chlamydia pneumoniae and severity of asthma
TRANSCRIPT
Scand J Infect Dis 34: 22–27, 2002
Chlamydia pneumoniae and Severity of AsthmaLEENA VON HERTZEN1, TUULA VASANKARI2, KARI LIIPPO2, EVA WAHLSTROÈ M3
and MIRJA PUOLAKKAINEN4
From the 1Finnish Lung Health Association, Helsinki, Finland, 2Department of Pulmonary Medicine, Turku Uni×ersityCentral Hospital, Preitila, Finland, 3National Public Health Institute, Vaccine De×elopment Laboratory, Helsinki, Finlandand Helsinki and 4Department of Virology, Uni×ersity of Helsinki, Helsinki, Finland
A substantial increase in the prevalence of asthma in the Western world during the last few decades has led to a continuoussearch for novel factors that might be involved in the development of the disease. We carried out a study to clarify whetherthere is a relationship between severity of asthma and Chlamydia pneumoniae-speci� c titres at the group level and whetherantibodies to the 60 kDa chlamydial heat shock protein (chsp60) are associated with asthma. A total of 116 (31 men, 85women) consecutive asthma patients from a chest clinic were recruited and divided into 3 groups according to the severity ofthe disease: there were 13 asthmatics with severe, 54 with moderate and 49 with mild asthma. In addition, 50 (31 men, 19women) consecutive blood donors were enrolled to serve as a control group. Sera for the measurements of speci� c IgG, IgAand IgM antibodies using a microimmuno� uorescence test and of chsp60 using an enzyme immunoassay were obtained uponenrolment and also 3–4 months later from the asthma patients. Severe and moderate asthma were found to be stronglyassociated with elevated IgA antibody levels to C. pneumoniae [odds ratio (OR) 5.58, 95% con� dence interval (CI)1.31–23.72 for severe and OR 5.65, 95% CI 2.05–15.53 for moderate asthma] in a logistic regression model. Furthermore,in women, the occurrence of elevated IgA antibody levels and the age-adjusted geometric mean titres of IgA antibodies weresigni� cantly higher among the asthmatics than the controls (p 5 0.003 and 0.04, respectively). Antibodies to chsp60 occurredmore frequently and in higher concentrations among the asthmatics than the controls, although the differences did not reachsigni� cance. In conclusion, severe and moderate asthma were signi� cantly associated with elevated IgA antibody levels to C.pneumoniae suggestive of chronic infection. Antibodies to chsp60 did not prove to be a useful marker of such an infectionamong the asthmatics studied here.
L. Øon Hertzen, MD, The Finnish Lung Health Association, Sibeliuksenkatu 11 A 1, FI -00250 Helsinki, Finland. Fax: »3589 454 21282; E-mail: leena.Øonhertzen@� lha.�
INTRODUCTION
Asthma is now recognized as a chronic in� ammatory disor-der, whatever its severity. This in� ammation is associatedwith changes in airway hyper-responsiveness, air� ow limita-tion, respiratory symptoms and disease chronicity (tissuedestruction and airway remodelling) (1). Probably morethan half of all asthmatics have no family history of atopyand show no skin prick-test positivity or elevated IgE titresto common allergens (2). The aetiology of this subtype ofasthma that characteristically has its onset in adulthood hasso far been enigmatic, although it has occasionally beenfound to follow a respiratory tract infection (3).
Chlamydia pneumoniae, a ubiquitous intracellular bac-terium prone to cause chronic infections, has been associ-ated with several chronic public health diseases, includingadult-onset and severe chronic asthma (4, 5). However, it isstill unclear whether C. pneumoniae is involved in eitherthe initiation or incitement of the disease, or in both.Persistent or repeated chlamydial infections may amplifythe in� ammation in the lungs (6) and lead to tissue scarringand airway remodelling, analogous to how repeated C.trachomatis infections in the Fallopian tubes lead to tubalocclusions and � nally to infertility [reviewed in Pearlmanand McNeeley (7)]. There is compelling evidence to indicatethat a 57–60 kDa protein of the stress protein family, thechlamydial heat shock protein (chsp)60, may play a crucialrole in the immunopathology of repeated or persistentchlamydial infection (8). Elevated levels of antibodies to
chsp60 have been shown to be signi� cantly associated withsevere sequelae of prolonged C. trachomatis infection, suchas scarring trachoma (9), pelvic in� ammatory disease andoccluded Fallopian tubes (10–12). Thus far data on anti-body levels to chsp60 and C. pneumoniae in relation todisease severity have been scanty. These data, however,may give some clue as to the possible involvement of thismicroorganism in the immunopathogenesis of the disease.
The aim of this study was to clarify whether there is arelationship between severity of asthma and C. pneumo-niae-speci� c titres at the group level, and whether antibod-ies to chsp60 are found in asthmatics more frequently thanin controls, suggesting a potential immunopathological rolefor chsp60 in asthma. We also wanted to evaluate theusefulness of this protein as a tentative marker of chronicC. pneumoniae infection.
MATERIALS AND METHODS
Study subjects
Patients were recruited to the study upon attendance at a chestclinic of Turku University Central Hospital. Patients attended theclinic for various reasons associated with asthma, but mainlybecause of veri� cation of the diagnosis, adjustment of medicationor worsening of breathlessness. A total of 116 consecutive patients(31 men, 85 women) who met the criteria of bronchial asthmaaccording to the guidelines of The American Thoracic Society (13)and who did not have concomitant chronic obstructive pulmonarydisease or chronic bronchitis (13) were enrolled. Atopy was de� nedby 1 or more positive skin prick tests to extracts of 25 commonallergens. All asthmatics were non-smokers or had stopped smok-
© 2002 Taylor & Francis. ISSN 0036-5548 DOI: 10.1080 00365540110077155
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Scand J Infect Dis 34 C. pneumoniae and se×erity of asthma 23
ing at least 10 y previously. The asthmatics were dividedinto 3 groups according to the severity of the disease basedon the following 3 criteria: (1) usage of corticosteroids; (2)number of acute exacerbations per year; and (3) reversibility oflung function after treatment. The 3 groups were comprised asfollows:
1. Severe asthma (Group I). Patients who were treated with oralcorticosteroids for \6 months per year and in addition had]3 acute exacerbations per year. Lung function was perma-nently and severely impaired [forced expiratory volume in 1 s(FEV1)B50% of the predicted value in most cases].
2. Moderate asthma (Group II). Patients who did not need oralcorticosteroids for most of the year, but used 0–3 short coursesper year and who additionally had regular inhalant steroidmedication (at doses of \500–1000 mg d, depending on thepreparation). FEV1 after treatment was \50% of the predictedvalue.
3. Mild asthma (Group III). Patients who did not use oralcorticosteroids, but used inhaled steroids at doses of 5500–1000 mg d. One oral steroid course per year was allowed.Reversibility of lung function after treatment was virtuallycomplete and FEV1 was ]80% of the predicted value.
The study protocol was approved by the Ethical Committee ofTurku University Central Hospital. Informed consent was ob-tained from all patients before enrolment. Blood samples werecollected upon enrolment and second samples were collected 3–4months later to obtain information about the nature of the infec-tion in each patient. The data in Tables II, III and IV are based onsera obtained upon enrolment to be comparable with those of thecontrols.
The control group comprised 50 consecutive non-smoking blooddonors (31 men, 19 women) enrolled from The Red Cross inFinland, Helsinki. The background characteristics of the cases andcontrols are presented in Table I.
Methods
C. pneumoniae-speci� c IgG, IgA and IgM antibodies were mea-sured by means of a microimmuno� uorescence (micro-IF) test (14)using C. pneumoniae strain K-6 as an antigen. Fluorescein-conju-gated anti-human IgG (Kallestad; Sano� Diagnostic Pasteur,Chaska, MN), anti-human IgA (Sigma Chemical Company, St.Louis, MO) and anti-human IgM (Dako A S, Copenhagen, Den-mark) were used as conjugates. Before the IgA and IgM measure-ments, all the sera were treated with Gullsorb Reagent (GullLaboratories, Salt Lake City, UT) to remove the IgG antibodies.Sera were tested blindly in serial 2-fold dilutions to the endpoint.
Based on a preliminary analysis of the data, which showed thefrequent occurrence of C. pneumoniae-speci� c antibodies alsoamong the controls, the criterion for elevated IgG antibodies wasde� ned as a titre of ]256, and that for IgA as ]320. For IgM,a titre of ]20 was considered positive. For an acute infection, thecriterion of a 4-fold titre change in any immunoglobulin classand or the presence of an IgM titre of ]20 was used.
Antibodies to C. pneumoniae-speci� c hsp60 were measured bymeans of an enzyme immunoassay (EIA). Polystyrene 96-wellplates (Nalge Ltd., Hereford, UK) were coated with recombinantC. pneumoniae hsp60H is, produced in 5 mg ml Bacillus subtilis (U.Airaksinen, unpublished work) overnight at room temperature.Residual binding was blocked by incubation with 3% bovine serumalbumin (BSA). Sera diluted 1 : 100 in PBS containing 1% BSAwere allowed to bind to the wells. The plates were washed andbinding was detected with horseradish peroxidase-labelled anti-body to human IgG and IgA (Dako A S, Denmark). After wash-ing, the substrate (BM Blue POD substrate; BoehringerMannheim, Germany) was added and the absorbance measured at450 nm. As some sera were found to bind strongly to uncoatedwells, all sera were tested using coated and uncoated wells, and theabsorbance values given by the latter were subtracted from thoseof the former (net absorbance¾Abscoa ted ¼Absu n coa ted) for � nalanalysis. Net absorbances ]0.25 were considered to be positive,
Table I. Background characteristics of cases and controls
Asthma
Severe ModerateCharacteristic Mild Controls
n 13 54 49 50No. of males females 3 10 17 37 11 38 31 19
46 (11.0)Mean age (y) (SD) 63 (12.6) 56 (17.2) 51 (16.1)41–81 21–63Range 19–8019–89
Mean lung functionFEV1 % predicted 48.5 75.6 89.9
97.9FVC % predicted 66.8 86.676.0FEV1 FVC % 65.5 71.823 (46.9)25 (46.3)4 (30.8)Atopy a n (%)
18.0 10.1 4.0Mean duration of asthma (y)
Concomitant disease, n (%)CHD 2 (4.1)9 (16.7)1 (7.7)
2 (3.7)AMI 0 (0)0 (0)Other b 3 (23.1) 13 (24.1) 12 (24.5)
Use of corticosteroids, n (%)47 (95.9)44 (81.5)Inhalant 13 (100)
Oral 13 (100) 16 (29.6) 4 (8.2)
a Skin prick-test positivity to at least 1 allergen.b Mostly hypertension (17 cases) and diabetes mellitus (5 cases).CHD¾coronary heart disease; AMI¾acute myocardial infarction.
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Table II. Occurrence of ele×ated IgG and IgA antibody le×els to C. pneumoniae and age -adjusted GMTs among the cases and controlsaccording to gender and group structure
Asthma
Moderate Mild Controls p aSevere
Elevated IgG levels (]256), n (%)20 (37.0) 11 (22.4)All 12 (24.0) 0.275 (38.5)
Men 1 (33.3) 9 (52.9) 1 (9.0) 8 (25.8) 0.0811 (29.7)Women 10 (26.3) 4 (21.1) 0.734 (40.0)
Elevated IgA levels (]320), n (%)25 (46.3) 13 (26.5)All 8 (16.0) 0.004*6 (46.2)
Men 1 (33.3) 11 (64.7) 3 (27.3) 8 (25.8) 0.044*14 (37.8) 10 (26.3) 0 (0) 0.003**Women 5 (50.0)
GMT-IgG81.1 81.1All 89.7 0.9593.2
Men 52.5 105.2 78.6 119.2 0.39Women 116.0 76.1 84.4 65.3 0.62
GMT-IgA92.9 69.8All 48.7 0.26100.0
Men 47.3 240.6 69.5 87.5 0.0964.1 71.7 23.0 0.04*Women 131.0
a For the difference between cases and controls.
based on our preliminary experiments with the Hsp60 EIA (unpub-lished work).
Statistical analysis
Fischer’s exact test was used for comparing categorical data, andthe F-test of the linear model was used for the continuous vari-ables. To assess the relationship between elevated titres andasthma, logistic regression analysis was used. Age, gender andgroup category were considered as explanatory variables and ele-vated speci� c IgG and IgA titres as dependent variables. The effectof each factor was expressed as odds ratios (ORs) and con� denceintervals (CIs) were calculated at the 95% level. The data wereprocessed using the S-Plus 4.0 Statistical package (MathSoft, Seat-tle, WA).
RESULTS
Elevated IgA antibody levels to C. pneumoniae occurredsigni� cantly more frequently among the asthmatics thancontrols (p¾0.04 for men, p¾0.003 for women). Amongwomen, the severity of asthma and the occurrence ofelevated IgA antibody levels showed a tendency towards alinear relationship at the group level (Table II).
Acute C. pneumoniae infection was found in 10 asthmat-ics and 1 control subject. Nearly all the asthmatics withacute infection were recently diagnosed cases: 8 asthmaticshad been diagnosed within the preceding few months; 1asthma patient within the previous 2 y; and only 1 patienthad long-lasting chronic asthma.
The age-adjusted geometric mean titre (GMT) of IgAantibodies among women was found to be highest amongthose with severe asthma, and a signi� cant difference be-tween the asthmatics and controls was obtained for women(p¾0.04). For IgG antibodies, no signi� cant differences
between the cases and controls, for either men or women,were found either in the occurrence of elevated antibodylevels or in the GMTs (Table II).
The occurrence of chsp60 seropositivity and mean ab-sorbances were found to be higher among the asthmaticsthan controls (25.9% vs. 12% for seropositivity; 0.15 vs.0.02 for mean absorbances), although the differences didnot reach signi� cance in this limited sample (Table III). Nopositive correlation between severity of asthma and theoccurrence of seropositivity at the group level wasobserved.
The association between asthma and elevated speci� cantibody levels was assessed using multiple logistic regres-sion analysis. This revealed that both severe and moderateasthma were strongly associated with elevated IgA titres toC. pneumoniae (OR 5.58, 95% CI 1.3–23.7 for severe andOR 5.65, 95% CI 2.1–15.5 for moderate asthma) (TableIV). No signi� cant associations were found for elevatedIgG titres and nor was any additional information obtainedif lower cut-off points for elevated titres (IgG]128; IgA]160) were used in this model. None of the potential con-founding factors among the cases, i.e. age, gender, skinprick-test positivity, use of oral corticosteroid drugs orconcomitant coronary heart disease, were found to haveany signi� cant effect on the elevated IgG or IgA antibodylevels to C. pneumoniae in the logistic regression model.
DISCUSSION
This study revealed that elevated IgA antibody levels to C.pneumoniae suggestive of chronic infection were stronglyassociated with severe and moderate asthma. Furthermore,
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Scand J Infect Dis 34 C. pneumoniae and se×erity of asthma 25
Table III. Occurrence of ele×ated IgG antibody le×els to chsp60 and mean titres among cases and controls according to gender and groupstructure
Asthma
Moderate Mild ControlsSevere p a
Chsp60 IgG absorbance ]0.25, n (%)13 (24.1) 15 (30.6) 6 (12.0)All 0.132 (15.4)4 (23.5) 3 (27.3) 5 (16.1)1 (33.3) 0.53Men9 (24.3) 12 (31.6)Women 1 (5.3) 0.111 (10.0)
Chsp60 IgG, mean (SD) absorbanceAll 0.12 (0.20) 0.14 (0.29) 0.16 (0.32) 0.02 (0.29) 0.53
0.18 (0.26) 0.19 (0.19) 0.04 (0.34)0.19 (0.13) 0.43Men0.21 (0.31) 0.16 (0.35) 0.00 (0.19) 0.91Women 0.10 (0.21)
a For the difference between cases and controls.
in women, both the occurrence of elevated IgA titres andthe GMTs of IgA antibodies to C. pneumoniae were shownto be signi� cantly higher among the asthmatics than thecontrols, and the highest GMT values in women wereobtained for severe asthma. In men, these associations wereweaker, although a very high GMT of IgA antibodies wasfound in men with moderate asthma. As antibodies to C.pneumoniae also occurred commonly among male controls,we cannot rule out the possibility that some of these menhad heart diseases or other chronic conditions associatedwith C. pneumoniae; data on possible chronic diseasescould not be obtained from the control group. We are alsoaware of the fact that blood donors may not be ideallyrepresentative of an unselected population. Owing to thesetting of this study, relatively high cut-off values forelevated titres were chosen. Similarly, high occurrences ofC. pneumoniae seropositivity by micro-IF and peripheralblood mononuclear cell positivity by PCR have also re-cently been reported in Swedish blood donors (15). Thepotential confounding effect of smoking has been excluded,because only non-smokers were recruited in our study.
Three groups of asthmatics of different disease severitywere enrolled. Inconsistency of criteria for the categoriza-tion of asthma severity still exists. We categorized thepatients on the basis of 3 objective measurements—steroidusage, number of acute exacerbations per year and lungfunction measurements—and found this categorization tobe useful for the purpose of this study. The average age atthe onset of asthma proved to be rather similar in allasthma groups (45–46 y), suggesting that the duration ofthe disease may be a more important factor than individualvulnerability in disease severity.
Indirect serological evidence of the association betweenC. pneumoniae and asthma has accumulated during the lastfew years. An association between newly diagnosed symp-tomatic asthma and elevated IgA antibody levels to C.pneumoniae suggestive of chronic infection has previouslybeen reported (16). Recently, elevated IgG antibody levelsto C. pneumoniae have been found to be signi� cantly
associated with severe chronic asthma (5) and intrinsicasthma in adults (17). However, direct evidence of thepresence of Chlamydia in the bronchi of adult asthmaticsobtained by PCR or by culture has so far been eitherlacking or very scarce. We were similarly unable to detectthe organism using PCR in any of the 150 sputa tested(data not shown), suggesting that sputum may not be arelevant sample type for PCR measurements in adult asth-matics. Bronchoscopic sampling of alveolar macrophagesor tissue from the lower parts of the bronchi (18) mayperhaps be more successful.
Serology, although widely used to diagnose C. pneumo-niae infection, does not give any information on the loca-tion of the infection or differentiate reliably between pastexposure and chronic ongoing infection. However, owing tothe short half-life of IgA antibodies (19), the constantpresence of elevated IgA levels has been widely used as amarker of persistent, chronic infection (16, 20, 21), whereasmore long-lived IgG antibodies alone may re� ect previousexposure to this organism (21). Little is known about therole of serum IgA in immune responses, but there isevidence that IgA, but not IgG, signi� cantly downregulatesthe release of the proin� ammatory cytokines tumour necro-sis factor-a and IL-6 in activated human monocytes in adose-dependent manner (22). IgA may thus interfere with
Table IV. Relationship between ele×ated IgA antibody le×els to C.pneumoniae and age, gender and group category deri×ed fromlogistic regression analysis
IgA titre]320
95% CICharacteristic OR
Age 1.01 0.98–1.03Gender 0.39 0.18–0.86Group
Severe 1.31–23.725.58Moderate 5.65 2.05–15.53Mild 2.68 0.92–7.77Control reference
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the development of chronic in� ammation in prolongedinfections (22). The high occurrence of elevated IgA anti-body levels found in the asthmatics in this study, particu-larly those with moderate to severe asthma, probablyre� ects this intrinsic mechanism of the body to amelioratein� ammation in advanced disease. However, the inductionof IgA production by human B cells has been found to beassociated with IL-10 (23), a cytokine that may furtherimpair the eradication of Chlamydia by downregulatingTh1 immune responses (24).
Although the occurrence of chsp60 seropositivity in thepresent study was clearly higher among the asthmatics thanthe controls, no positive correlation between chsp60seropositivity and asthma severity was found; rather, asth-matics with mild disease showed frequently higher valuesthan those with severe or moderate disease. Our � ndingthat no correlation between hsp60 and micro-IF antibodylevels was observed (data not shown) is in accordance withearlier C. trachomatis studies by Toye et al. (12) and byPeeling et al. (10). However, in those studies a strongassociation between chsp60 antibodies and chronic diseasewas found (10, 12), in contrast to the present study. As ahighly conserved molecule, there is remarkable homologyin hsp60 sequences between different bacteria and evenhumans (25, 26), which inevitably leads to cross-reactionsand obviously also to autoimmune reactions throughmolecular mimicry (26). Although the role of chsp60 inasthma pathogenesis cannot be wholly ruled out on thebasis of this study, chsp60 seropositivity appears not to bea useful diagnostic tool for suspected chronic chlamydialinfection in asthmatics.
Similarly, the ultimate role of C. pneumoniae inasthma—whether it is an inducer or inciter of the dis-ease—cannot be decisively determined on the basis of thiscross-sectional study. It appears, as has been reportedearlier (17), that both roles are possible. Some cases ofasthma observed in this study were obviously initiated byan acute infection, as most of the 10 such infections de-tected were observed in asthmatics with newly diagnoseddisease. However, as the highest values of elevated IgAantibody levels and GMTs of IgA antibodies were generallyfound in asthmatics with severe or moderate asthma, andas mild asthma was not signi� cantly associated with ele-vated IgA antibody levels in the logistic regression model, itis highly probable that C. pneumoniae, when chronically orrecurrently present, is primarily an inciter of the disease byamplifying in� ammation in the bronchi and enhancing thedevelopment of tissue scarring and airway remodelling. Theuse of corticosteroids may further favour the establishmentand subsequent reactivation of a chronic C. pneumoniaeinfection (27, 28). In agreement with our results are thevery recent data reported by Black et al. (29). They foundthat the use of inhaled steroids at high doses was associatedwith signi� cant elevation of IgG and IgA antibodies to C.pneumoniae. Moreover, an inverse relationship between
elevated antibody levels to C. pneumoniae—and not toother common respiratory pathogens—and markers ofasthma severity was shown, suggesting a role for C. pneu-moniae as an inciter of disease progression.
We conclude that constantly elevated IgA antibody levelsto C. pneumoniae suggestive of chronic infection are associ-ated with moderate and severe asthma, supporting the viewthat the major role of this organism in asthma may be inperpetuating or inciting disease progression. Seropositivityto chsp60 was not found to be a useful marker for sus-pected chronic C. pneumoniae infection among the asth-matics. Whether antimicrobial drugs will have any role inthe prevention or delay of asthma progression remains tobe clari� ed.
ACKNOWLEDGEMENTS
This study was supported by a grant from The Finnish Anti-Tu-berculosis Association and partially by the contract BIO4-CT96-0152 of the Biotechnology Programme of the Commission of theEuropean Union.
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Submitted March 16, 2001; accepted July 24, 2001
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