help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on February 8, 2007, doi:10.1164/rccm.200607-926OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200607-926OCv1
175/8/791    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Droemann, D.
Right arrow Articles by Dalhoff, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Droemann, D.
Right arrow Articles by Dalhoff, K.
American Journal of Respiratory and Critical Care Medicine Vol 175. pp. 791-797, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200607-926OC

Original Article

Disparate Innate Immune Responses to Persistent and Acute Chlamydia pneumoniae Infection in Chronic Obstructive Pulmonary Disease

Daniel Droemann1, Jan Rupp2, Torsten Goldmann3, Ulrike Uhlig4, Detlev Branscheid5, Ekkehard Vollmer3, Peter Kujath6, Peter Zabel1,7 and Klaus Dalhoff7

1 Medical Clinic, Research Center Borstel, Borstel, Germany; 2 Institute of Medical Microbiology and Hygiene, University of Schleswig-Holstein, Campus Lübeck, Lübeck, Germany; 3 Clinical and Experimental Pathology, Research Center Borstel, Borstel, Germany; 4 Division of Pulmonary Pharmacology, Research Center Borstel, Borstel, Germany; 5 Department of Thoracic Surgery, Krankenhaus Großhansdorf, Großhansdorf, Germany; 6 Department of Thoracic Surgery, University of Schleswig-Holstein, Campus Lübeck, Lübeck, Germany; and 7 Medical Clinic III, University of Schleswig-Holstein, Campus Lübeck, Lübeck, Germany

Correspondence and requests for reprints should be addressed to Daniel Droemann, M.D., Medical Clinic, Research Center Borstel, Parkallee 35, D-23845 Borstel, Germany. E-mail: ddroemann{at}fz-borstel.de


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Chlamydia pneumoniae (Cpn) infection may play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available comparing persistent and acute infection of this pathogen in the human respiratory tract.

Objectives: To study Cpn-induced innate immune responses in lung tissue from patients with COPD and control subjects ex vivo and in vitro.

Methods: Cpn detection was done by nested polymerase chain reaction, in situ hybridization, and immunohistochemistry ex vivo in unstimulated tissue and in vitro using an acute Cpn infection model. As main endpoints for the assessment of early cellular responses, nuclear factor (NF)-{kappa}B activation and CXC chemokine ligand (CXCL)-8 expression were evaluated. The role of Toll-like receptors (TLRs) as recognition molecules in Cpn-induced innate responses was tested by blocking experiments.

Measurements and Main Results: Fifteen percent of patients with COPD were chronically infected with Cpn in contrast to 0% of control subjects (p < 0.05). There were no differences in CXCL-8 and NF-{kappa}B expression between infected and noninfected COPD tissue ex vivo. In contrast, acute in vitro infection induced an intense innate immune response including up-regulation of TLR2. Blocking experiments demonstrated the predominant role of TLR2 in induction of the early immune response, whereas no influence on chlamydial infection rates was observed.

Conclusions: Acute in vitro infection of human lung tissue with Cpn elicited a marked innate response via TLR2, whereas chronic chlamydial infection in patients with COPD was not associated with enhanced cellular activation. These findings suggest different roles of Cpn during acute and chronic stages of pulmonary infection.

Key Words: Chlamydia pneumoniae • innate immunity • pulmonary host defense • Toll-like receptor 2 • chronic obstructive pulmonary disease



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Chlamydia pneumoniae (Cpn) infection may play a role in the pathogenesis of chronic obstructive pulmonary disease. Few data are available comparing persistent and acute infection of this pathogen in the human respiratory tract.

What This Study Adds to the Field
Although acute infection with Cpn elicited an inflammatory response via Toll-like receptor 2, chronic infection in patients with chronic obstructive pulmonary disease was not associated with enhanced lung pathology, suggesting different roles of Cpn.

 
Chlamydophila (Chlamydia) pneumoniae (Cpn) is an obligate intracellular gram-negative pathogen that is detected in 5 to 10% of community-acquired pneumonia and other lower respiratory tract infections. The high seroprevalence of 70 to 80% indicates that most adults are at least once infected during their lifetime (1). Several studies have suggested that Cpn may play a role in the pathogenesis of bronchial asthma and chronic obstructive pulmonary disease (COPD) (2–4). Highly different Cpn infection rates in patients with COPD have been reported, ranging from 4 to 34% during exacerbations (3, 5, 6) and 24 to 38% in exacerbation-free intervals (7, 8). This may be due to differences in study populations, epidemiologic variation, and the limitations of the main diagnostic techniques, such as serology (which is inaccurate in distinguishing acute and chronic infection) and polymerase chain reaction (PCR) (which lacks international standardization). Interestingly, chronic chlamydial infection in patients with stable COPD was observed to be related to a higher rate of exacerbations (9), which is in line with the link between bacterial colonization and chronic airway inflammation and the decline of lung function described in recent years (10, 11). In contrast, Seemungal and colleagues did not find an association between Cpn detection in the airways at exacerbations and exacerbation frequency (8). Thus, the possible role of Cpn in the pathogenesis of COPD remains controversial, and the consequences of chronic tissue infection with Cpn are not clear.

Few data are available concerning the presence of Cpn in human lung tissue. Wu and associates reported immunohistochemical detection of Cpn in 54% of alveolar macrophages (AMs) from patients with COPD and in 29% of cells from control subjects (12). We recently have detected Cpn in 13% of COPD lung tissue by in situ hybridization (ISH) (13). Regarding the consequences of acute chlamydial infection, we and other groups have shown that AMs respond to Cpn infection in vitro with a strong release of inflammatory mediators (14, 15).

Toll-like receptors (TLRs) are central molecules for the recognition of pathogens and the induction of inflammation (16). TLR2 responds preferentially to components of gram-positive bacteria, such as peptidoglycan and lipoteichoic acid (16), whereas TLR4 responds to components of gram-negative bacteria, such as lipopolysaccharide (LPS) (17). Interestingly, TLR2 has been described to predominantly interact with Cpn in vitro (18, 19). TLR2 is expressed on AMs and alveolar epithelial cells (AECs) (20). We recently described an altered AM phenotype of patients with COPD and smokers with reduced TLR2 expression compared with nonsmokers (21). Thus, patients with COPD may be more susceptible to Cpn infection due to impaired pathogen recognition. TLR4 is functionally expressed on AECs (22) and may also play a role in Chlamydia-induced immune responses (23).

The aim of the present study was to comparatively evaluate the role of Cpn during chronic infection in patients with stable COPD and in an acute human lung tissue infection model. Nuclear factor (NF)-{kappa}B and CXC chemokine ligand (CXCL)-8 were chosen as markers of early cellular activation and innate response because they are critical components of the airway inflammation associated with COPD. In addition, the role of TLR2 or TLR4 in pathogen-induced innate responses was evaluated.

Some of the results of this study have been previously reported in abstract form (24).


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Protocol
Cpn-induced innate immune responses in lung tissues obtained from patients with COPD and control subjects were studied ex vivo and in vitro. Detection of Cpn was done ex vivo in unstimulated tissue using nested PCR, ISH, and immunohistochemistry (IHC), and in vitro by using an acute Chlamydia infection model (ACIM). For the assessment of early cellular responses, NF-{kappa}B activation using electrophoretic mobility shift assay and CXCL-8 expression using enzyme-linked immunosorbent assay (ELISA) and ISH were evaluated. The role of TLR2 and TLR4 in Cpn-induced innate responses was tested by blocking experiments with monoclonal antibodies.

Lung Tissues
The specimens were tumor-free material at least 5 cm away from the tumor front. For in vitro infection experiments, lung specimens (1 cm3) were cultured in RPMI 1640 medium (Sigma, Taufkirchen, Germany) at 37°C and 5% CO2 for 24 hours and incubated with 500 µl chlamydial suspensions (107 inclusion-forming units per one milliliter [IFU/ml]) as described previously (13). Tissues were fixed using the HOPE (HEPES-glutamic acid buffer mediated organic solvent protection effect) technique (25). See the online supplement for additional details.

Culture of Cpn
The respiratory isolate Cpn CWL029 (American Type Culture Collection [ATCC] VR-1310) was grown on HEp-2 monolayers as described previously (26).

ISH
Hybridization targeting the Cpn-specific PstI fragment and a 272-bp fragment of human CXCL-8 mRNA was performed overnight in moist chambers at 46°C (27). All samples were analyzed by two independent investigators (T.G. and D.D.). See the online supplement for additional details.

Real-Time Reverse Transcriptase–PCR of TLR2 and TLR4 mRNA Expression
Reverse transcriptase (RT)–PCR was performed using the NucleoSpin RNA II kit (Macherey-Nagel, Dueren, Germany) and reverse transcribed into complementary DNA (Roche First-Strand PCR kit; Roche Diagnostics, Mannheim, Germany), PCR amplification was performed using the LightCycler Detection System (Roche Molecular Biochemicals, Mannheim, Germany).

Immunohistochemical Staining
Primary antibodies (anti-human TLR2, clone TL2.1; kindly provided by Genentech, San Francisco, CA; anti-human TLR2, clone TL2.1, anti-human TLR4; eBioscience, San Diego, CA; anti–NF-{kappa}B p65 subunit; Chemicon International, Temecula, CA; antichlamydial LPS, CF-2; Washington Research Foundation, WA; and major outer membrane protein, clone RR402; Dako, Glostrup, Denmark) were applied in a dilution of 1:100 as described elsewhere (20).

NF-{kappa}B Activation by Electrophoretic Mobility Shift Assay
Preparation of nuclear extracts was done as described elsewhere (28). Oligonucleotides containing the NF-{kappa}B consensus sequence element were obtained from Geneka Biotech, Inc. (Montreal, PQ, Canada).

Cell Culture
A549 cells obtained from the European collection of cell cultures were grown in 25 cm2 polystyrene flasks with Dulbecco's modified Eagle's medium (Sigma). Cells were seeded in 35-mm tissue culture dishes (Nunc, Weisbaden, Germany). A549 cells were preincubated (1 h, 37°C) with TLR2 and TLR4 monoclonal antibodies (1 µg/ml) before stimulation with Cpn. Analogous lung specimens were preincubated before stimulation with Cpn and cultured.

Cytokine Assays
Measurement of CXCL-8 levels in supernatants was performed using commercially available ELISA kits (Biosource, Solingen, Germany).

Statistical Analysis
Data are presented as the mean ± SD. Statistics were performed with nonparametric tests. For independent samples, Student's t test was used. For categorical variables, 2x2 tables were analyzed using a chi-square test. p values less than 0.05 were considered statistically significant. Calculations were performed with Statistica for Windows (version 5; Statsoft, Hamburg, Germany), 1997.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients and Lung Tissues
The study population consisted of 42 patients with COPD (mean age, 62 yr; 29 males, 13 females; mean pack-years, 59; GOLD [Global Initiative for Chronic Obstructive Lung Disease] stage I: 4 patients, stage II: 23 patients, stage III: 15 patients) who had an indication for lung surgery of peripheral nodules (Table 1). No patient had undergone antimicrobial treatment before the operation. Systemic steroid treatment was administered preoperatively in 13 of 42 patients in doses of less than 20 mg/day of prednisone equivalent. Nevertheless, we found no differences of NF-{kappa}B and CXCL-8 expression between patients with COPD treated and not treated with steroids (data not shown). Eleven patients without chronic airway diseases served as control subjects (mean age, 59 yr; 6 males, 5 females). Lung tissue samples were obtained from lobectomy or atypical resections (patients with COPD, n = 42: lung cancer, n = 35; metastases of extrapulmonary tumors, n = 5; benign nodules, n = 2; control subjects, n = 11: lung cancer, n = 2; metastases of extrapulmonary tumors, n = 7; benign nodules, n = 2).


View this table:
[in this window]
[in a new window]

  		TABLE 1. DEMOGRAPHIC DATA AND CHLAMYDIA PNEUMONIAE DETECTION IN LUNG TISSUES FROM PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE AND CONTROL SUBJECTS

 
Detection of Cpn Infection in Lung Tissue Ex Vivo
Fifteen percent (n = 6 of 42) of the COPD lung tissue proved to be Cpn-DNA positive as detected by nested PCR. In all Cpn-positive lungs, PCR detection was confirmed by positive signals using ISH targeting chlamydial DNA and IHC for major outer membrane proteins and chlamydial LPS. Cpn-DNA–negative lungs did not show positive signals using ISH and IHC for chlamydial LPS.

Representative findings in COPD lung tissue are displayed in Figure 1. Chlamydial infection was primarily detected in AMs (Figures 1A, 1C, and 1D) and to a lesser degree also in AECs (Figure 1B). There was no difference regarding infection rates between tissue from patients with early (GOLD I/II) or late (GOLD III) stage COPD. The distribution of Cpn-positive cells in the lung tissue showed a nonhomogeneous, patchy pattern. In lung tissues of patients without COPD, there was no detection of Cpn using PCR (p < 0.05, Table 1).


Figure 1
View larger version (96K):
[in this window]
[in a new window]

  		Figure 1. In situ hybridization (A and B) targeting Chlamydia pneumoniae (Cpn) DNA and immunohistochemical staining for chlamydial major outer membrane protein (C) and chlamydial LPS (D) in chronic obstructive pulmonary disease (COPD) lung tissue. Cpn is detected in alveolar macrophages (A, original magnification, x400; C and D, original magnification, x600) and alveolar epithelial cells (B, original magnification, x400). Cpn detection was not associated with increased inflammation level in COPD lungs infected with Cpn (E, hematoxylin-eosin stain, original magnification, x400).

 
Pathogen-induced Cellular Activation and Immune Response in Acute and Chronic Chlamydial Infection
Histologic assessment of lung tissue by investigators blinded to the results of Cpn detection did not reveal increased inflammation levels in lungs with persistent Cpn infection (Figure 1E). To evaluate more specifically whether Cpn detection is associated with increased cellular activation, NF-{kappa}B translocation was concomitantly assessed in adjacent tissue sections because NF-{kappa}B activation is known to be an important step in Cpn-induced innate responses. Figure 2A shows that NF-{kappa}B is up-regulated in COPD lungs as compared with control lungs without an underlying pulmonary condition. However, no difference in NF-{kappa}B expression between patients with COPD and those without Cpn detection ex vivo was observed. In contrast, after Cpn in vitro infection using the ACIM, a marked cellular activation indicated by NF-{kappa}B translocation was consistently found. The infection pattern in the ACIM showed primarily infection of AMs (Figure 3A), and positive signals for the NF-{kappa}B p65 subunit expression were demonstrated in these cells (Figure 3B). In addition, the expression of CXCL-8 as an important early innate response mediator was evaluated in both models. Again, enhanced unstimulated expression was seen in patients with COPD compared with control subjects, but there was no increase in the Cpn-positive lungs ex vivo. In contrast, marked increase of CXCL-8 expression was found in the ACIM (Figure 2B).


Figure 2
View larger version (97K):
[in this window]
[in a new window]

  		Figure 2. Detection of nuclear factor (NF)-{kappa}B activation (A) by electrophoretic mobility shift assay in the acute chlamydial infection model, nonsmokers, Chlamydia pneumoniae (Cpn)–positive, and Cpn-negative COPD lung tissues. In situ hybridization targeting CXCL-8 mRNA in nonsmokers (B), Cpn-negative chronic obstructive pulmonary disease (COPD) (C), Cpn-positive COPD (D), and in vitro infected lung tissues (E) (representative results of nonsmokers, n = 8; COPD Cpn+, n = 6; COPD Cpn–, n = 15; in vitro infection, n = 6 experiments).

 

Figure 3
View larger version (65K):
[in this window]
[in a new window]

  		Figure 3. In situ hybridization targeting Chlamydia pneumoniae (Cpn) DNA (A) and immunohistochemical staining for the nuclear factor-{kappa}B p65 subunit (B) in in vitro–infected COPD lung tissue (original magnification, x400). Positive signals are mainly visible in alveolar macrophages.

 
TLR Expression in Cpn-infected Lung Tissue
Comparing TLR2 and TLR4 protein expression (IHC) in COPD lung tissue, we found a strong expression of TLR2 in contrast to TLR4 ex vivo (Figure 4). TLR2 expression was seen in AMs and AECs, whereas TLR4 expression was demonstrable mainly in AMs and rarely in alveolar epithelia. Cpn infection detected ex vivo did not influence TLR2 and TLR4 expression (data not shown). Comparing detection of TLR2 and TLR4 mRNA, there were again no differences according to Cpn infection status. In contrast, in the in vitro infection model, we found a marked up-regulation of TLR2 but not TLR4 after incubation with Cpn. Data are shown for TLR2 (Figure 4C).


Figure 4
View larger version (81K):
[in this window]
[in a new window]

  		Figure 4. Immunohistochemical staining of Toll-like receptor (TLR)-2 (A) and TLR4 (B) in chronic obstructive pulmonary disease (COPD) lung tissue (original magnification, x400). (C) TLR2 mRNA expression in controls, Chlamydia pneumoniae (Cpn)+ and Cpn– COPD (Cpn+, n = 4; Cpn–, n = 4), and in vitro–infected COPD lung tissue (n = 4). *p < 0.05 compared with controls, Cpn+, and Cpn– COPD lung tissue. (D) IL-8 secretion in lung tissue after Cpn infection over 24 hours ± blocking of TLR2 and TLR4 by monoclonal antibody. *p < 0.05 compared with Cpn infection, Cpn infection + isotype antibody and Cpn + anti–TLR4 antibody.

 
Modulation of TLR Expression
Cell culture and tissue experiments using a human alveolar type II epithelium-like cell line (A549) and the ACIM revealed that inhibition of TLR2 signaling by a neutralizing antibody results in a significantly reduced innate response to Cpn infection as evidenced by CXCL-8 release from cell and tissue culture (shown for tissue culture, Figure 4D). In contrast, blocking of TLR4 had no effect. In contrast to the effects on the immune response, TLR blockade had no influence on infection rates in the tissue model (Table 2).


View this table:
[in this window]
[in a new window]

  		TABLE 2. EFFECTS OF TOLL-LIKE RECEPTOR BLOCKADE ON INFLAMMATION (CXCL-8 EXPRESSION) AND INFECTION RATES (IN SITU HYBRIDIZATION TARGETING CHLAMYDIAL DNA) IN THE ACUTE INFECTION MODEL (n = 6) IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE LUNG TISSUE

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrate that chronic Cpn infection is detected in 15% of lungs from patients with COPD but is not associated with enhanced host cell activation. This is in contrast to acute Cpn in vitro infection of lung tissue, which elicits a marked innate immune response dependent on TLR2—but not TLR4—signaling. In lung tissue of patients without COPD, Cpn infection was not detected.

Detection of Cpn in respiratory tissue has previously been reported by Wu and colleagues who described the localization of Chlamydia in AMs using IHC, and found that all tested lungs from patients with and without COPD were Cpn positive in contrast to PCR results. The immunohistochemical staining was more intense in lung tissue from patients with COPD (12). We confirmed the increased detection of Cpn in COPD as compared with control patients, but observed chlamydial infection in a clearly lower proportion of patients than did Wu and colleagues, which may be due to differences in the experimental protocols. The specificity of our detection methods is underlined by the fact that positive results obtained in the PCR were all confirmed in the ISH and IHC procedures. There was no association of Cpn detection to COPD stage and lung function, which is in line with data from serological profiles and PCR in peripheral blood mononuclear cells (29, 30).

A trigger role of Cpn infection in chronic airway inflammation is suggested by the marked proinflammatory response elicited by this microorganism in vitro (15, 31) and by the antiapoptotic effect of chlamydial infection on immune cells (32, 33). In the ACIM, we demonstrated an increased CXCL-8 release after Cpn infection and the expression of the p65 subunit mainly in AMs, indicating the activated form of NF-{kappa}B. This is in line with previous data from cell culture experiments in endothelial cells infected with Cpn (34) as well as in human airway epithelial cells (35). Krull and colleagues demonstrated the activation of different signal transduction pathways such as protein tyrosine phosphorylation and up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase. In addition, enhanced expression of endothelial adhesion molecules was shown (32). Interaction among different lung cells may play an additional role because coincubation of leukocytes with AECs facilitates the infection of epithelial cells (36).

What are the implications of these findings for chronic chlamydial lung infection in vivo? Detection of Cpn in lung tissue from patients with COPD was not associated with enhanced cellular activation in our study as evaluated by conventional histology, NF-{kappa}B expression, and CXCL-8 expression. These findings suggest that detection of this pathogen in human lung tissue is not necessarily associated with an active immune response. This has also been experienced in cardiovascular diseases where frequent demonstration of Cpn in atherosclerotic plaques was not consistently associated with disease. Moreover, large intervention trials with antimicrobial agents active against chlamydiae have not shown significant benefits (37). Regarding respiratory disease, a recent trial of ketolide treatment in patients with asthma found a significant reduction of asthma symptoms in the verum group, but no relationship between chlamydia or mycoplasma infection status and the response to treatment (38).

The discrepancies between the acute in vitro infection model and the ex vivo findings may be due to the difference between acute and persistent Cpn infection. Persistence is a well-known phenomenon of chlamydial infection. It is facilitated by the intracellular life cycle of this microorganism and may explain a reduced inflammatory potential in vivo. Subinhibitory concentrations of antibiotics, cytokine treatment, induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase, and iron depletion have been reported to induce chlamydial persistence in epithelial cell cultures (39–41). In addition, an incomplete developmental cycle and restricted growth of Cpn have been described in AMs (14). Although distinct morphologic characteristics, like a decrease in typical inclusions and an increase of smaller, less dense atypical inclusions, are observed in these models, transcriptome and proteome analyses are not as consistent. Several alterations in pathogen and host genes, such as chlamydial major outer membrane protein, the cysteine-rich outer membrane protein omcB, and chlamydial heat shock protein-60 (14, 42, 43), have been described in persistence models, but none of them has been attributed so far to the state of chlamydial infection found in chronic airway diseases. A higher rate of acute exacerbations in patients with chronic bronchitis with Cpn colonization (determined by Cpn-DNA detection) is one of the few clinical markers to show an association with long-term Cpn infection (9). Physiologic or pharmacologic mediators influencing the switch between the acute and the persistent phase of chlamydial infection in vivo are unknown, but could be of interest to better understand the host–pathogen interaction and to modulate persistent chlamydial infections.

Regarding the role of TLRs for the recognition of chlamydia and induction of the innate response, the majority of data describe a predominant role of TLR2. Netea and colleagues demonstrated that stimulation of proinflammatory cytokines by Cpn in human mononuclear cells is a TLR2-dependent event and TLR4 as well as CD14 do not play important roles in this process (18). Prebeck showed similar results indicating a predominant role of TLR2 for detection of Cpn in dendritic cells in contrast to TLR4 (19). In addition, TLR-independent, but MyD88-dependent signaling with stimulation of IL-18 has recently been demonstrated (44). The expression of TLR2 in human lung was recently characterized by our group not only in AMs but also in AECs, which are involved in the immune response to Cpn (20). Our results from in vitro–infected lung tissue suggest a prominent role of TLR2 in the innate response to Cpn with markedly diminished IL-8 release after selective receptor blockade. Considering the altered phenotype of AMs from patients with COPD and smokers (21) with decreased TLR2 expression, this may explain the increased susceptibility of these persons to Cpn infection (5). On the other hand, recent data suggest that Cpn infection is associated with an unbalanced proinflammatory cytokine response in patients with COPD as compared with healthy control subjects (31). The combination of impaired host defense and enhanced cytokine response could contribute to the development of chronic airway inflammation in COPD. TLR4 has also been implicated in Cpn-induced cell activation (23, 45) and has been shown to be functional when expressed on AECs (22). However, we could only demonstrate a very weak expression of TLR4 protein in human lung tissue and no effect of TLR4 blockade in the context of Cpn infection.

Surprisingly, there was no concomitant effect of anti-TLR2 treatment on chlamydial infection rates. The dissociation between effects on the immune response and on bacterial clearance also has been observed recently in knockout models after chlamydial infection (23) as well as in pneumococcal disease (46). The reasons for this phenomenon are not clear. Possible explanations include TLR-independent defense mechanisms, multicomponent receptor clusters, which may partly compensate for blockade of single components, and interactions between cytokine concentrations and bacterial metabolism (47). Further studies are needed to elucidate the role of these mechanisms in the interplay of inflammation and bacterial growth.

In conclusion, our findings suggest that chronic Cpn infection is frequent in patients with COPD. Pathogen recognition in acute infection is mainly mediated by TLR2, leading to a marked innate immune response in vitro. However, detection of Cpn from clinical samples is not associated with enhanced lung cell activation and may solely indicate persistence. Further studies are needed to clarify which environmental or pathogen-induced factors are responsible for the initiation of productive chlamydial infection.


   Acknowledgments
 
The authors thank H. Kühl, W. Martens, J. Hofmeister, and S. Ross for excellent technical assistance.


   FOOTNOTES
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200607-926OC on February 8, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 8, 2006; accepted in final form January 26, 2007


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Leinonen, M. Pathogenetic mechanisms and epidemiology of Chlamydia pneumoniae. Eur Heart J 1993;14(Suppl K):57–61.[Abstract/Free Full Text]
  2. Black PN, Scicchitano R, Jenkins CR, Blasi F, Allegra L, Wlodarczyk J, Cooper BC. Serological evidence of infection with Chlamydia pneumoniae is related to the severity of asthma. Eur Respir J 2000;15:254–259.[Abstract]
  3. Miyashita N, Niki Y, Nakajima M, Kawane H, Matsushima T. Chlamydia pneumoniae infection in patients with diffuse panbronchiolitis and COPD. Chest 1998;114:969–971.[Medline]
  4. von Hertzen LC. Chlamydia pneumoniae and its role in chronic obstructive pulmonary disease. Ann Med 1998;30:27–37.[Medline]
  5. Blasi F, Legnani D, Lombardo VM, Negretto GG, Magliano E, Pozzoli R, Chiodo F, Fasoli A, Allegra L. Chlamydia pneumoniae infection in acute exacerbations of COPD. Eur Respir J 1993;6:19–22.[Abstract]
  6. Karnak D, Beng-sun S, Beder S, Kayacan O. Chlamydia pneumoniae infection and acute exacerbation of chronic obstructive pulmonary disease (COPD). Respir Med 2001;95:811–816.[CrossRef][Medline]
  7. Blasi F, Cosentini R, Tarsia P. Chlamydia pneumoniae respiratory infections. Curr Opin Infect Dis 2000;13:161–164.[Medline]
  8. Seemungal TA, Wedzicha JA, MacCallum PK, Johnston SL, Lambert PA. Chlamydia pneumoniae and COPD exacerbation. Thorax 2002; 57:1087–1088. [Author reply, 1088–1089.][Free Full Text]
  9. Blasi F, Damato S, Cosentini R, Tarsia P, Raccanelli R, Centanni S, Allegra L. Chlamydia pneumoniae and chronic bronchitis: association with severity and bacterial clearance following treatment. Thorax 2002; 57:672–676.[Abstract/Free Full Text]
  10. Seemungal TA, Donaldson GC, Bhowmik A, Jeffries DJ, Wedzicha JA. Time course and recovery of exacerbations in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;161: 1608–1613.[Abstract/Free Full Text]
  11. Wilkinson TM, Patel IS, Wilks M, Donaldson GC, Wedzicha JA. Airway bacterial load and FEV1 decline in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;167: 1090–1095.[Abstract/Free Full Text]
  12. Wu L, Skinner SJ, Lambie N, Vuletic JC, Blasi F, Black PN. Immunohistochemical staining for Chlamydia pneumoniae is increased in lung tissue from subjects with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;162:1148–1151.[Abstract/Free Full Text]
  13. Rupp J, Droemann D, Goldmann T, Zabel P, Solbach W, Vollmer E, Branscheid D, Dalhoff K, Maass M. Alveolar epithelial cells type II are major target cells for C. pneumoniae in chronic but not in acute respiratory infection. FEMS Immunol Med Microbiol 2004;41:197–203.[CrossRef][Medline]
  14. Haranaga S, Yamaguchi H, Ikejima H, Friedman H, Yamamoto Y. Chlamydia pneumoniae infection of alveolar macrophages: a model. J Infect Dis 2003;187:1107–1115.[CrossRef][Medline]
  15. Redecke V, Dalhoff K, Bohnet S, Braun J, Maass M. Interaction of Chlamydia pneumoniae and human alveolar macrophages: infection and inflammatory response. Am J Respir Cell Mol Biol 1998;19:721–727.[Abstract/Free Full Text]
  16. Imler JL, Hoffmann JA. Toll receptors in innate immunity. Trends Cell Biol 2001;11:304–311.[CrossRef][Medline]
  17. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 1999;274:10689–10692.[Abstract/Free Full Text]
  18. Netea MG, Kullberg BJ, Galama JM, Stalenhoef AF, Dinarello CA, Van der Meer JW. Non-LPS components of Chlamydia pneumoniae stimulate cytokine production through Toll-like receptor 2-dependent pathways. Eur J Immunol 2002;32:1188–1195.[CrossRef][Medline]
  19. Prebeck S, Kirschning C, Durr S, da Costa C, Donath B, Brand K, Redecke V, Wagner H, Miethke T. Predominant role of Toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells. J Immunol 2001;167:3316–3323.[Abstract/Free Full Text]
  20. Droemann D, Goldmann T, Branscheid D, Clark R, Dalhoff K, Zabel P, Vollmer E. Toll-like receptor 2 is expressed by alveolar epithelial cells type II and macrophages in the human lung. Histochem Cell Biol 2003;119:103–108.[CrossRef][Medline]
  21. Droemann D, Goldmann T, Tiedje T, Zabel P, Dalhoff K, Schaaf B. Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients. Respir Res 2005;6:68.[CrossRef][Medline]
  22. Armstrong L, Medford AR, Uppington KM, Robertson J, Witherden IR, Tetley TD, Millar AB. Expression of functional Toll-like receptor-2 and -4 on alveolar epithelial cells. Am J Respir Cell Mol Biol 2004; 31:241–245.[Abstract/Free Full Text]
  23. Mueller M, Postius S, Thimm JG, Gueinzius K, Muehldorfer I, Hermann C. Toll-like receptors 2 and 4 do not contribute to clearance of Chlamydophila pneumoniae in mice, but are necessary for the release of monokines. Immunobiology 2004;209:599–608.[CrossRef][Medline]
  24. Wu G, Droemann D, Goldmann T, Rupp J, Vollmer E, Zabel P, Dalhoff K. Antiapoptotic and proinflammatory function of Toll-like receptor 2 in acute C. pneumoniae infection of COPD lung tissue. Eur Respir J 2006;28:575s.
  25. Olert J, Wiedorn KH, Goldmann T, Kuhl H, Mehraein Y, Scherthan H, Niketeghad F, Vollmer E, Muller AM, Muller-Navia J. HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues. Pathol Res Pract 2001;197:823–826.[CrossRef][Medline]
  26. Maass M, Harig U. Evaluation of culture conditions used for isolation of Chlamydia pneumoniae. Am J Clin Pathol 1995;103:141–148.[Medline]
  27. Goldmann T, Wiedorn KH, Kuhl H, Olert J, Branscheid D, Pechkovsky D, Zissel G, Galle J, Muller-Quernheim J, Vollmer E. Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues. Pathol Res Pract 2002;198:91–95.[CrossRef][Medline]
  28. Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 1991;19:2499.[Free Full Text]
  29. Lieberman D, Ben Yaakov M, Lazarovich Z, Ohana B, Boldur I. Chlamydia pneumoniae infection in acute exacerbations of chronic obstructive pulmonary disease: analysis of 250 hospitalizations. Eur J Clin Microbiol Infect Dis 2001;20:698–704.[CrossRef][Medline]
  30. Smieja M, Leigh R, Petrich A, Chong S, Kamada D, Hargreave FE, Goldsmith CH, Chernesky M, Mahony JB. Smoking, season, and detection of Chlamydia pneumoniae DNA in clinically stable COPD patients. BMC Infect Dis 2002;2:12.[CrossRef][Medline]
  31. Rupp J, Kothe H, Mueller A, Maass M, Dalhoff K. Imbalanced secretion of IL-1beta and IL-1RA in Chlamydia pneumoniae-infected mononuclear cells from COPD patients. Eur Respir J 2003;22:274–279.[Abstract/Free Full Text]
  32. Krull M, Klucken AC, Wuppermann FN, Fuhrmann O, Magerl C, Seybold J, Hippenstiel S, Hegemann JH, Jantos CA, Suttorp N. Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae. J Immunol 1999;162:4834–4841.[Abstract/Free Full Text]
  33. van Zandbergen G, Gieffers J, Kothe H, Rupp J, Bollinger A, Aga E, Klinger M, Brade H, Dalhoff K, Maass M, et al. Chlamydia pneumoniae multiply in neutrophil granulocytes and delay their spontaneous apoptosis. J Immunol 2004;172:1768–1776.[Abstract/Free Full Text]
  34. Dechend R, Maass M, Gieffers J, Dietz R, Scheidereit C, Leutz A, Gulba DC. Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kappaB and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis. Circulation 1999;100:1369–1373.[Abstract/Free Full Text]
  35. Jahn HU, Krull M, Wuppermann FN, Klucken AC, Rosseau S, Seybold J, Hegemann JH, Jantos CA, Suttorp N. Infection and activation of airway epithelial cells by Chlamydia pneumoniae. J Infect Dis 2000; 182:1678–1687.[CrossRef][Medline]
  36. Rodriguez N, Fend F, Jennen L, Schiemann M, Wantia N, Prazeres da Costa CU, Durr S, Heinzmann U, Wagner H, Miethke T. Polymorphonuclear neutrophils improve replication of Chlamydia pneumoniae in vivo upon MyD88-dependent attraction. J Immunol 2005;174:4836–4844.[Abstract/Free Full Text]
  37. Ngeh J, Anand V, Gupta S. Chlamydia pneumoniae and atherosclerosis: what we know and what we don't. Clin Microbiol Infect 2002;8:2–13.[CrossRef][Medline]
  38. Johnston SL, Blasi F, Black PN, Martin RJ, Farrell DJ, Nieman RB. The effect of telithromycin in acute exacerbations of asthma. N Engl J Med 2006;354:1589–1600.[Abstract/Free Full Text]
  39. Gieffers J, Rupp J, Gebert A, Solbach W, Klinger M. First-choice antibiotics at subinhibitory concentrations induce persistence of Chlamydia pneumoniae. Antimicrob Agents Chemother 2004;48:1402–1405.[Abstract/Free Full Text]
  40. Pantoja LG, Miller RD, Ramirez JA, Molestina RE, Summersgill JT. Characterization of Chlamydia pneumoniae persistence in HEp-2 cells treated with gamma interferon. Infect Immun 2001;69:7927–7932.[Abstract/Free Full Text]
  41. Wehrl W, Meyer TF, Jungblut PR, Muller EC, Szczepek AJ. Action and reaction: Chlamydophila pneumoniae proteome alteration in a persistent infection induced by iron deficiency. Proteomics 2004;4: 2969–2981.[CrossRef][Medline]
  42. Mathews S, George C, Flegg C, Stenzel D, Timms P. Differential expression of ompA, ompB, pyk, nlpD and Cpn0585 genes between normal and interferon-gamma treated cultures of Chlamydia pneumoniae. Microb Pathog 2001;30:337–345.[CrossRef][Medline]
  43. Molestina RE, Klein JB, Miller RD, Pierce WH, Ramirez JA, Summersgill JT. Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells. Infect Immun 2002;70:2976–2981.[Abstract/Free Full Text]
  44. Netea MG, Kullberg BJ, Jacobs LE, Verver-Jansen TJ, van der Ven-Jongekrijg, Galama JM, Stalenhoef AF, Dinarello CA, Van der Meer JW. Chlamydia pneumoniae stimulates IFN-gamma synthesis through MyD88-dependent. J Immunol 2004;173:1477–1482.[Abstract/Free Full Text]
  45. Costa CP, Kirschning CJ, Busch D, Durr S, Jennen L, Heinzmann U, Prebeck S, Wagner H, Miethke T. Role of chlamydial heat shock protein 60 in the stimulation of innate immune cells by Chlamydia pneumoniae. Eur J Immunol 2002;32:2460–2470.[CrossRef][Medline]
  46. Knapp S, Wieland CW, Van't Veer C, Takeuchi O, Akira S, Florquin S, van der Poll T. Toll-like receptor 2 plays a role in the early inflammatory response to murine pneumococcal pneumonia but does not contribute to antibacterial defense. J Immunol 2004;172:3132–3138.[Abstract/Free Full Text]
  47. Mehta SJ, Miller RD, Ramirez JA, Summersgill JT. Inhibition of Chlamydia pneumoniae replication in HEp-2 cells by interferon-gamma: role of tryptophan catabolism. J Infect Dis 1998;177:1326–1331.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
W. MacNee
Update in Chronic Obstructive Pulmonary Disease 2007
Am. J. Respir. Crit. Care Med., April 15, 2008; 177(8): 820 - 829.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200607-926OCv1
175/8/791    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Droemann, D.
Right arrow Articles by Dalhoff, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Droemann, D.
Right arrow Articles by Dalhoff, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2007 American Thoracic Society