Abstract
Infection with Chlamydia pneumoniae can trigger acute asthma and is associated with severe chronic asthma. The aim of the present study was to examine the relationship between airway inflammation and serological response to C. pneumoniae in acute severe asthma.
Subjects (n=54) were recruited within 4 h of presentation to the emergency department with an acute exacerbation of asthma. Clinical history taking, sputum induction (0.9% saline), spirometry and acute and convalescent serology for C. pneumoniae immunoglobulins A and G were performed.
At presentation, 47% of subjects had antibodies directed against C. pneumoniae, and 38% (20) demonstrated an increase in C. pneumoniae antibody levels, with 15 demonstrating a rise in immunoglobulin A concentration. C. pneumoniae responders exhibited significantly higher sputum neutrophil levels (4.6×106 cells·mL−1) compared to nonresponders (1.2×106 cells·mL−1, p=0.02) and elevated sputum eosinophil cationic protein concentration (3,981 versus 1,122 ng·mL−1, p=0.02).
An acute antibody response to Chlamydia pneumoniae is common in exacerbations of asthma. The serological features suggest that Chlamydia pneumoniae reactivation may trigger neutrophilic airway inflammation in acute asthma.
This study was supported by the National Health and Medical Research Council, Canberra, New South Wales, Australia.
Chlamydia pneumoniae is an intracellular pathogen 1, 2 that has recently been associated with chronic airway diseases such as asthma. Acute C. pneumoniae infection can cause exacerbation of asthma 3, and chronic/persistent infection has been linked to chronic persistent asthma, and adult onset asthma 4. Children with increased levels of secretory immunoglobulin (Ig) A directed against C. pneumoniae were found to have more frequent clinical exacerbations of asthma 5, whereas, in adults with asthma, higher titres of antibodies directed against C. pneumoniae were associated with more severe clinical disease 6, 7. These data suggest that C. pneumoniae infection may influence the clinical course of asthma.
The mechanism of these effects is not known, but may involve a modification of the airway inflammatory response. Typically, asthma is characterised by a type 2 T-helper lymphocyte (Th)-driven eosinophil response that proceeds to airway mucosal damage and airway hyperresponsiveness. Recurrent or persistent infection with Chlamydia in trachoma and salpingitis causes a chronic lymphocytic infiltrate and tissue remodelling 8. Persistent or recurrent infection with C. pneumoniae may also amplify asthmatic inflammation. In the present study, the aim was to investigate this hypothesis by studying C. pneumoniae infection and airway inflammation in subjects with an acute exacerbation of asthma. Characterisation of the serological response to C. pneumoniae and its relationship to airway inflammation in subjects who presented to the emergency department with acute asthma was sought.
Methods
Subjects
Adults aged 16–74 yrs who presented to the emergency department of John Hunter Hospital, Newcastle, Australia, with an acute exacerbation of asthma were included if they were recruited within 4 h of presentation, provided an adequate sputum sample and attended for follow-up serological testing. Asthma was defined using American Thoracic Society criteria and an acute exacerbation of asthma by presentation to the emergency department for treatment of acute asthma characterised by deterioration of symptoms, increase in use of short-acting β2-agonist for relief of symptoms or deterioration in self-monitored peak expiratory flow leading to presentation to the emergency department. Subjects who presented to the emergency department over an 11-month period were reviewed. Of 84 individuals who agreed to participate, 31 were not included for the following reasons: 10 had received parenteral corticosteroids in the emergency department >4 h before interview, one showed consolidation consistent with pneumonia on chest radiography, nine had diagnosed chronic obstructive pulmonary disease (COPD), one had a pneumothorax complicating their asthma, three were pregnant, one was found not to have asthma on objective testing at follow-up, one yielded an inadequate sputum specimen at Visit 2 and a further five subjects did not return for follow-up and convalescent serology was not obtained.
Design
After initial medical assessment and treatment, the investigator obtained informed consent and performed clinical assessment, spirometry (Vitalograph Ltd, Buckingham, UK), sputum induction and venepuncture for acute phase serology. Treatment was administered according to current asthma guidelines and included aerosol β2-agonists and systemic corticosteroids. Subjects were asked to return 4–5 weeks later, when they had symptomatically returned to baseline and were no longer on parenteral corticosteroids (Visit 2). Subjects underwent clinical assessment, allergy skin-prick tests, spirometry, bronchial provocation challenge, sputum induction and venepuncture for convalescent phase serology.
Clinical assessment
Standardised history taking elicited asthma triggers, atopic disease, medications taken when stable and since symptom deterioration, the time course and cause of the exacerbation, and previous asthma severity 9. Atopy was determined by the presence of at least one immediate reaction to skin-prick testing (weal >3 mm), carried out using a 1:10 weight/volume dilution of 20 common allergens (from Bayer Australia Ltd, Pymble, Australia) at Visit 2.
Sputum induction
Sputum induction was only performed if subjects could perform an adequate forced expiratory volume in one second (FEV1) or peak flow manoeuvre, and if this was >25% of the predicted value after pretreatment with salbutamol 200 µg delivered via Volumatic spacer (Allen and Hanbury's, Melbourne, Australia). All subjects received supplemental oxygen during sputum induction. Sputum was induced using sterile normal saline (0.9%) delivered from an ultrasonic nebuliser (ULTRA-NEB®; DeVilbiss Health Care Inc., Somerset, PA, USA) with a Hans Rudolph 2700 two-way nonrebreathing valve box (Hans Rudolph, Inc., Kansas City, MO, USA), as described previously 10. Spirometry was performed 1 min after each nebulisation period, and supplemental salbutamol was administered if there was a fall of ≥20% in spirometric results from baseline. Sputum induction was stopped once an adequate sample was obtained, if spirometric results fell below 20% of baseline and did not recover within 10 min, at the subject's request or at the investigators' discretion. At Visit 2, sputum was induced using ultrasonically nebulised hypertonic saline (4.5%) as described previously 11.
Sputum analysis
Sputum portions were selected from saliva and processed as described previously 12. The sputum was dispersed using 0.1% dithiothreitol (Sputolysin 10%; Calbiochem Corp., La Jolla, CA, USA), and total cell count (TCC) of nonsquamous cells and viability were determined. Supernatant was aspirated and stored at −70°C, and cytocentrifuge slides prepared from the resuspended cell pellet (Shandon Cytospin, Sewickley, PA, USA). A differential count was obtained by counting 400 cells from May-Grünwald Giemsa-stained cytopreparations. Eosinophils were enumerated from slides stained with Chromotrope 2R in the same fashion. Sputum supernatant eosinophil cationic protein (ECP) levels were determined by radioimmunoassay (ECP RIA; Kabi Pharmacia Diagnostics AB, Uppsala, Sweden), and interleukin (IL)-8 levels by enzyme-linked immunosorbent assay (ELISA) (Quantikine Human IL-8 ELISA; R&D systems, Minneapolis, MN, USA).
Serology
Acute and convalescent sera were tested for C. pneumoniae IgG and IgA, C. psittaci IgG and Helicobacter pylori IgG and IgA. C. psittaci serology was investigated to ensure that there were no subjects with a false positive C. pneumoniae result due to C. psittaci infection. H. pylori serology was tested to control for a nonspecific antibody response. Antibodies directed against the C. pneumoniae outer membrane complex were detected in serum using ELISA (Bioclone Pty Ltd, Sydney, Australia). A test index was calculated from optical densities relative to that of control material, where an index of <0.9 is negative, 0.9–1.09 is equivocal, 1.10–2.99 is positive and >2.99 is strongly positive. A rise in antibody response was regarded as a change in index of ≥1.35 for C. pneumoniae IgG and ≥1.0 for IgA, which corresponds to a four-fold rise in titre by microimmunofluorescent assay 13. C. psittaci IgG was also detected using a complement fixation test. H. pylori IgG and IgA were detected by ELISA (Genesis Diagnostics, Littleport, UK). If a subject had a specific rise in C. pneumoniae IgG or IgA between Visits 1 and 2 in the absence of an antibody response to C. psittaci or a rise in either IgG or IgA directed against H. pylori, this was regarded as serological evidence of either acute or reactivating infection with C. pneumoniae.
Statistical methods
Statistical analysis was carried out using STATA (Stata Corporation, College Station, TX, USA). Where variables were not normally distributed, they were log transformed and the results reported as mean and sem or 95% confidence interval. If this was not possible, variables were analysed using nonparametric tests. Differences between groups were analysed using a two-sided unpaired t-test. Differences in proportions between groups were analysed using Fisher's exact test or the Chi-squared statistic. Univariate relationships between continuous variables were analysed using Spearman's rank correlation coefficient, r. Significance was accepted when p was <0.05.
Results
Subject characteristics
Subjects had a mean age of 45 yrs (range 16–74 yrs), 26% were male and 13% current smokers. Most subjects had at least moderate persistent asthma with 37 (70%) having Aas scores of ≥4 9. At presentation with acute asthma, the subjects had moderate airflow obstruction with a mean±sem FEV1 of 65.2±5.5% pred. Subsequently 63% were admitted to hospital and the median length of stay was 1.5 days. The subjects with an acute response to C. pneumoniae were similar to nonresponders in terms of age, sex, smoking status, and clinical asthma severity and treatment (table 1⇓).
Subject characteristics
Serological response
At Visit 1, 25 (47%) subjects had a C. pneumoniae IgG index of ≥0.9, indicating past exposure to C. pneumoniae. Fifteen of these showed a rise in IgA index, indicating serological reactivation or reinfection with C. pneumoniae. Of the 28 subjects who were serologically negative for C. pneumoniae, five (9%) showed a rise in IgG index, indicating acute infection. Overall, 20 (38%) subjects exhibited a significant rise in C. pneumoniae antibody levels (antibody responders).
There were no subjects with IgG directed against C. psittaci at Visit 1 or 2. Of those who showed either an IgA or IgG response to C. pneumoniae at Visit 1, five (15%) had IgG directed against H. pylori and seven (23%) IgA. Only one subject exhibited a rise in H. pylori IgA between visits, but not in C. pneumoniae IgA, and no subjects showed a rise in H. pylori IgG between visits. All subjects who showed a rise in IgA and IgG directed against C. pneumoniae had a specific antibody response, consistent with acute or reactivating infection.
Antibody response to Chlamydia pneumoniae and airway inflammation
At presentation with acute asthma, the sputum TCC was increased in C. pneumoniae antibody responders (mean 8.1×106 cells·mL−1) compared to nonresponders (3.1×106 cells·mL−1, p=0.03) (table 2⇓, fig. 1⇓). C. pneumoniae responders had significantly more sputum neutrophils (4.6×106 cells·mL−1) compared to nonresponders (1.2×106 cells·mL−1, p=0.02) (table 2⇓, fig. 2⇓). In addition, they had significantly more macrophages/monocytes (2.1×106 cells·mL−1) compared to nonresponders (0.8×106 cells·mL−1, p=0.02) (table 2⇓). There were no significant differences in sputum eosinophil and lymphocyte numbers (table 2⇓). Those with a C. pneumoniae antibody response had higher sputum ECP levels (3,981 ng·mL−1) compared to nonresponders (1,122 ng·mL−1, p=0.02) (table 2⇓). There was a trend towards higher sputum IL-8 levels in responders (38 ng·mL−1) compared to nonresponders (16.2 ng·mL−1, p=0.08) (table 2⇓).
Sputum total cell count (TCC) at Visits 1 and 2 in subjects with (♦) and without (▪) an acute rise in Chlamydia pneumoniae antibody levels. Data have been log transformed and are presented as mean and 95% confidence interval (for clarity, antilog values are presented on the axis). *: p<0.05 versus subjects without rise in antibody levels.
Sputum neutrophil count at Visits 1 and 2 in subjects with (♦) and without (▪) an acute rise in Chlamydia pneumoniae antibody levels. Data have been log transformed and are presented as mean and 95% confidence interval (for clarity, antilog values are presented on the axis). *: p<0.05 versus subjects without rise in antibody levels.
Induced sputum cell counts and fluid phase marker levels in acute asthma with Chlamydia pneumoniae infection
By Visit 2, those who showed an antibody response to C. pneumoniae exhibited a significant fall in sputum neutrophil number (p=0.045) (fig. 2⇑) and sputum ECP level (p=0.01) (fig. 3⇓). By Visit 2, there were no significant differences in sputum inflammatory markers between those who showed an increase in C. pneumoniae antibody levels and those who did not (table 3⇓). The subjects with an acute antibody response to C. pneumoniae tended to have had more asthma exacerbations requiring oral corticosteroids in the previous 12 months (60 versus 36%, Chi-squared 2.8, p=0.09).
Sputum eosinophil cationic protein (ECP) concentration at Visits 1 and 2 in subjects with (♦) and without (▪) an acute rise in Chlamydia pneumoniae antibody levels. Data have been log transformed and are presented as mean and 95% confidence interval (for clarity, antilog values are presented on the axis). *: p<0.05 versus subjects without rise in antibody levels.
Induced sputum cell counts and fluid phase marker levels at Visit 2
Discussion
In the present study, the relationship between airway inflammation and infection with C. pneumoniae in acute asthma was examined. It was found that over one-third of adults presenting with acute severe asthma showed a rise in C. pneumoniae-specific antibodies consistent with acute infection, reinfection or reactivation of latent infection with C. pneumoniae. These subjects exhibited a more intense inflammatory response during the acute exacerbation, with an increase in sputum TCC, neutrophil count and ECP level compared to subjects with acute asthma who did not show an increase in C. pneumoniae antibody levels.
Subjects were recruited from patients presenting to the emergency department with acute asthma, who had at least moderate airflow obstruction, representing a group with more severe acute asthma. This group was selected for study based on previous work linking C. pneumoniae infection with asthma exacerbations and severe asthma 6, 7, 14. The sputum induction time tended to be shorter at Visit 1 compared to Visit 2, but there were no differences in nebuliser time between C. pneumoniae responders and nonresponders at either visit that would be sufficient to account for the difference in neutrophil count or ECP level that was seen 15.
C. pneumoniae is an intracellular pathogen that is an important cause of acute respiratory tract infection 16 and there is evidence to suggest that it may persist within macrophages and cause chronic infection. In subjects with COPD who have undergone lung resection, the organism has been identified immunohistochemically within lung tissue at a far greater rate than in controls 17. Thus persistence of C. pneumoniae may be important in the pathogenesis of these chronic inflammatory disorders. Hahn et al. 18 were the first to describe an association between C. pneumoniae infection and the development of adult onset asthma or exacerbations of asthma, and they later demonstrated an increased risk of development of adult onset asthma in association with elevated levels of IgA directed against C. pneumoniae 4. Although the prevalence of C. pneumoniae infection does not appear to be greater in asthma 14, three studies have associated C. pneumoniae infection with increased asthma severity in adults 6, 7, 14 and increased numbers of exacerbations in children 5. These results imply that C. pneumoniae may be an acute precipitant of exacerbations of asthma, whereas, in chronic asthma, reinfection or persistent infection may be associated with more severe disease.
The high frequency of a C. pneumoniae-specific antibody response is consistent with the high prevalence of C. pneumoniae infection reported in severe asthma 7. In addition, reactivation of C. pneumoniae infection by corticosteroids has been reported in experimental models 19, and could be relevant since each of the subjects with acute asthma was treated with systemic corticosteroids. Laitinen et al. 19 reported that corticosteroid treatment could reactivate C. pneumoniae infections in up to 60% of mice. The clinical implications of this warrant further study in view of the widespread use of systemic corticosteroids in acute asthma, and the high seroprevalence of C. pneumoniae reactivation observed in the present study.
A potential limitation of the present study and most others mentioned is that infection with C. pneumoniae has been inferred serologically without isolation of the organism. C. pneumoniae is difficult to culture and serology cannot be regarded as the gold standard for diagnosis. When detection of the organism has been attempted using the polymerase chain reaction and immunohistochemistry, the likelihood of detection was greater in those with elevated titres; however, these methods are not standardised between centres. The present study used an ELISA to detect antibody response to an outer membrane protein complex that is highly specific to C. pneumoniae 13. This test has been shown to be highly sensitive and specific when compared to Western-blot analysis and to show very high concordance with results obtained by microimmunofluorescence 13. In order to improve specificity, complement fixation was also performed for C. psittaci, which was negative in all subjects. To ensure that the antibody response seen was limited to C. pneumoniae and not a marker of generalised immune activation in acute asthma, an acute antibody response to another unrelated pathogen, H. pylori, was also sought and no increase in levels of antibodies directed against this organism were seen. The results indicate that the present observations reflect a specific C. pneumoniae antibody response. It was found that subjects with this antibody response exhibited increased airway inflammation.
The specific antibody response to C. pneumoniae and heightened airway inflammation could have occurred in response to infection or reactivation of infection with C. pneumoniae, or, alternatively, may represent a B-cell response with increased C. pneumoniae IgA production triggered by another stimulus. It was determined that subjects who had an antibody response were reacting to specific C. pneumoniae antigens, as there was no increase in levels of antibodies directed against the unrelated organism H. pylori. Repeated infection with C. pneumoniae leads to expression of internal heat shock proteins (hsps), particularly hsp-60, which closely reacts with human hsp-60 and triggers autoimmune-directed inflammation that is thought to play a role in the pathogenesis of trachoma 20. One possible explanation of the present results is that a nonspecific inflammatory reaction occurred due to acute asthma or as part of a viral infection, and this may have triggered a C. pneumoniae antibody response that further exacerbated airway inflammation. IgA is a potent stimulus for eosinophil activation, and eosinophil degranulation can lead to neutrophil recruitment to the airway 21. Therefore, increased levels of IgA could trigger ECP release and increase sputum neutrophil numbers. This possibility requires further evaluation in a study with concurrent assessment of viral infection and C. pneumoniae antibody responses.
An alternative possibility is that the C. pneumoniae antibody response reflects reinfection or reactivation of infection with C. pneumoniae, and the sputum neutrophil response has occurred in response to this. There is limited data on the normal host immune response to C. pneumoniae. A case report of an acute infection in asthma found an acute lymphocytic infiltrate 3. Infection with C. trachomatis results in an acute neutrophilic infiltrate followed by an increase in plasma cell numbers. Murine models of C. pneumoniae infection support a similar pattern of inflammation in the lung, with acute neutrophilia followed by an increase in monocyte numbers. Reinfection results in a strong cell-mediated immune response with elevated interferon gamma levels, indicative of a Th1-mediated response which is felt to be important in clearing the organism 22–24. Subjects with asthma that respond with a Th2-dominant response may therefore be less able to clear the organism, possibly leading to persistence of the organism. It has been shown that production of IL-10 by Th2 leads to increased B-cell production of IgA 25, suggesting that elevated IgA levels or an increase in levels of IgA directed against C. pneumoniae may be an important marker of subjects who are less able to clear C. pneumoniae 25. The present results are in keeping with this, as the majority of responders to C. pneumoniae demonstrated an increase in IgA levels, and this was associated with an increase in sputum neutrophil count at presentation with acute asthma. Neutrophils are potentially a potent source of proteolytic enzymes with the ability to damage and activate the airway epithelium 26, whereas neutrophil elastase can cause eosinophil degranulation 27. In addition, IgA receptors have been detected on activated eosinophils 28 and activation of IgA can cause eosinophil degranulation 29. The inflammatory changes caused by infection with C. pneumoniae, therefore, have the potential to amplify the inflammation and airway damage present in asthma. Furthermore, there is also the potential for asthma therapy to potentiate this response given the ability of corticosteroids to reactivate C. pneumoniae infection 19.
To conclude, the present study has demonstrated that subjects with acute asthma and an acute rise in Chlamydia pneumoniae antibody levels exhibit more intense airway inflammation with elevated sputum neutrophil counts and sputum eosinophil cationic protein levels compared to subjects with no antibody response. It remains to be determined whether this is a response to acute infection, reactivation of chronic Chlamydia pneumoniae infection or a result of nonspecific airway inflammation triggering the antibody response in susceptible asthmatics with previous exposure to Chlamydia pneumoniae. Such an intense immune response in acute asthma may have important implications for the development of more severe chronic asthma.
- Received January 15, 2002.
- Accepted May 2, 2002.
- © ERS Journals Ltd
References
