- Email: [email protected]
Chlamydia pneumoniae and atherosclerosis: Links to the disease process
Bacterial Agents and Atherosclerosis Chairperson: David Taylor-Robinson
Chlamydia pneumoniae and atherosclerosis: Links to the disease process Gerald I. Byrne, PhD, and Murat V. Kalayoglu, PhD Madison, Wis
Chlamydia pneumoniae is an obligate intracellular prokaryotic human pathogen responsible for a significant portion of atypical pneumonia and associated with a variety of chronic sequelae, the most significant of which is atherosclerosis. The organism is endowed with several attributes that may contribute to the development of atherosclerotic lesions or promote tissue damage at the site of an existing lesion. Two key events that are directly involved in the atherogenic process include the development of foam cells from macrophages and the oxidation of lipoproteins at the site of lesion development. The former process allows for deposition of cholesterol-containing low-density lipoprotein (LDL) and the latter can contribute directly to tissue damage locally. We have hypothesized that C pneumoniae may interact with mononuclear phagocytes in ways that are consistent with the view that this organism contributes to atherosclerotic lesion development. We have demonstrated that the presence of C pneumoniae causes macrophage foam cell formation and lipid oxidation with murine and human cells cocultured in the presence of LDL. In addition, we have provided evidence that implicates 2 putative chlamydial virulence factors in the development of these pathologic processes. Chlamydial lipopolysaccharide has been shown to cause macrophages to develop into foam cells in the presence of LDL, and the 60-kDa chlamydial heat shock protein (cHsp60), a known pathogenesis-inducing protein, has been found to contribute to oxidation of LDL in the presence of macrophages. Work is currently underway to define mechanisms involved in these processes and to further refine the putative role of C pneumoniae in atherogenesis and atherosclerotic lesion development. (Am Heart J 1999;138:S488-S490.) Infectious causes for diseases with clear inflammatory components, such as cardiovascular disease,1-3 have been proposed for more than 100 years, but interest in this topic has recently reemerged because of a body of evidence implicating specific infectious agents in the development and progression of atherosclerosis and related cardiovascular disease (CVD) processes in people.4-6 One agent that has attracted particular attention is the obligate intracellular prokaryotic human pathogen, Chlamydia pneumoniae, because of a wealth of serologic associations in individuals with CVD, its identification and isolation from human atheromatous tissue, its causal associations with atherosclerotic lesion development in rabbits and lesion progression in mice, and the putative efficacy of secondary prevention antibiotic treatment trials in people (see other contributions to this supplement for details and references). To further substantiate or refute a role for C pneumoniae in the development or progression of CVD, we initiated a series of cell culture experiments From the Department of Medical Microbiology and Immunology, University of Wisconsin, Madison. Reprint requests: Gerald I. Byrne, PhD, Department of Medical Microbiology and Immunology, 1300 University Ave, University of Wisconsin–Madison, School of Medicine, Madison, WI 53706. E-mail: [email protected] Copyright © 1999 by Mosby, Inc. 0002-8703/99/$8.00 + 0 4/0/101754
focused on determining if the presence of C pneumoniae could influence changes in macrophage physiology consistent with pathologic events associated with atheroma development in the presence of low-density lipoprotein (LDL). We focused on measuring LDL uptake by macrophages (foam cell formation) and oxidative modification of LDL to products with demonstrable toxic activity (Figure 1). We also set out to determine if specific C pneumoniae virulence factors could be associated with the development of foam cells and oxidative modification of LDL.
C pneumoniae, foam cell formation, and uptake of LDL by macrophages Recently, we published a study that provides evidence that C pneumoniae has the capacity to induce foam cell formation by human monocyte–derived macrophages.7 We found that exposure of macrophages to C pneumoniae, followed by incubation in the presence of LDL (100 µg/mL) isolated from normolipidemic donors with the use of density gradient ultracentrifugation8 caused a marked increase in the number of foam cells. This was shown by oil-red-O staining of neutral lipids9 and cellassociated accumulation of cholesteryl esters.10 Use of various inhibitors provided evidence that classic scavenger receptor usage was not involved in LDL uptake by
American Heart Journal Volume 138, Number 5, Part 2
Byrne and Kalayoglu S489
Figure 1
Proposed role for C pneumoniae infection in the pathogenesis of atherosclerosis.
Table I. C pneumoniae–Mφ interactions as pathogenic mechanisms in atherosclerosis Event Macrophage foam cell formation C pneumoniae–infected macrophages exposed to high concentrations of LDL accumulate lipopolysaccharide esters LDL-dependent increase in foam cell formation Cellular oxidation of LDL C pneumoniae–infected monocytes enhance LDL oxidation Inhibitable by vitamin E LDL-dependent increase in LDL oxidation
Predominant virulence determinant c- Lipopolysaccharide c-Lipopolysaccharide is sufficient to induce foam cell formation and LDL uptake Lipid X inhibits C pneumoniae–induced and c-Lipopolysaccharide–induced foam cell formation chsp60 chsp60 is sufficient to induce monocyte LDL oxidation Inhibitable by vitamin E
Possible mechanism
Does not involve classic native apolipoprotein B/E LDL receptor ?Increased accumulation of cholesterol by enhanced expression of promiscuous lipoprotein receptors Superoxide independent
Mφ, Mononuclear phagocytes; c-Lipopolysaccharide, chlamydial lipopolysaccharide; chsp60, chlamydial heat shock protein 60; LDL, low-density lipoprotein.
macrophages in the presence of C pneumoniae but rather native LDL was taken up in an apparent specific fashion. This potentially unique finding is controversial and will require additional studies to more precisely measure if minimal modification of LDL has occurred. In addition, it will be important to assess what contribution bulk LDL uptake makes in the presence of C pneumoniae and definitively determine a role for specific receptors in the process. Subsequent studies11 were done to provide evidence that induction of foam cell formation and cholesteryl ester accumulation by C pneumoniae occurred in the presence of LDL even after the pathogen was heated at 95° C for 60 minutes, whereas treatment with 25 mmol/L periodate in acetate buffer significantly impaired activity. When C pneumoniae was replaced by chlamydial lipopolysaccharide in the assay systems, it was found that this chlamydia-derived molecule was sufficient to induce foam cell formation and cholesteryl ester accumulation by macrophages incubated in the presence of LDL. Significantly, lipid X, a known lipopolysaccharide antago-
nist,12 interfered with the capacity of chlamydial lipopolysaccharide to induce foam cell formation in a dose-dependent way. These data are summarized in Table I and indicate how chlamydial lipopolysaccharide may serve as a virulence factor in a developing atherosclerotic lesion. Given that lipopolysaccharide is a commonly encountered bacterial product, it is important to identify agents that can not only induce cellular lipid accumulation but also be associated with atherosclerosis and be localized to atheromas. The scheme presented in Figure 2 shows how C pneumoniae exhibits attributes consistent with atheroma development and is known to be present at the site of disease.
C pneumoniae and the cellular oxidation of LDL Oxidized LDL can lead directly to tissue damage in atherosclerosis by a variety of mechanisms. To study the effects of C pneumoniae on macrophage-mediated oxidation, infected and uninfected human monocytes
American Heart Journal November 1999
S490 Byrne and Kalayoglu
Figure 2
genic potentiating form. Results presented here demonstrate that C pneumoniae can cause both of these activities when incubated in the presence of macrophages and LDL. Furthermore, specific chlamydial molecules (lipopolysaccharide, Hsp60) have been associated with each of these processes (Table I and Figure 1). Taken together, these data support a role for C pneumoniae in the development and progression of atherosclerosis and suggest that this organism may be playing an active role in tissue damage associated with atheroma development rather than being a mere innocent bystander. Work is currently underway to identify more precisely the mechanisms involved in lipopolysaccharide-mediated foam cell formation and Hsp60-mediated oxidation of LDL.
References
C pneumoniae and induction of cellular lipid accumulation.
were cultured in the presence of native LDL, and oxidation was quantified by measuring the accumulation of thiobarbituric reactive substances (TBARS) using a standard fluorometric procedure13 modified for use in microtiter assays.14 Results of this work suggest that in the presence of C pneumoniae and macrophages, oxidation of LDL into its highly atherogenic form occurred in a manner independent of superoxide radical generation. Additionally, it was found that heat treatment of C pneumoniae rendered it incapable of contributing to the induction of LDL oxidation, ruling out lipopolysaccharide as the inducing agent. Interestingly, at the time that these studies were being done, Kol et al15 reported that chlamydial heat shock protein 60 (cHsp60), a protein with known pathogenic potential in well-defined chronic chlamydial diseases,16 was present in a relatively high portion (47%) of atheromatous tissue collected from human surgical specimens. When experiments were repeated with the use of recombinant cHsp60 in place of whole organisms, it was found that cHsp60 was capable of inducing LDL oxidation in a manner similar to that observed with C pneumoniae itself. These data are summarized in Table I.
Conclusions Two key events in the initiation and development of atherosclerotic lesions are production of foam cells from macrophages and the oxidation of LDL into an athero-
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801-9. 2. Mendall M, Patel P, Ballam L, et al. C-reactive protein and its relation to cardiovascular risk factors: a population based cross sectional study. BMJ 1996;313:1061-5. 3. Ridker P, Cushman M, Stampfer M, et al. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. 1997. N Engl J Med 1997;336:973-9. 4. Libby P, Egan D, Skarlatos S. Roles of infectious agents in atherosclerosis and restenosis: an assessment of the evidence and need for future research. Circulation 1997;96:4095-103. 5. Coles KA, Plant AJ, Riley TV, et al. Cardiovascular disease: an infectious aetiology? Rev Med Microbiol 1998;9:17-27. 6. Gura T. Infections: a cause of artery-clogging plaques? Science 1998;281:35-6. 7. Kalayoglu MV, Byrne GI. Induction of macrophage foam cell formation by Chlamydia pneumoniae. J Infect Dis 1998;177:725-9. 8. Redgrave T, Roberts D, West C. Separation of plasma lipoproteins by density gradient ultracentrifugation. Anal Biochem 1975;65:42-9. 9. Huh H, Pearce S, Yesner L, et al. Regulated expression of CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam cell formation. Blood 1996;87:2020-8. 10. Gamble W, Vaughn M, Kruth H, et al. Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. J Lipid Res 1978;19:1068-70. 11. Kalayoglu MV, Byrne GI. A Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccharide. Infect Immun 1998;66:5067-72. 12. Raetz C. Biochemistry of endotoxins. Ann Rev Biochem 1990;59: 129-70. 13. Janero D. Malondialdehyde and thiobarbituric acid reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515-40. 14. Kalayoglu MV, Hoerneman B, LaVerda D, et al. Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae. J Infect Dis 1999;180:780-90. 15. Kol A, Sukhova GK, Lichtman AH, et al. Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-α and matrix metalloproteinase expression. Circulation 1998;98:300-7. 16. Brunham RC, Peeling RW. Chlamydia trachomatis antigens: role in immunity and pathogenesis. Infect Agents Dis 1994;3:218-33.
Chlamydia pneumoniae and atherosclerosis: Links to the disease process Gerald I. Byrne, PhD, and Murat V. Kalayoglu, PhD Madison, Wis
Chlamydia pneumoniae is an obligate intracellular prokaryotic human pathogen responsible for a significant portion of atypical pneumonia and associated with a variety of chronic sequelae, the most significant of which is atherosclerosis. The organism is endowed with several attributes that may contribute to the development of atherosclerotic lesions or promote tissue damage at the site of an existing lesion. Two key events that are directly involved in the atherogenic process include the development of foam cells from macrophages and the oxidation of lipoproteins at the site of lesion development. The former process allows for deposition of cholesterol-containing low-density lipoprotein (LDL) and the latter can contribute directly to tissue damage locally. We have hypothesized that C pneumoniae may interact with mononuclear phagocytes in ways that are consistent with the view that this organism contributes to atherosclerotic lesion development. We have demonstrated that the presence of C pneumoniae causes macrophage foam cell formation and lipid oxidation with murine and human cells cocultured in the presence of LDL. In addition, we have provided evidence that implicates 2 putative chlamydial virulence factors in the development of these pathologic processes. Chlamydial lipopolysaccharide has been shown to cause macrophages to develop into foam cells in the presence of LDL, and the 60-kDa chlamydial heat shock protein (cHsp60), a known pathogenesis-inducing protein, has been found to contribute to oxidation of LDL in the presence of macrophages. Work is currently underway to define mechanisms involved in these processes and to further refine the putative role of C pneumoniae in atherogenesis and atherosclerotic lesion development. (Am Heart J 1999;138:S488-S490.) Infectious causes for diseases with clear inflammatory components, such as cardiovascular disease,1-3 have been proposed for more than 100 years, but interest in this topic has recently reemerged because of a body of evidence implicating specific infectious agents in the development and progression of atherosclerosis and related cardiovascular disease (CVD) processes in people.4-6 One agent that has attracted particular attention is the obligate intracellular prokaryotic human pathogen, Chlamydia pneumoniae, because of a wealth of serologic associations in individuals with CVD, its identification and isolation from human atheromatous tissue, its causal associations with atherosclerotic lesion development in rabbits and lesion progression in mice, and the putative efficacy of secondary prevention antibiotic treatment trials in people (see other contributions to this supplement for details and references). To further substantiate or refute a role for C pneumoniae in the development or progression of CVD, we initiated a series of cell culture experiments From the Department of Medical Microbiology and Immunology, University of Wisconsin, Madison. Reprint requests: Gerald I. Byrne, PhD, Department of Medical Microbiology and Immunology, 1300 University Ave, University of Wisconsin–Madison, School of Medicine, Madison, WI 53706. E-mail: [email protected] Copyright © 1999 by Mosby, Inc. 0002-8703/99/$8.00 + 0 4/0/101754
focused on determining if the presence of C pneumoniae could influence changes in macrophage physiology consistent with pathologic events associated with atheroma development in the presence of low-density lipoprotein (LDL). We focused on measuring LDL uptake by macrophages (foam cell formation) and oxidative modification of LDL to products with demonstrable toxic activity (Figure 1). We also set out to determine if specific C pneumoniae virulence factors could be associated with the development of foam cells and oxidative modification of LDL.
C pneumoniae, foam cell formation, and uptake of LDL by macrophages Recently, we published a study that provides evidence that C pneumoniae has the capacity to induce foam cell formation by human monocyte–derived macrophages.7 We found that exposure of macrophages to C pneumoniae, followed by incubation in the presence of LDL (100 µg/mL) isolated from normolipidemic donors with the use of density gradient ultracentrifugation8 caused a marked increase in the number of foam cells. This was shown by oil-red-O staining of neutral lipids9 and cellassociated accumulation of cholesteryl esters.10 Use of various inhibitors provided evidence that classic scavenger receptor usage was not involved in LDL uptake by
American Heart Journal Volume 138, Number 5, Part 2
Byrne and Kalayoglu S489
Figure 1
Proposed role for C pneumoniae infection in the pathogenesis of atherosclerosis.
Table I. C pneumoniae–Mφ interactions as pathogenic mechanisms in atherosclerosis Event Macrophage foam cell formation C pneumoniae–infected macrophages exposed to high concentrations of LDL accumulate lipopolysaccharide esters LDL-dependent increase in foam cell formation Cellular oxidation of LDL C pneumoniae–infected monocytes enhance LDL oxidation Inhibitable by vitamin E LDL-dependent increase in LDL oxidation
Predominant virulence determinant c- Lipopolysaccharide c-Lipopolysaccharide is sufficient to induce foam cell formation and LDL uptake Lipid X inhibits C pneumoniae–induced and c-Lipopolysaccharide–induced foam cell formation chsp60 chsp60 is sufficient to induce monocyte LDL oxidation Inhibitable by vitamin E
Possible mechanism
Does not involve classic native apolipoprotein B/E LDL receptor ?Increased accumulation of cholesterol by enhanced expression of promiscuous lipoprotein receptors Superoxide independent
Mφ, Mononuclear phagocytes; c-Lipopolysaccharide, chlamydial lipopolysaccharide; chsp60, chlamydial heat shock protein 60; LDL, low-density lipoprotein.
macrophages in the presence of C pneumoniae but rather native LDL was taken up in an apparent specific fashion. This potentially unique finding is controversial and will require additional studies to more precisely measure if minimal modification of LDL has occurred. In addition, it will be important to assess what contribution bulk LDL uptake makes in the presence of C pneumoniae and definitively determine a role for specific receptors in the process. Subsequent studies11 were done to provide evidence that induction of foam cell formation and cholesteryl ester accumulation by C pneumoniae occurred in the presence of LDL even after the pathogen was heated at 95° C for 60 minutes, whereas treatment with 25 mmol/L periodate in acetate buffer significantly impaired activity. When C pneumoniae was replaced by chlamydial lipopolysaccharide in the assay systems, it was found that this chlamydia-derived molecule was sufficient to induce foam cell formation and cholesteryl ester accumulation by macrophages incubated in the presence of LDL. Significantly, lipid X, a known lipopolysaccharide antago-
nist,12 interfered with the capacity of chlamydial lipopolysaccharide to induce foam cell formation in a dose-dependent way. These data are summarized in Table I and indicate how chlamydial lipopolysaccharide may serve as a virulence factor in a developing atherosclerotic lesion. Given that lipopolysaccharide is a commonly encountered bacterial product, it is important to identify agents that can not only induce cellular lipid accumulation but also be associated with atherosclerosis and be localized to atheromas. The scheme presented in Figure 2 shows how C pneumoniae exhibits attributes consistent with atheroma development and is known to be present at the site of disease.
C pneumoniae and the cellular oxidation of LDL Oxidized LDL can lead directly to tissue damage in atherosclerosis by a variety of mechanisms. To study the effects of C pneumoniae on macrophage-mediated oxidation, infected and uninfected human monocytes
American Heart Journal November 1999
S490 Byrne and Kalayoglu
Figure 2
genic potentiating form. Results presented here demonstrate that C pneumoniae can cause both of these activities when incubated in the presence of macrophages and LDL. Furthermore, specific chlamydial molecules (lipopolysaccharide, Hsp60) have been associated with each of these processes (Table I and Figure 1). Taken together, these data support a role for C pneumoniae in the development and progression of atherosclerosis and suggest that this organism may be playing an active role in tissue damage associated with atheroma development rather than being a mere innocent bystander. Work is currently underway to identify more precisely the mechanisms involved in lipopolysaccharide-mediated foam cell formation and Hsp60-mediated oxidation of LDL.
References
C pneumoniae and induction of cellular lipid accumulation.
were cultured in the presence of native LDL, and oxidation was quantified by measuring the accumulation of thiobarbituric reactive substances (TBARS) using a standard fluorometric procedure13 modified for use in microtiter assays.14 Results of this work suggest that in the presence of C pneumoniae and macrophages, oxidation of LDL into its highly atherogenic form occurred in a manner independent of superoxide radical generation. Additionally, it was found that heat treatment of C pneumoniae rendered it incapable of contributing to the induction of LDL oxidation, ruling out lipopolysaccharide as the inducing agent. Interestingly, at the time that these studies were being done, Kol et al15 reported that chlamydial heat shock protein 60 (cHsp60), a protein with known pathogenic potential in well-defined chronic chlamydial diseases,16 was present in a relatively high portion (47%) of atheromatous tissue collected from human surgical specimens. When experiments were repeated with the use of recombinant cHsp60 in place of whole organisms, it was found that cHsp60 was capable of inducing LDL oxidation in a manner similar to that observed with C pneumoniae itself. These data are summarized in Table I.
Conclusions Two key events in the initiation and development of atherosclerotic lesions are production of foam cells from macrophages and the oxidation of LDL into an athero-
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801-9. 2. Mendall M, Patel P, Ballam L, et al. C-reactive protein and its relation to cardiovascular risk factors: a population based cross sectional study. BMJ 1996;313:1061-5. 3. Ridker P, Cushman M, Stampfer M, et al. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. 1997. N Engl J Med 1997;336:973-9. 4. Libby P, Egan D, Skarlatos S. Roles of infectious agents in atherosclerosis and restenosis: an assessment of the evidence and need for future research. Circulation 1997;96:4095-103. 5. Coles KA, Plant AJ, Riley TV, et al. Cardiovascular disease: an infectious aetiology? Rev Med Microbiol 1998;9:17-27. 6. Gura T. Infections: a cause of artery-clogging plaques? Science 1998;281:35-6. 7. Kalayoglu MV, Byrne GI. Induction of macrophage foam cell formation by Chlamydia pneumoniae. J Infect Dis 1998;177:725-9. 8. Redgrave T, Roberts D, West C. Separation of plasma lipoproteins by density gradient ultracentrifugation. Anal Biochem 1975;65:42-9. 9. Huh H, Pearce S, Yesner L, et al. Regulated expression of CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam cell formation. Blood 1996;87:2020-8. 10. Gamble W, Vaughn M, Kruth H, et al. Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. J Lipid Res 1978;19:1068-70. 11. Kalayoglu MV, Byrne GI. A Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccharide. Infect Immun 1998;66:5067-72. 12. Raetz C. Biochemistry of endotoxins. Ann Rev Biochem 1990;59: 129-70. 13. Janero D. Malondialdehyde and thiobarbituric acid reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515-40. 14. Kalayoglu MV, Hoerneman B, LaVerda D, et al. Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae. J Infect Dis 1999;180:780-90. 15. Kol A, Sukhova GK, Lichtman AH, et al. Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-α and matrix metalloproteinase expression. Circulation 1998;98:300-7. 16. Brunham RC, Peeling RW. Chlamydia trachomatis antigens: role in immunity and pathogenesis. Infect Agents Dis 1994;3:218-33.