(Circulation. 2000;101:1760.)
© 2000 American Heart Association, Inc.
Brief Rapid Communications |
Hydroxymethylglutaryl Coenzyme A Reductase Inhibitors Modify the Inflammatory Response of Human Macrophages and Endothelial Cells Infected With Chlamydia pneumoniae
From the Department of Internal Medicine II (H.K., K.D., J.R., H.A.K.), the Institute of Microbiology (M.M.), the Department of Rheumatology (A.M.), Medical University of Lübeck; and the Department for Internal Medicine III (J.K.), University of Heidelberg, Germany.
Correspondence to K. Dalhoff, Med Klinik II, Medizinische Universität, Ratzeburger Allee 160, D-23538 Lübeck, Germany.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—In patients with atherosclerosis, hepatic hydroxymethylglutaryl coenzyme A reductase (CSE) inhibitors may reduce the activation of inflammation. Because Chlamydia pneumoniae infection has been linked to coronary artery disease through the induction of plaque inflammation, we investigated whether cerivastatin affects the infection rate of human macrophages and endothelial cells (ECs) and their proinflammatory activation after chlamydial infection.
Methods and Results—Macrophages were collected from the
alveolar compartment of 6 volunteers and 10 patients with chronic
bronchitis. ECs were obtained from 10 umbilical cords. The C.
pneumoniae strain CWL was incubated with macrophages or
ECs in the presence and absence of the CSE inhibitor
cerivastatin. The infection rate was determined by
immunofluorescence microscopy. The release of
monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and
tumor necrosis factor (TNF)-
was quantified by ELISA. The release of
oxygen radicals was determined by ferricytochrome assay. Infection
rates were tendentially lower after the preincubation of
macrophages with CSE inhibitors (17.2% versus
9.3% and 18.2% versus 10.4%, respectively; P=NS). The
secretion of MCP-1, IL-8, and TNF- by infected macrophages
from volunteers increased. Coincubation with cerivastatin resulted in
significantly lower MCP-1 and IL-8 production, whereas the
release of TNF- remained unaffected. Similar effects regarding
chemokine release were observed in ECs.
Conclusions—CSE inhibitors modify the inflammatory response of human immune cells to C. pneumoniae. This finding could be relevant for the therapeutic potential of CSE statins in patients with atherosclerosis and C. pneumoniae infection.
Key Words: inflammation • endothelium • atherosclerosis • infection
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Chlamydia pneumoniae is an obligate intracellular bacterium that is associated with bronchopulmonary infections and that is capable of multiplying in a wide range of host cells.1 Alveolar macrophages seem to form the major reservoir of pulmonary infection. They respond to challenge with C. pneumoniae by releasing inflammatory mediators,2 and they may serve as a vector for dissemination.3
Inflammation is a major factor in the pathogenesis of atherosclerosis. Atheromatous plaques are characterized by infiltrates of inflammatory cells, which release reactive oxygen species, cytokines, and chemokines. Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are expressed at increased levels in atheromatous tissue.4 C. pneumoniae is of particular interest as a potential stimulus because viable organisms can be cultured from coronary atheromas.5
Hepatic hydroxymethylglutaryl coenzyme A reductase inhibitors reduce cardiovascular morbidity in patients with atherosclerosis. Growing evidence indicates that statins modulate cellular immune functions, in addition to their cholesterol-lowering effects.6
We investigated the effects of hydroxymethylglutaryl coenzyme A reductase inhibition on infection rate and the production of inflammatory mediators by human macrophages and endothelial cells (ECs) after infection with Chlamydia pneumoniae in vitro.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Culture of C. pneumoniae
Human epithelial cell-2 monolayers containing the C. pneumoniae strain CWL-029 (CWL; ATCC VR-1310) were grown as previously described.2 Chlamydial concentrations of 10 µg/mL and 100 µg/mL were equivalent to 4 or 40 elementary bodies per milliliter, respectively. In preliminary experiments with uninfected human ECs subjected to the same treatment, no mediator release was observed (data not shown).
Infection of Macrophages With CWL
Alveolar macrophages were collected from 6 nonsmoking
male volunteers (mean age, 27.2 years) and 10 patients (mean age,
58.4±18 years) with stable, chronic bronchitis. Bronchoalveolar lavage
was performed as previously described.2 The proportion of
macrophages was 94.4±2.8% in the volunteers and 93.4±5.1%
in the patients. Cells were cultured at a concentration of
105/mL in tissue culture plates with medium 199,
which contained 5% fetal calf serum (FCS), 1% L-glutamine, and
1% penicillin/streptomycin (GIBCO), for 3 hours at 37°C with an air
concentration of 5% CO2. Cells were then washed
twice and randomly assigned to coincubation with or without CWL (10 to
100 µg/mL) and cerivastatin (0.48 µg/mL).
Cerivastatin was provided by Bayer AG, Leverkusen, FRG. The optimal dosage and timing were determined in other experiments (data not shown). Macrophages from patients with chronic bronchitis were also coincubated with mevalonate (0.13 mg/mL; Sigma).
At 48 and 96 hours, the infected monolayers were methanol-fixed and stained for CWL with a fluorescein isothiocyanate-conjugated antibody (DAKO). Cell viability was assessed before and after culture by trypan blue exclusion.
Infection of Human ECs With CWL
Human ECs were obtained from 10 umbilical cords using the method
of Jaffe et al.7 Cells were suspended in
endothelial cell growth medium (Promo Cell), added to
culture flasks (Greiner) precoated with fibronectin, and cultured at
concentrations of 2x105/well at 37°C and 5%
CO2. Purity was assessed by morphology and factor
VIII staining. Confluent monolayers from the second passage were
coincubated as described above.
Cytokines
Tumor necrosis factor (TNF)-, IL-8, and MCP-1 levels were
determined by commercially available ELISAs (Bender and BIOSOURCE) in
cell supernatants that were harvested at 48 and 96 hours and stored
frozen at -70°C.
Superoxide Anion
Superoxide production was determined by the
ferricytochrome C reduction assay (340 ATTC-Photometer, Fa.SLT). This
analysis was restricted to supernatants of macrophages
from volunteers because of the limited cell numbers obtained with
bronchoalveolar lavage.
Nuclear Factor 
B Assay
The extraction of nuclear proteins was performed in a subset of
4 patients, with or without CWL (10 µg/mL, 100 µg/mL) coincubation,
using the same time points and cell concentrations as for
cytokine measurements and according to the "mini extraction
method."8
Statistics
Data are expressed as mean±SD and were analyzed with
the Wilcoxon test for comparison of nonparametric,
paired data and the Mann-Whitney U test for nonparametric,
unpaired data. Results were corrected with the Bonferroni adjustment
for multiple comparisons. P<0.05 was considered
significant.
Ethics
The protocol was approved by the Ethical Committee of the
Medical University of Lübeck. All volunteers gave written,
informed consent.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Infection Rate of Macrophages and ECs
After the incubation of cells with C. pneumoniae, elementary bodies were readily ingested. Inclusion bodies appeared in macrophages and ECs at 48 hours, indicating productive infection. Macrophages showed a time-dependent mean infection rate between 17.2% and 34% (CWL 10 µg/mL); the infection rate for ECs was between 45% and 60% at 48 and 96 hours, respectively.
Coincubation of macrophages with cerivastatin resulted in a nonsignificant reduction of the infection rate (patients without cerivastatin, 18.2±3% versus 10.4±3% with cerivastatin; volunteers: 17.2±5% versus 9.3±4%, respectively); no changes were found after the coincubation of ECs with cerivastatin.
Superoxide Anion Production of Macrophages
To evaluate the superoxide anion production,
macrophages from volunteers were used. Four hours after
infection, superoxide production was significantly increased in
infected versus noninfected cells (0.98±0.1 versus 0.63±0.1 nmol/L
per 105 cells). Pretreatment with cerivastatin
reduced superoxide production by 20.4% in infected cells and
by 19% in noninfected cells (P<0.05).
Cytokine Release of Macrophages and ECs
Infection with C. pneumoniae resulted in a significant
increase of cytokine release
(Table
). The relative increase at 96
hours was 7.5-fold for TNF-, 3-fold for MCP-1, and 2.7-fold for
IL-8. Coincubation with cerivastatin reduced the MCP-1 release
3.2-fold and the IL-8 release 1.3-fold
(Figure); TNF- release was not
altered.
|
|
Macrophages from patients with bronchitis revealed higher
spontaneous MCP-1 secretion than macrophages from volunteers,
indicating in vivo activation (Table
). Levels were not different
for IL-8 and TNF-. Infection of preactivated
macrophages with CWL (10 µg/mL) resulted in significant
increases of cytokine release. At higher chlamydial
concentrations (100 µg/mL), a further stimulation was not detectable.
Treatment of activated macrophages with cerivastatin
resulted in a reduction of MCP-1 secretion, but the other
cytokines remained unaffected. Inhibition of MCP-1 release was
completely abolished by mevalonic acid (61 452 pg/mL with mevalonic
acid versus 16 438 pg/mL without mevalonic acid, mean of 10
experiments).
Infection of ECs with C. pneumoniae (10 µg/mL) resulted in significant increases of chemokine release (increase at 96 hours was 1.4-fold for MCP-1 and 3.9-fold for IL-8; P<0.05), whereas at higher chlamydial concentrations (100 µg/mL), no further increase was seen. Coincubation with cerivastatin resulted in a 3.2-fold reduction in MCP-1 release (P<0.05) and a 2.8-fold reduction in IL-8 release (P<0.05).
Activation of Nuclear Factor B Transcription Factor
Translocated nuclear factor B (NFB) p65 increased
significantly in macrophages infected with CWL (10 µg/mL)
compared with uninfected cells (1.2±0.3 versus 4.3±0.6 absorbance
at 450 nm/nuclear protein; P<0.05), whereas the
higher CWL (100 µg/mL) concentration was associated with a reduction
of translocated NFB (3.2±0.6 EU 450 nm/nuclear protein).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The major finding of this study was a stimulation of the IL-8 and MCP-1 release of human immune cells by C. pneumoniae and an inhibition of the chemokine release after pretreatment with cerivastatin, independent of CWL infection status. These data extend previous findings regarding the release of TNF-
, IL-1, and IL-8 from
macrophages and the enhanced MCP-1 production of ECs
after chlamydial infection.1 2 For these
cytokines, a number of proatherogenic effects have been
reported. MCP-1 plays an important role in the recruitment of
monocytes, and it has been detected in human
atheromas.4 An increased release of MCP-1 by
macrophage-derived foam cells and smooth muscle cells was
demonstrated. MCP-1 secretion after CWL infection may be due to
the activation of NFB, which increased in parallel with MCP-1
release. NFB is a key regulator of a wide range of proinflammatory
genes, including TNF-, IL-8, and MCP-1, and it has been detected in
atherosclerotic lesions.9 Coincubation with cerivastatin reduced the chemokine release from infected and noninfected macrophages and ECs. This effect provides additional evidence for the anti-inflammatory properties of statins, which include an altered regulation of DNA transcription, suppressed natural-killer-cell activity, chemotaxis, enhanced apoptosis, and impaired cytokine production.6
In macrophages, the inhibition of MCP-1 release by
cerivastatin was not associated with concomitant changes of TNF-
concentrations. These results agree with a previous study, which could
not demonstrate an effect of statins on the
lipopolysaccharide-induced TNF- production by
monocytes.10 Alternatively, the inhibition of chemokine
release may be induced by a blockade of the mevalonate pathway and a
modification of regulatory intracellular proteins.11 This
possibility is supported by the reversal of chemokine inhibition after
the addition of mevalonate in our study. The effects of cerivastatin
seem to be nonspecific because decreased chemokine release was equally
observed in infected and noninfected cells. However, this effect was
preserved in situations of cell activation by chronic inflammation
(chronic bronchitis) and intracellular infection (C.
pneumoniae), which alters multiple cell responses to exogenous
stimuli.
In summary, we have demonstrated the stimulation of cytokine release from human immune cells infected with C. pneumoniae and an inhibitory effect of cerivastatin on the chemokine release in vitro. Further studies are needed to evaluate the clinical relevance of these data and the potential therapeutic consequences.
|
Acknowledgments |
|---|
This study was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Ma 2070/2-1).
Received August 30, 1999; revision received February 23, 2000; accepted February 28, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
-
Gaydos CA, Summersgill JT, Sahney NN, et
al. Replication of Chlamydia pneumoniae in vitro
in human macrophages, endothelial cells and
aortic smooth muscle cells. Infect Immun. 1996;64:1614–1620.[Abstract]
-
Redecke V, Dalhoff K, Bohnet S, et al. Interaction of
Chlamydia pneumoniae and human alveolar macrophages
and inflammatory response. Am J Respir Cell Mol
Biol. 1998;19:721–727.
[Abstract/Free Full Text]
-
Moazed TC, Kuo C, Grayston T. Evidence of systemic
dissemination of C. pneumoniae via macrophages in
the mouse. J Infect Dis. 1997;177:1322–1325.
-
Capers Q, Alexander RW, Lou P, et al. Monocyte
chemoattractant protein-1 in aortic tissues of hypertensive rats.
Hypertension. 1997;30:1397–1402.
[Abstract/Free Full Text]
-
Maass M, Bartels C, Engel PM, et al. Endovascular
presence of viable Chlamydia pneumoniae is a common
phenomenon in coronary artery disease. J Am Coll
Cardiol. 1998;31:827–832.
[Abstract/Free Full Text]
-
Rosenson RS, Tangney CC, Casey CL. Inhibition of
proinflammatory cytokine production by
pravastatin. Lancet. 1999;353:983–984.[Medline]
[Order article via Infotrieve]
-
Jaffe EA, Nachmann NL, Becker CG, et al. Culture of
human endothelial cells derived from umbilical veins.
J Clin Invest. 1973;52:2745–2756.
-
Gabriel C, Tanner A. Transcription factor binding
assays on 96-well plates. Tools Tech. 1997:36.
-
Brand K, Page S, Walli AK, et al. Role of NF-kappa B
in atherogenesis. Exp Physiol. 1997;82:297–304.[Abstract]
-
De Bont N, Netea MG, Rovers C, et al. LPS-induced
cytokine production and expression of LPS-receptors by
peripheral blood mononuclear cells of patients with
familial hypercholesterolemia and the effect of
HMG-CoA reductase inhibitors.
Atherosclerosis. 1998;139:147–152.[Medline]
[Order article via Infotrieve]
-
Goldstein JL, Brown MS. Regulation of mevalonate
pathway. Nature. 1990;343:425–430.[Medline]
[Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  F. Gao, L. Linhartova, A. McD. Johnston, and D. R. Thickett Statins and sepsis Br. J. Anaesth., March 1, 2008; 100(3): 288 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.B. J. Mancini, M. Etminan, B. Zhang, L. E. Levesque, J. M. FitzGerald, and J. M. Brophy Reduction of Morbidity and Mortality by Statins, Angiotensin-Converting Enzyme Inhibitors, and Angiotensin Receptor Blockers in Patients With Chronic Obstructive Pulmonary Disease J. Am. Coll. Cardiol., June 20, 2006; 47(12): 2554 - 2560. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Erkkila, M. Jauhiainen, K. Laitinen, K. Haasio, T. Tiirola, P. Saikku, and M. Leinonen Effect of Simvastatin, an Established Lipid-Lowering Drug, on Pulmonary Chlamydia pneumoniae Infection in Mice Antimicrob. Agents Chemother., September 1, 2005; 49(9): 3959 - 3962. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Fessler, S. K. Young, S. Jeyaseelan, J. G. Lieber, P. G. Arndt, J. A. Nick, and G. S. Worthen A Role for Hydroxy-Methylglutaryl Coenzyme A Reductase in Pulmonary Inflammation and Host Defense Am. J. Respir. Crit. Care Med., March 15, 2005; 171(6): 606 - 615. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Han, M. Parsons, X. Zhou, A. C. Nicholson, A. M. Gotto Jr, and D. P. Hajjar Functional Interplay Between the Macrophage Scavenger Receptor Class B Type I and Pitavastatin (NK-104) Circulation, November 30, 2004; 110(22): 3472 - 3479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P E Lazzerini, P L Capecchi, F Nerucci, A Fioravanti, F Chellini, M Piccini, S Bisogno, R Marcolongo, and F Laghi Pasini Simvastatin reduces MMP-3 level in interleukin 1{beta} stimulated human chondrocyte culture Ann Rheum Dis, July 1, 2004; 63(7): 867 - 869. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sabbatini, A. Pisani, F. Uccello, V. Serio, R. Seru, R. Paterno, B. Cianciaruso, G. Fuiano, and M. Andreucci Atorvastatin Improves the Course of Ischemic Acute Renal Failure in Aging Rats J. Am. Soc. Nephrol., April 1, 2004; 15(4): 901 - 909. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. McKay, B. P. Leung, I. B. McInnes, N. C. Thomson, and F. Y. Liew A Novel Anti-Inflammatory Role of Simvastatin in a Murine Model of Allergic Asthma J. Immunol., March 1, 2004; 172(5): 2903 - 2908. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Lindsberg and A. J. Grau Inflammation and Infections as Risk Factors for Ischemic Stroke Stroke, October 1, 2003; 34(10): 2518 - 2532. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Dechend, J. Gieffers, R. Dietz, A. Joerres, J. Rupp, F. C. Luft, and M. Maass Hydroxymethylglutaryl Coenzyme A Reductase Inhibition Reduces Chlamydia pneumoniae-Induced Cell Interaction and Activation Circulation, July 22, 2003; 108(3): 261 - 265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kawashima, T. Yamashita, Y. Miwa, M. Ozaki, M. Namiki, T. Hirase, N. Inoue, K.-i. Hirata, and M. Yokoyama HMG-CoA Reductase Inhibitor Has Protective Effects Against Stroke Events in Stroke-Prone Spontaneously Hypertensive Rats Stroke, January 1, 2003; 34(1): 157 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. V. Kalayoglu, P. Libby, and G. I. Byrne Chlamydia pneumoniae as an Emerging Risk Factor in Cardiovascular Disease JAMA, December 4, 2002; 288(21): 2724 - 2731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Di Napoli, A.A. Taccardi, M. Oliver, and R. De Caterina Statins and stroke: evidence for cholesterol-independent effects Eur. Heart J., December 2, 2002; 23(24): 1908 - 1921. [PDF]
|
|
|
|
 |
|
 S. Zeuke, A. J Ulmer, S. Kusumoto, H. A Katus, and H. Heine TLR4-mediated inflammatory activation of human coronary artery endothelial cells by LPS Cardiovasc Res, October 1, 2002; 56(1): 126 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Palinski and S. Tsimikas Immunomodulatory Effects of Statins: Mechanisms and Potential Impact on Arteriosclerosis J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1673 - 1681. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Stollberger and J. Finsterer Role of Infectious and Immune Factors in Coronary and Cerebrovascular Arteriosclerosis Clin. Vaccine Immunol., March 1, 2002; 9(2): 207 - 215. [Full Text] [PDF]
|
|
|
|
|
|
L. Wilhelmsen Inflammation, infection, and coronary heart disease Eur. Heart J., March 1, 2002; 23(5): 343 - 344. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Wassmann, U. Laufs, K. Muller, C. Konkol, K. Ahlbory, A. T. Baumer, W. Linz, M. Bohm, and G. Nickenig Cellular Antioxidant Effects of Atorvastatin In Vitro and In Vivo Arterioscler. Thromb. Vasc. Biol., February 1, 2002; 22(2): 300 - 305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Takemoto and J. K. Liao Pleiotropic Effects of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Arterioscler. Thromb. Vasc. Biol., November 1, 2001; 21(11): 1712 - 1719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Blankenberg, H. J. Rupprecht, C. Bickel, C. Espinola-Klein, G. Rippin, G. Hafner, M. Ossendorf, K. Steinhagen, and J. Meyer Cytomegalovirus Infection With Interleukin-6 Response Predicts Cardiac Mortality in Patients With Coronary Artery Disease Circulation, June 19, 2001; 103(24): 2915 - 2921. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Wassmann, U. Laufs, A. T. Baumer, K. Muller, K. Ahlbory, W. Linz, G. Itter, R. Rosen, M. Bohm, and G. Nickenig HMG-CoA Reductase Inhibitors Improve Endothelial Dysfunction in Normocholesterolemic Hypertension via Reduced Production of Reactive Oxygen Species Hypertension, June 1, 2001; 37(6): 1450 - 1457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Palinsk New Evidence for Beneficial Effects of Statins Unrelated to Lipid Lowering Arterioscler. Thromb. Vasc. Biol., January 1, 2001; 21(1): 3 - 5. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||