute to the antiproliferative effects of conditioned medium. The results of our proliferation assays seem to be in contrast to those of other studies that investigated effects of C. pneumoniae infection on SMC proliferation. Coombes et al. described an intimal thickening of aortic tissue in rabbits following infection with C. pneumoniae (4). The intimal thickening of the aortas correlated with the detection of both PDGF-BB and chlamydial antigen (4). The same group also reported that the conditioned medium from Chlamydia-infected endothelial cells stimulates the growth of SMC (3). The factor responsible for these effects was not identified, although increased expression of PDGF-BB by infected endothelial cells may be involved (4). It has previously been shown that C. pneumoniae stimulates SMC to produce bFGF (18). However, the findings of the present study indicate a positive net balance of growth inhibitory PGE2 versus potential growth stimulatory factors in the conditioned medium of infected SMC. In atherosclerotic plaques, PGE2 released from Chlamydia-infected SMC may inhibit the proliferation of neighboring SMC. On the other hand, it has to be considered that C. pneumoniae may also directly affect SMC growth (19, 20). Sasu et al. reported that
4904
NOTES
INFECT. IMMUN.
chlamydial heat shock protein 60 can activate the proliferation of SMC via Toll-like receptor 4 (19). The proliferation of SMC in the fibrous cap of atherosclerotic plaques is accompanied by the deposition of extracellular matrix proteins, thereby promoting plaque stability (11). Acute ischemic syndromes are usually due to the rupture of instable atherosclerotic plaques and subsequent thrombosis (11, 21). In symptomatic lesions, the fibrous cap can rupture as a result of matrix degradation caused by matrix metalloproteinases and the reduction in the number of SMC following increased apoptosis and decreased proliferation (11, 21, 22). Since COX-2 and PGE2 synthase are overexpressed in symptomatic lesions, the up-regulation of PGE2 is discussed as playing a role in decreased SMC proliferation and in plaque destabilization (2, 5). Recently, Ezzahiri et al. reported that the infection of low-density lipoprotein receptor/ApoE⫺/⫺ mice with C. pneumoniae results in a significant decrease in the SMC content of the fibrous cap of atherosclerotic lesions in the aortic arch (6). The finding that C. pneumoniae infection of SMC decreases SMC proliferation via stimulation of PGE2 synthesis suggests that the interaction of vascular SMC with C. pneumoniae may contribute to mechanisms promoting the destabilization of atherosclerotic plaques.
9.
10.
11. 12. 13. 14. 15.
16. 17.
This work was supported by grant CAPNETZ 01KL0104 from the Bundesministerium fu ¨r Bildung und Forschung, Berlin, Germany.
18.
REFERENCES
19.
1. Boman, J., and M. R. Hammerschlag. 2002. Chlamydia pneumoniae and atherosclerosis: critical assessment of diagnostic methods and relevance to treatment studies. Clin. Microbiol. Rev. 15:1–20. 2. Cipollone, F., C. Prontera, B. Pini, M. Marini, M. Fazia, D. De Cesare, A. Iezzi, S. Ucchino, G. Boccoli, V. Saba., F. Chiarelli, F. Cuccurullo, and A. Mezzetti. 2001. Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E2-dependent plaque instability. Circulation 104:921– 927. 3. Coombes, B. K., and J. B. Mahony. 1999. Chlamydia pneumoniae infection of human endothelial cells induces proliferation of smooth muscle cells via an endothelial cell-derived soluble factor(s). Infect. Immun. 67:2909–2915. 4. Coombes, B. K., B. Chiu, I. W. Fong, and J. B. Mahony. 2002. Chlamydia pneumoniae infection of endothelial cells induces transcriptional activation of platelet-derived growth factor-B: a potential link to intimal thickening in a rabbit model of atherosclerosis. J. Infect. Dis. 185:1621–1630. 5. Dhume, A. S., and D. K. Agrawal. 2003. Inability of vascular smooth muscle cells to proceed beyond S phase of cell cycle, and increased apoptosis in symptomatic carotid artery disease. J. Vasc. Surg. 38:155–161. 6. Ezzahiri, R., F. R. M. Stassen, H. A. J. M. Kurvers, M. M. L. van Pul, P. J. E. H. M. Kitslaar, and C. A. Bruggeman. 2003. Chlamydia pneumoniae infection induces an unstable atherosclerotic plaque phenotype in LDLreceptor, ApoE double knockout mice. Eur. J. Vasc. Endovasc. Surg. 26:88– 95. 7. Futaki, N., S. Takahashi, M. Yokoyama, I. Arai, S. Higuchi, and S. Otomo. 1994. NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro. Prostaglandins 47:55–59. 8. Gouni-Berthold, I., and A. Sachinidis. 2002. Does the coronary risk factor
Editor: J. N. Weiser
20.
21. 22.
23.
24.
25. 26.
low density lipoprotein alter growth and signalling in vascular smooth muscle cells? FASEB J. 16:1477–1487. Jahn, H. U., M. Kru ¨ll, F. N. Wuppermann, A. C. Klucken, S. Rosseau, J. Seybold, J. H. Hegemann, C. A. Jantos, and N. Suttorp. 2000. Infection and activation of airway epithelial cells by Chlamydia pneumoniae. J. Infect. Dis. 182:1678–1687. Kozaki, K., W. E. Kaminski, J. Tang, S. Hollenbach, P. Lindhal, C. Sullivan, J.-C. Yu, K. Abe, P. J. Martin, R. Ross, C. Betsholtz, N. A. Giese, and E. W. Raines. 2002. Blockade of platelet-derived growth factor or its receptors transiently delays but does not prevent fibrous cap formation in ApoE null mice. Am. J. Pathol. 161:1395–1407. Libby, P. 2000. Changing concepts of atherogenesis. J. Intern. Med. 247: 349–358. Libby, P., S. J. Warner, and G. B. Friedman. 1988. Interleukin 1: a mitogen for human vascular smooth muscle cells that induces the release of growthinhibitory prostanoids. J. Clin. Investig. 81:487–498. Mahony, J. B., and B. K. Coombes. 2001. Chlamydia pneumoniae and atherosclerosis: does the evidence support a causal or contributory role? FEMS Microbiol. Lett. 197:1–9. Newby, A. C., and A. B. Zaltsman. 2000. Molecular mechanisms in intimal hyperplasia. J. Pathol. 190:300–309. Padro ´, T., R. M. Mesters, B. Dankbar, H. Hintelmann, R. Bieker, M. Kiehl, W. E. Berdel, and J. Kienast. 2002. The catalytic domain of endogenous urokinase-type plasminogen activator is required for the mitogenic activity of platelet-derived and basic fibroblast growth factors in human vascular smooth muscle cells. J. Cell Sci. 115:1961–1971. Proudfoot, D., C. Fitzsimmons, J. Torzewski, and D. E. Bowyer. 1999. Inhibition of human arterial smooth muscle cell growth by human monocyte/ macrophages: a co-culture study. Atherosclerosis 145:157–165. Ro ¨del, J., D. Prochnau, K. Prager, E. Pentcheva, M. Hartmann, and E. Straube. 2003. Increased production of matrix metalloproteinases 1 and 3 by smooth muscle cells upon infection with Chlamydia pneumoniae. FEMS Immunol. Med. Microbiol. 38:159–164. Ro ¨del, J., M. Woytas, A. Groh, K.-H. Schmidt, M. Hartmann, M. Lehmann, and E. Straube. 2000. Production of basic fibroblast growth factor and interleukin 6 by human smooth muscle cells following infection with Chlamydia pneumoniae. Infect. Immun. 68:3635–3641. Sasu, S., D. LaVerda, N. Qureshi, D. T. Golenbock, and D. Beasley. 2001. Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation. Circ. Res. 89:244–250. Selzman, C. H., M. G. Netea, M. A. Zimmermann, A. Weinberg, L. L. Reznikov, F. I. Grover, and C. A. Dinarello. 2003. Atherogenic effects of Chlamydia pneumoniae: refuting the innocent bystander hypothesis. J. Thorac. Cardiovasc. Surg. 126:688–693. Shah, P. K. 2003. Mechanisms of plaque vulnerability and rupture. J. Am. Coll. Cardiol. 41:15S–22S. Sukhova, G. K., U. Scho ¨nbeck, E. Rabkin, F. J. Schoen, A. R. Poole, R. C. Billinghurst, and P. Libby. 1999. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation 99:2503–2509. Walton, L. J., I. J. Franklin, T. Bayston, L. C. Brown, R. M. Greenhalgh, G. W. Taylor, and J. T. Powell. 1999. Inhibition of prostaglandin E2 synthesis in abdominal aortic aneurysms: implications for smooth muscle cell viability, inflammatory processes, and the expansion of abdominal aortic aneurysms. Circulation 100:48–54. Wong, S. T., L. P. Baker, K. Trinh, M. Hetman, L. A. Suzuki, D. R. Storm, and K. E. Bornfeldt. 2001. Adenylyl cyclase 3 mediates prostaglandin E2induced growth inhibition in arterial smooth muscle cells. J. Biol. Chem. 276:34206–34212. Yamashita, K., K. Ouchi, M. Shirai, T. Gondo, T. Nakazawa, and H. Itoh. 1998. Distribution of Chlamydia pneumoniae infection in the atherosclerotic carotid artery. Stroke 29:773–778. Yoneda, H., K. Miura, H. Matsushima, K. Sugi, T. Murakami, K. Ouchi, K. Yamashita, H. Itoh, T. Nakazawa, M. Suzuki, and M. Shirai. 2003. Aspirin inhibits Chlamydia pneumoniae-induced NFB activation, cyclo-oxygenase-2 expression and prostaglandin E2 synthesis and attenuates chlamydial growth. J. Med. Microbiol. 52:409–415.
