INTRODUCTION

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Chlamydia pneumoniae is an intracellular pathogen and a common cause of respiratory tract infections (1). There is increasing evidence that persistent infection with C. pneumoniae is linked to chronic diseases including atherosclerosis. There is an increased risk for myocardial infarction in subjects who are seropositive for C. pneumoniae (2), and C. pneumoniae can be demonstrated in atherosclerotic plaques through immunohistochemistry and the polymerase chain reaction (PCR) (3, 4). Less attention has been devoted to the relationship between C. pneumoniae and chronic obstructive pulmonary disease (COPD).

Blasi and coworkers compared 142 outpatients with exacerbations of COPD and 114 healthy controls (5). They found that the prevalence of IgG antibodies to C. pneumoniae was 63% in the subjects with COPD and 46% in the control subjects (p = 0.007). In addition, the geometric mean titer of antibodies to C. pneumoniae was higher in the subjects with COPD. The presence of antibodies to C. pneumoniae does not, however, prove that the subjects have persistent infection. Von Hetzen and colleagues performed PCR for C. pneumoniae in sputum from 54 subjects with COPD (6). Forty-one of the 54 subjects were classified as having severe COPD (FEV₁ < 50% predicted), with the others classified as having mild disease. Twenty-three subjects hospitalized with pneumonia served as a control group. Fifty-nine percent of the subjects with severe COPD, 40% of the subjects with mild COPD, and 21% of the control group were positive for C. pneumoniae through PCR. Although Van Hetzen and colleagues did not repeat the PCR several months later to confirm that this represented chronic infection, only one subject had a change in serum antibodies suggestive of acute infection. Van Hetzen and colleagues proposed that persistent infection with C. pneumoniae may amplify the inflammation that occurs in COPD (7).

We investigated the relationship between C. pneumoniae and COPD by using immunohistochemistry on lung tissue resected at surgery from a group of subjects with COPD and a matching group of past or present smokers with normal lung function.

	METHODS

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Subjects

We obtained blocks of archival, formalin-fixed lung tissue from two groups of subjects. In the first group were patients who had had one or more lobes resected for bronchial carcinoma. The specimens were identified through the computerized records of the Department of Pathology, Green Lane Hospital. The operations were performed between January 1992 and September 1996. All of the blocks used were from sites remote from the bronchial carcinoma. Further information, including smoking history, past medical history, medication, and preoperative lung function, were obtained from the patient's hospital notes. Patients were excluded if they had a history of asthma. Patients were classified as having COPD if their FEV₁ was < 80% predicted and the ratio of FEV₁ to FVC was < 70%. Patients with FEV₁  80% and FEV₁/FVC  70% served as controls.

A second group of subjects consisted of individuals who had had a postmortem following accidental death from causes such as road traffic accidents or carbon monoxide poisoning. Again, archival blocks of lung tissue from this group were used in the study.

The study was approved by the North Health Ethics Committee, Auckland, New Zealand.

Immunohistochemistry

Sections 5 µm thick were cut on a microtome and mounted on poly-L-lysine-treated microscope slides. Immunohistochemistry for C. pneumoniae was done with a monoclonal antibody (RR-402; Washington Research Foundation, Seattle, WA) that is directed against the major outer membrane protein of C. pneumoniae (8). Staining was done with the avidin-streptavidin-alkaline phosphatase method.

Immediately before staining, the slides were warmed to 37° C in an incubator for at least 1 h, and were then deparaffinized in xylene and rehydrated in a grading series of alcohol dilutions and 0.01 M phosphate-buffered saline (PBS). Normal horse serum was applied for 1 h at room temperature (RT) in order to block nonspecific binding. The primary antibody (RR-402, 1:1,000 dilution) was applied to one section on each slide, and mouse ascites fluid (1:1,000 dilution; Sigma, St. Louis, MO) or PBS was added to the other section on the slide, which acted as a negative control. The sections were incubated for 18 h at 4° C and were then washed with PBS, after which the secondary antibody (antimouse IgG; Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) was applied for 45 min at RT. The sections were washed again with PBS, and the streptavidin-alkaline phosphatase conjugate reagent (1:1,000 dilution) was applied for 1 h at RT. After a washing with PBS, Fast Red substrate (Sigma) was applied to each section for 3 to 5 min at RT. The sections were then washed in water and counterstained in Gill's hematoxylin.

Immunostaining was also done on positive and negative tissue controls. The negative tissue controls were 21-d-old rat brain sections (a gift from A. Scheepens of the Department of Paediatrics, University of Auckland), and the positive tissue controls were mouse lung tissue infected by C. pneumoniae (a gift from Professor P. Saikku of the Public Health Institute, Oulu, Finland) (9).

Double staining for both macrophages and C. pneumoniae was done on the sections from the patients who had undergone a lobectomy. The staining for macrophages was done with an antihuman mouse monoclonal antibody directed against CD68 (Dako, Glostrup, Denmark). The avidin-biotin-peroxidase system with diaminobenzidine (DAB) and metal enhancer was used. After being stained for C. pneumoniae with the alkaline phosphatase method as described earlier, the slides were immersed in 200 ml of PBS and 5 ml of hydrogen peroxide for 1 h at RT to inhibit endogenous peroxidase. The slides were then washed with PBS and the same steps were followed as described earlier, up to and including the application of the secondary antibody. The slides were then washed with PBS, and the substrate (DAB with metal enhancer) was applied for 3 to 5 min. This was followed by washing in water to stop the reaction. Cells staining for C. pneumoniae were red, those staining for CD68 were blue, and doubly staining cells were purple.

Polymerase Chain Reaction

The polymerase chain reaction (PCR) for C. pneumoniae was performed on tissue from selected subjects. Ten 10-µm-thick sections from each of six subjects who had undergone lobectomy were used. DNA was retrieved with a kit set from Boehringer Mannheim (Mannheim, Germany). Primers were used as described by Campbell and colleagues (10). The primers were HL-1: 5'-GTTGTTCATGAAGGCCTACT-3'; HM-1: 5'-GTGTCATTCGCCAAGGTTAA-3'; and HR-1: 5'-TGCATAACCTACGGTGTGTT-3'. The sections were deparaffinized, and the first PCR reaction was performed as follows: the samples were pretreated at 94° C for 5 min and amplified for 35 cycles. Each cycle consisted of denaturation at 94° C for 1 min, annealing at 55° C for 1 min, and primer extension at 72° C for 1 min. DNA was amplified in 50-ml volumes containing 50 mmol of deoxynucleotide triphosphate (dNTP), 0.5 mmol of each primer (HL-1 and HR-1), 1 U Taq polymerase, 5 mmol MgCl₂, 1 mmol Tris-HCl, and 50 mmol KCl, pH 8.3. The second PCR was performed with 2 ml of the first amplification, as follows: samples were pretreated at 94° C for 1 min, followed by annealing at 48° C for 1 min and primer extension at 72° C for 1 min. DNA was amplified in 50-ml volumes containing 200 mmol dNTP, 0.5 mmol of each primer (HR-1 and HM-1), 1 U Taq polymerase, 3 mmol MgCl₂, 1 mmol Tris-Cl, and 50 mmol KCl, pH 8.3. The amplifications were performed in an automated thermocycler (Robocycler; Stratagene, La Jolla, CA).

Microscopy

For microscopy, the top edge of each section was located. Cells stained for C. pneumoniae were then counted in successive fields at a magnification of ×400, starting at the center of the top edge of the section and proceeding down to the bottom of the section until a vertical column had been counted. An average of 16 fields were counted for each section.

Statistical Analysis

Results are expressed as mean ± SD. Groups were compared by using Student's t test.

	RESULTS

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Subjects

The characteristics of the subjects are shown in Table 1. The subjects who had a lobectomy for bronchial carcinoma were divided into those with COPD (n = 16) and control subjects (n = 21). The subjects with COPD were similar to the controls with respect to age, sex, and smoking history (Table 1). In the subjects with COPD, the FEV₁ was 64 ± 8% (mean ± SD), as compared with 95 ± 11% for the controls. The FEV₁/FVC values were 57 ± 6% and 90 ± 16%, respectively. Only a small number of the subjects with COPD and none of the controls were receiving treatment with inhaled medicines (Table 1).

The second group of subjects were individuals who had undergone a postmortem because they had died accidentally (n = 19). These subjects were younger (32 ± 14 yr) than those in the first group. None of these individuals had a history of respiratory disease or evidence of respiratory disease on macroscopic or microscopic examination of the lungs. We did not have the results of spirometry for these individuals.

Immunohistochemistry

Positive staining for C. pneumoniae was detected both in the alveolar wall and in the small airways (none of the sections examined included large airways). Examples of positively staining cells are shown in Figure 1. Positive staining was seen in all of the subjects who had tissue removed at surgery. In the subjects with COPD, an average of 14.5 ± 8.4 cells were seen in each ×400 field, compared with 9.3 ± 5.1 cells in the controls. This difference was statistically significant (p = 0.02).

View larger version (140K): [in this window] [in a new window]   Figure 1.   Section of lung parenchyma showing immunohistochemical staining for Chlamydia pneumoniae. The cells staining positively for C. pneumoniae are red (original magnification: ×1,000).

Positive staining was seen in only 8 of the 18 subjects (44%) in the second (i.e., the postmortem) group. In this group, the average number of cells per field in subjects with positive staining was only 0.4. No staining was seen in the negative tissue controls or in negative controls in which mouse ascitic fluid was used in place of the monoclonal antibody to C. pneumoniae.

Macrophages were identified through staining for CD68. The mean number of CD68-positive cells was 18.9 per ×400 field in the control subjects who had had a lobectomy and 12.9 per ×400 field in those who had COPD. This difference was not significant (p = 0.15). The proportion of macrophages infected with C. pneumoniae was identified through double staining (Figure 2). In the control subjects, 29 ± 27% of macrophages stained positively for C. pneumoniae, as compared with 54 ± 20% of macrophages in the subjects with COPD (p < 0.001).

View larger version (136K): [in this window] [in a new window]   Figure 2.   Section of lung parenchyma showing double staining for Chlamydia pneumoniae and macrophages (CD68-positive cells). Macrophages are stained blue. Cells infected by Chlamydia pneumoniae are stained pink. Cells that are double stained are shown by arrows (original magnification: ×400).

PCR for C. pneumoniae was performed on tissues from six of the subjects who had a lobectomy. The investigator who performed the PCR was blinded to the results of the immunohistochemical examination. Three of the subjects were definitely positive for C. pneumoniae on PCR, one was borderline, and two were negative (all of these subjects were positive for C. pneumoniae on immunohistochemistry).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

All of the subjects in this study who had a lobectomy for bronchial carcinoma were positive for C. pneumoniae by immunohistochemistry. In addition, the subjects with COPD had significantly more cells that stained positive for C. pneumoniae than did the control subjects. A difference between the two groups was also evident in the proportion of macrophages that stained positively for C. pneumoniae (54% versus 29%, respectively).

We had not expected to find all of the lobectomy samples positive for C. pneumoniae, but seropositivity for C. pneumoniae does increase with age, smoking (1, 11), and the diagnosis of bronchial carcinoma (12). We did not have the opportunity to measure antibodies to C. pneumoniae in the lobectomy group, but on the basis of published series we would anticipate that more than 80% of the subjects would have been seropositive (6, 13). Evidence that we were indeed observing true positive staining came from the use of appropriate positive and negative controls. Furthermore, only 44% of the younger subjects (average age: 32 yr), who had died accidentally showed positive staining for C. pneumoniae. Epidemiologic studies would suggest that approximately 50% of subjects of this age would be seropositive. There is uncertainty about whether C. pneumoniae is cleared after acute infection or whether it persists in the body indefinitely, but our findings suggest that the latter may be the case.

In a subgroup of patients we found that four of six were positive for C. pneumoniae on PCR. This provided further evidence, in addition to our immunohistochemical results, of the presence of C. pneumoniae in the lungs of these subjects. It may appear surprising that immunohistochemistry was more sensitive than PCR for C. pneumoniae, but this may simply reflect the difficulty of detecting C. pneumoniae with PCR in formalin-fixed, paraffin-embedded tissues. In studies using atherosclerotic tissue, immunohistochemistry for C. pneumoniae has also proved to be more sensitive than PCR (3).

Adenovirus is another organism that can cause persistent infection in the respiratory tract. Matsue and colleagues looked for adenoviral DNA in lung tissue resected from patients having surgery for lung cancer (14). They used PCR to look for the E1A region of the adenoviral genome, and compared subjects with and without airflow obstruction. Using paraffin-embedded tissues, they found that 88% of sections from subjects with COPD were positive on PCR for E1A, compared with 85% of sections from controls. However, densitometric analysis showed three times as much of the E1A product in the samples from subjects with COPD than in those from the controls. Our findings and those of Matsue and colleagues suggest that infection with intracellular pathogens may occur to a greater extent in COPD.

If there is increased infection with C. pneumoniae in COPD, it raises the question of whether or not such infection contributes to the pathogenesis of COPD or whether the organism is simply a "bystander." A similar uncertainty surrounds the role of C. pneumoniae in atherosclerosis (15). There is evidence that the infection of monocytes (16) with C. pneumoniae leads to the increased formation of cytokines such as tumor necrosis factor- and interleukin-8, both of which are increased in the sputum of subjects with COPD (17, 18). One could speculate that chronic infection with C. pneumoniae could promote inflammation in the lungs of subjects with COPD, but proof of this would require studies with antibiotics directed against C. pneumoniae.