Anti-Chlamydia pneumoniae heat shock protein 10 antibodies in asthmatic adults

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1 FEMS Immunology and Medical Microbiology 35 (2003) 107^111 Anti-Chlamydia pneumoniae heat shock protein 10 antibodies in asthmatic adults Abstract Fotini Betsou, Jean Marie Sueur, Jeanne Or la Biobanque de Picardie, 16 rue Fernel, Amiens, France Received 28 June 2002; received in revised form 25 September 2002; accepted 29 November 2002 First published online 20 January 2003 A multicenter prospective study was performed on 160 asthmatic adults suffering from acute episodes of bronchitis and 88 nonasthmatic controls, to investigate potential associations among Chlamydia pneumoniae infection and/or anti-c. pneumoniae heat shock protein 10 antibodies, and asthma. We used micro-immunofluorescence to detect serum anti-c. pneumoniae IgG, IgA and IgM antibodies and enzyme-linked immunosorbent assay to detect serum anti-chsp10 peptide IgG antibodies. The serological prevalence of C. pneumoniae was 73.1%. An association was observed between the presence of anti-chsp10 antibodies and adult onset asthma. The humoral immune responses were not confined to any particular region of the Chsp10 protein. ß 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Asthma; Bronchitis; Chsp10; Chlamydia pneumoniae 1. Introduction Chlamydia pneumoniae is a common cause of respiratory infections worldwide and it has been estimated that most people are infected with C. pneumoniae two or three times during their lifetime [1]. C. pneumoniae has been implicated in a number of chronic disease processes, including adult onset asthma and atherosclerosis [2^6]. Asthma is an important cause of morbidity in all age groups, a ecting 2^10% of the population. There is an association between exacerbations of asthma and acute C. pneumoniae infections, and antibiotic treatment has been found to alleviate the symptoms of asthma [2]. Other groups have shown that anti-chlamydial treatment can lead to the improvement or total remission of asthma [7,8]. C. pneumoniae is susceptible to several antibiotics used to treat respiratory infections [9], but it is often necessary to use prolonged therapy to achieve clinical cure [10] and C. pneumoniae may persist in the lungs after treatment [11]. A positive association has been found between immune responses to C. pneumoniae and the frequency of asthma exacerbations * Corresponding author. Tel.: +33 (3) ; Fax: +33 (3) address: fay.betsou@biobanque-picardie.com (F. Betsou). in children [12]. It has been suggested that the immune response resulting from chronic C. pneumoniae infection interacts with allergic in ammation, thus exacerbating the symptoms of asthma. Micro-immuno uorescence (MIF) is usually used to monitor immune responses to C. pneumoniae [13]. Bacterial heat shock proteins (Hsp) are widely conserved and immunodominant antigens are frequently found following infection. Chlamydia heat shock proteins (Chsp) have been implicated in chronic disease processes, and detection of antibodies to Hsp in Chlamydia infections is not unusual. There is some evidence that Chlamydia heat shock protein 60 is present in human atheromata and that it plays a role in the development of lesions by directly activating macrophages [14]. Antibodies to Chsp60 are generated during Chlamydia trachomatis infections complicated by acute reactive arthritis [15] and infertility [16]. Furthermore, a signi cant association has been found between persistent anti-chsp60 antibodies and adult onset asthma [17]. Correspondingly, the detection of anti-chsp10 antibodies is associated with severity of C. trachomatis genital tract infection [18]. We used synthetic overlapping peptides to study the humoral immune response to C. pneumoniae Chsp10 in asthmatic adults su ering from episodes of bronchitis and to evaluate the association between asthma or acute asthma exacerba / 03 / $22.00 ß 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi: /s (03)

2 108 F. Betsou et al. / FEMS Immunology and Medical Microbiology 35 (2003) 107^111 Table 1 Serological markers of C. pneumoniae infection in asthmatic adults (N = 160) and non-asthmatic control individuals (N = 88) Serological marker Percentage [C.I. 95%] a Percentage [C.I. 95%] a Control group Asthmatic group MIF IgG v 1024 or IgM v [7.5^22.5] 6.9 [3^10.8] MIF IgG v [63.7^82.3] 66.2 [58.9^73.5] anti-chsp10 IgG 15.9 [8.3^23.7] 43.1 [35.3^50.7] a C.I., con dence intervals (95% C.I. are indicated in brackets). tions and the presence of anti-c. pneumoniae Chsp10 antibodies. 2. Materials and methods 2.1. Study population We studied 160 asthmatic adults (median age = 40 years; range = 18^68 years; 67 males and 93 females) a ected by acute bronchitis. Acute bronchitis was diagnosed on the basis of an infectious syndrome accompanied by productive coughing raising mucoid or mucopurulent expectorations, and slight bronchial rale. None of the patients took inhaled steroids. None of the patients had serological evidence of C. trachomatis infection. Informed consent was obtained from all patients. The control group included 88 healthy donors without serological evidence of C. trachomatis infection (median age = 43 years; range = 17^58; 45 males and 43 females) Sample collection One serum sample was obtained from each healthy donor. Two serum samples were obtained at 3-week intervals (one before antibiotic treatment and one after) from each asthmatic patient. The samples were kept at 370 C until biological investigation Antibiotic treatment Patients with asthma were treated twice per day with 150 mg roxithromycin for 10 days Detection of serum C. pneumoniae antibodies C. pneumoniae-speci c IgG, IgA and IgM were measured by the MIF test using elementary bodies (EB) from C. pneumoniae IOL 207 as the antigen. These EB were prepared from egg yolk sacs as described previously [13,19]. Patients were classi ed into three mutually exclusive titer categories: (1) patients with an IgM titer v 12, an IgG titer v 1024 or who had undergone IgG or IgA seroconversion (a di erence of at least four dilution factors between the two serum samples); (2) patients with an IgG titer v 64; (3) patients with an IgG titer 6 64 and an IgA titer 6 16, who were considered to be seronegative for C. pneumoniae. A positive reaction to egg yolk antigens meant that the result could not be interpreted Detection of anti-chsp10 antibodies Six overlapping N-biotinylated peptides, HSPW1^ HSPW6, were synthesized (Neosystem, Pont de Claix, France). The peptides had the following amino acid sequences: HSPW1: M-S-D-Q-A-T-T-L-R-I-L-P-L-G-D-R-I-L-V (aa1^19). HSPW2: I-L-V-K-R-E-E-E-E-A-T-A-R-G-G-I-I-L (aa17^34). HSPW3: I-I-L-P-D-T-A-K-K-K-Q-D-R-A-E-V-L-V-L (aa32^50). HSPW4: L-V-L-G-T-G-K-R-T-D-D-G-T-L-L-P-F-E- V-Q-V (aa48^68). HSPW5: V-Q-V-G-D-I-I-L-M-D-K-Y-A-G-Q-E-I-T-I (aa66^84). HSPW6: I-T-I-D-D-E-E-Y-V-I-L-Q-S-S-E-I-M-A-V-L (aa82^101). Microtiter plates that had been pre-coated with streptavidin were coated with 1 Wg ml 31 of each of the six peptides HSPW1^HSPW6. Aliquots of sera diluted 1:80 in PBS (phosphate-bu ered saline)^tween 0.2% were added to the wells and the plates were incubated at 37 C for 60 min. The wells were then washed three times with PBS^ MIF IgG>=64 MIF IgG>=1024 or IgM>=12 MIF IgG<64 and IgA<12 control asthmatic Fig. 1. Percentage of anti-chsp10 seroprevalence in asthmatic and nonasthmatic control adults. Vertical error bars indicate 95% con dence intervals.

3 Table 2 Distribution of anti-chsp10 antibodies in the patient- and in the control group MIF serological status Percentage of control individuals harboring anti-chsp10 IgG v 1024 or IgM v [22^50] n =13 n =11 IgG v [10.2^29.8] 35.8 [27^45] n =64 n = 106 IgG 6 64 and IgA [0^26] 65.8 [52^80] n =11 n =41 Uninterpretable n =2 95% con dence intervals are indicated in brackets. F. Betsou et al. / FEMS Immunology and Medical Microbiology 35 (2003) 107^ Percentage of individuals in the asthmatic group harboring anti-chsp10 Tween 20 and incubated with a 1:1000 dilution of alkaline phosphatase-conjugated goat anti-human IgG antibody. After 60 min at 37 C, the wells were washed three times and the alkaline phosphatase substrate, paranitrophenylphosphate, was added. After incubation at room temperature for 30 min, the reaction was stopped with 3 M NaOH and the optical densities (OD) of plates were read at 405 nm. For each peptide, a positive sample was de ned as one yielding an OD value that was at least 2S.D. above the mean value obtained with a panel of 30 MIF-negative samples, that is an OD s 0.26 for HSPW1, s 0.28 for HSPW2, s 0.33 for HSPW3, s 0.29 for HSPW4, s 0.26 for HSPW5 and s 0.31 for HSPW Statistical analysis The M 2 test was used to calculate inter-group di erence. The 95% con dence intervals were calculated. Multivariate analyses for sex (block analysis) and age (covariance analysis) were performed. Maximum likelihood estimation of a binormal ROC (receiver operating characteristic) curve from categorical rating data was performed; for this purpose, data were classi ed into six categories, representing level of con dence that a case is positive or negative [20]. 3. Results 3.1. Serological prevalence of C. pneumoniae Serological MIF studies in the asthmatic group showed that 11 patients had an IgM titer v 12or an IgG titer v 1024 (6.9%) and 106 patients had an IgG titer v 64 (66.2%). Two patients had uninterpretable results. The total seroprevalence of C. pneumoniae in the asthmatic group was 73.1%. The total seroprevalence in the control group was 87.5%; 13 of the controls had an IgM titer v 12or an IgG titer v 1024 (14.8%), and 64 had an IgG titer v 64 (72.7%). Therefore, there was no signi cant difference between the asthmatic group and control group with regard to anti-c. pneumoniae antibody (Table 1) Detection of anti-hspw1^6 antibodies About one-third (36.4% þ 14%) of the patients with a MIF IgM titer v 12or IgG titer v 1024 had detectable anti-chsp10 antibodies that were speci c for at least one of the HSPW peptides. These antibodies were also detected in 35.8% ( þ 9.1%) of the patients with a MIF IgG titer v 64. About two-thirds (65.8% þ 14%) of the asthmatic patients who were serologically negative for C. pneumoniae had detectable anti-chsp10 IgG antibodies. The total seroprevalence of anti-chsp10 IgG antibodies in the asthmatic population studied was 43.1% ( þ 7.7%). The total seroprevalence in the control group was 15.9% ( þ 7.7%). Therefore, a signi cant di erence in the prevalence of anti-chsp10 was observed between healthy and asthmatic individuals (P ). Only 9.1% ( þ 17%) of the control individuals who were serologically negative for C. pneumoniae had detectable anti-chsp10 antibodies. Anti-Chsp10 antibodies were not detected in any of the controls with a MIF IgM titer v 12or IgG titer v 1024, but were detected in 20.3% ( þ 9.8%) of those with a MIF IgG titer v 64 (Table 2). Therefore, the pattern of anti- Chsp10 reactivity was di erent in asthmatic patients and non-asthmatic healthy adults. Globally, a higher proportion of anti-chsp10-positive individuals was observed in the asthmatic group, both in the C. pneumoniae-infected group and in the group that was not infected with C. pneumoniae (Fig. 1). However, when subjected to ROC curve analysis, Chsp10 seroreactivity was not highly discriminating between presence and absence of asthma (Fig. 2). Sensitivity 1 0,8 0,6 0,4 0, ,040,090,14 0,4 0,9 1-Specificity True Positive Fraction 95% Confidence Interval Fig. 2. Maximum likelihood estimation of a binormal ROC curve showing correlation between all sera and the presence or absence of asthma. The tted ROC area is equal to 0.58.

4 110 F. Betsou et al. / FEMS Immunology and Medical Microbiology 35 (2003) 107^111 O.D.405nm HSPW1 HSPW2 HSPW3 HSPW4 HSPW5 HSPW Patterns of anti-hspw1^6 antibody reactivity No particular Chsp10 sequential immunodominant region was detected by scanning the protein with the six overlapping peptides. In most of the seropositive patients, all six peptides elicited the same titer of reactive antibodies. Two cases of seroconversion were found. In the rst, seroconversion was observed for all six HSPW peptides, whereas in the second one, seroconversion was observed only for HSPW1, HSPW2and HSPW3. Five cases of differential reactivity against the six HSPW peptides were found; one of these cases corresponded to the second case of seroconversion mentioned above, in two cases only the HSPW1 peptide was reactive and in the other two cases only the HSPW3 peptide was reactive. Similarly, only three of the healthy controls showed di erential reactivity against the HSPW peptides. In one case, only the HSPW1 peptide was reactive, in another case, only the HSPW3 peptide was reactive, and in the last case, only HSPW1, HSPW5 and HSPW6 were reactive. The di erent serum reactivity pro les against the six peptides scanning the Chsp10 sequence are shown in Fig Discussion 1,2 1,1 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 D1 D2 D3 P1 P2 P3 P4 P5 Individual sera Fig. 3. Di erential serum reactivities against the six overlapping peptides HSPW1^6 scanning the Chsp10 sequence. Ordinate scale represents OD 405 nm values. D, donor; P, asthmatic patient. C. pneumoniae infections are very frequent in asthmatic patients and C. pneumoniae may play a role in acute exacerbations of asthma, or induce persistent chronic asthma [2,3]. Therefore, we examined the serological response to overlapping C. pneumoniae Chsp10 peptides in a group of asthmatic adults a ected by acute bronchitis or acute exacerbation of asthma. None of the patients included in this study had either clinical symptoms or serological evidence of C. trachomatis genital tract infection. Hence, the presence of anti-chsp10 antibodies was not due to C. trachomatis infections. Nevertheless, Chsp10 belongs to a family of proteins that is highly conserved among di erent species of bacteria and eukaryotes, and cross-reactivity due to other pathogens, such as Bordetella pertussis (42% identity) or Escherichia coli (39% identity), or to human Hsp10 (32% identity) cannot be excluded. Interestingly, we found an association between the detection of anti-chsp10 antibodies and asthma. Signi cantly more asthmatic patients, with or without serological evidence of C. pneumoniae infection, had detectable serum IgG anti-chsp10 antibodies (43.1% versus 15.9% of control subjects). Our results are consistent with results obtained by Huittinen et al. These researchers found a signi cant association between anti-chsp60 IgA antibodies and asthma [17]. The anti-chsp10 antibodies detected in our study were of the IgG isotype. No anti-chsp10 IgA responses were detected. Approximately one-third of the patients with a MIF IgG titer v 64, and one-third of those with a MIF IgG titer v 1024 or IgM titer v 12, harbored anti-chsp10 antibodies. A surprisingly high percentage (65.8%) of asthmatic patients without serological evidence of C. pneumoniae infection harbored anti-chsp10 antibodies. This might be the result of non-speci c reactivity, in response to an infection other than C. pneumoniae. Non-speci c pro-in ammatory stimuli might induce either non-speci c polyclonal B cell stimulation or increased expression of the endogenous Hsp10 gene and autoimmune-mediated crossreactions. It was recently shown that the Hsp70 gene is expressed in peripheral blood mononuclear cells in patients su ering from acute asthmatic episodes, but not in patients in the convalescent period [21]. All of the patients included in our study were su ering from acute asthmatic episodes and upregulation of the endogenous Hsp10 gene expression cannot be excluded. Most of the sera used in our study had the same titers against all six overlapping synthetic peptides (HSPW1^6). Thus, the sequential epitopes recognized by anti-chsp10 antibodies are generally not con ned to a particular region of the Chsp10 protein. There were eight cases of di erential reactivity against the six HSPW peptides and in all cases the reactivity was against the N-terminal portion of the molecule, and more particularly, against regions comprised between amino acids (aa) 1^19 and/or 32^50. It is noteworthy that the corresponding regions of the E. coli GroES protein, a member of the Hsp10 protein family, are exposed [22]. Little is known about the role of Chsp10 in the pathophysiology of chlamydial infections. C. pneumoniae infections are gaining interest, because they have been implicated in coronary heart disease [6]. This is only conceivable in cases of infection or antigen persistence, and Hsp might be such persistent antigens. Our study shows that the presence of serum anti-chsp10 IgG antibodies is associated with a MIF IgG titer v 64 in a control group. Reactivity against Chsp10 is signi cantly higher in asth-

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