HLA DQ alleles and interleukin-10 polymorphism associated with Chlamydia trachomatis-related tubal factor infertility: a case–control study

A.H. Kinnunen1, H-M. Surcel1,7, M. Lehtinen2, J. Karhukorpi3, A. Tiitinen4, M. Halttunen4, A. Bloigu1, R.P. Morrison5, R. Karttunen6 and J. Paavonen4

1 National Public Health Institute, 90101 Oulu, 2 National Public Health Institute, Helsinki, 3 Clinical Microbiology Laboratory, University Hospital of Oulu, 4 Department of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland, 5 Department of Microbiology, Montana State University, Bozeman, MT, USA and 6 Department of Medical Microbiology, University of Oulu, Oulu, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The relationship between Chlamydia trachomatis tubal factor infertility (TFI) and the host’s immunoregulatory genes was studied. METHODS: Cell-mediated immune responses to C. trachomatis and chlamydial heat shock protein (CHSP60) were determined by lymphocyte proliferation assay. HLA-DQ alleles and interleukin-10 (IL-10) promoter polymorphism (–1082 A/G) were analysed in 52 TFI cases and in 61 controls by PCR. RESULTS: HLA-DQB1 or DQA1 alleles did not significantly differ between the TFI group and the control group. However, DQA1*0102 and DQB1*0602 alleles together with IL-10 –1082AA genotype were found significantly more frequently in the TFI patients than in the controls (0.18 and 0.02 respectively; P = 0.005). Five (22%) of the 23 patients who had a positive lymphocyte proliferative response to CHSP60 were positive also for IL-10 –1082AA and for the HLA-DQA1*0102 and HLA-DQB1*0602 alleles. CONCLUSIONS: Our results reveal an association of a cellular immune response to CHSP60, HLA class II alleles and IL-10 promoter genotypes in patients with chlamydial TFI.

Key words: Chlamydia trachomatis/CHSP60/HLA alleles/infertility/interleukin-10


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chlamydia trachomatis is one of the most common causes of sexually transmitted diseases (STD) in women (Paavonen and Lehtinen, 1996Go). It is an obligate intracellular gram-negative-like bacterium that replicates in membrane-bound vacuoles (inclusions) in the cytoplasm of the host cells. Many chlamydial infections are asymptomatic, and reinfections are common. If left untreated, Chlamydia has a high tendency to remain persistent in inflamed tissues such as the upper genital tract of patients with pelvic inflammatory disease (PID) (Cohen and Brunham, 1999Go). Prolonged inflammation may lead to tissue scarring and sometimes occlusion of the Fallopian tubes. However, only a certain proportion of the infected women develop tubal factor infertility (TFI), suggesting that genetic factors associated with the host modulate the immune defence mechanisms and thereby the pathogenesis of chlamydial diseases.

Protective immunity and eradication of chlamydial infection is dependent on cell-mediated immunity and the presence of interferon-{gamma} (IFN-{gamma}), a typical product of Th1-type immune response (Brunham, 1999Go). However, dominant production of interleukin (IL)-10 at the site of the infection/inflammation seems to mediate suppression of the Th1-type immune response (Wang et al., 1999Go; Yang et al., 1999Go) thus giving more room to a Th2-type of immune response. This suggests that the balance between IFN-{gamma} and IL-10 regulates the final course of chlamydial infection (Arno et al., 1990Go). Cytokine gene polymorphisms and secreted levels of IFN-{gamma} and IL-10 can be highly variable between individuals (Turner et al., 1997Go; Gibson et al., 2001Go). It is also possible that the balance between the Th1 and Th2 types of responses, characterized by IFN-{gamma} or IL-10 secretion respectively (Surcel et al., 1994Go), may be controlled by epitopes selected by the human leukocyte antigen (HLA) class II molecules from processed microbial antigen.

Chlamydial heat shock protein 60 (CHSP60) plays an important role in the immunopathogenic mechanisms of the adverse sequelae of chlamydial infections (Neuer et al., 2000Go; Witkin et al., 2000Go). Furthermore, an association between CHSP60 antibodies and HLA class II alleles has been described in humans (Gaur et al., 1999Go). The CHSP60-induced immune response is associated with the levels of both IFN-{gamma} and IL-10 in mice (Yi et al., 1997Go). We have recently found that IL-10 is frequently secreted by the CHSP60-specific T cells in women with TFI (Kinnunen et al., 2002Go). CHSP60-induced IL-10 secretion is more vigorous in women with TFI than in controls without TFI (A.H.Kinnunen et al., unpublished data). To investigate further the association of CHSP60-directed immune response with IL-10 production in chlamydial TFI, IL-10 gene polymorphism, HLA class II alleles (DQA1 and DQB1) and CHSP60 specific lymphoproliferative responses were analysed in women with TFI and in healthy controls.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cases and controls
The population consisted of 52 women (mean age 34 years, range 23–40 years) with laparoscopically verified TFI attending IVF treatment at the Infertility Clinic, Department of Obstetrics and Gynecology, University of Helsinki. Tubal infertility was confirmed by diagnostic laparoscopy, or in some cases by operative laparoscopy performed due to extrauterine pregnancy or hydrosalpinges. The group contained women with hydrosalpinges, with proximally occluded tubes or with severe tubo-ovarian adhesions. Patients with endometriosis were not included in this group. Peripheral blood samples (20 ml) were taken for immunological studies and HLA genotyping, and transferred to the laboratory at room temperature. The control group for HLA genotyping and IL-10 polymorphism analysis consisted of 61 Finnish female adults (age range 23–69 years) who were collected in the context of another study on HLA-association and Helicobacter carriage and whose HLA allele frequencies were found to be equal to those in general Finnish population (Ikäheimo et al., 1996Go).

Serology
C. trachomatis-specific IgG and IgA serum antibodies were determined using commercially available enzyme immunoassay kits (Labsystems, Helsinki, Finland) (Närvänen et al., 1997Go) according to the manufacturer’s instructions. Specific immunoglobulin (Ig) antibodies present in the serum specimens (diluted 1:10) became attached to C. trachomatis peptides bound to the polystyrene surface of the Microstrip® wells and were detected using horse-radish peroxidase-conjugated anti-human IgG or IgA. The results were obtained in terms of the mean absorbance (optical density, OD) of duplicated samples at 450 nm, and with a positive cut-off level of OD >=1, as recommended by the manufacturer. Serum samples were not available for five subjects.

Lymphocyte cultures
Peripheral blood lymphocytes (PBL) were isolated from heparinized blood by Ficoll-Paque® (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. Cells were washed three times with Hanks’ balanced salt solution (Sigma, St Louis, MO, USA) and suspended in Roswell Park Memorial Institute 1640 medium (Sigma) containing 10% heat-inactivated human AB serum (Finnish Red Cross, Helsinki, Finland) for the lymphocyte proliferative assay.

C. trachomatis (serovar E and F) elementary body (EB) antigens and recombinant CHSP60, also known as GroEL-1, were used as the antigens. The recombinant CHSP60 (LaVerda et al., 2000Go) contained <0.03 ng/ml endotoxin, as determined by the Limulus assay. Optimum concentrations of antigens were determined in preliminary experiments as the minimum concentrations giving maximal lymphocyte proliferation.

The PBL (5x104 cells/well) proliferative reactivity against C. trachomatis EB antigen (3 µg/ml) and CHSP60 (2.5 µg/ml) was assessed in vitro by culture stimulation in round-bottomed 96-well plates with or without antigen in a total volume of 200 µl. The cultures were incubated in humidified 5% CO2 at 37°C for 6 days, and [methyl-3H]thymidine (0.2 µCi/well; Amersham Life Science, Buckinghamshire, UK) was added to the cultures for the last 18 h of incubation (Surcel et al., 1993Go). The lymphocyte proliferative responses were measured as counts per minute (c.p.m.) of incorporated [methyl-3H]thymidine with a liquid scintillation counter (Wallac, Turku, Finland), and the results were expressed as stimulation indices (SI = mean c.p.m. in the presence of the antigen divided by mean c.p.m. in its absence) for triplicate cultures. SI >2.5 was considered a positive response to an antigen. The viability and reactivity of the cultured PBL were controlled in each experiment by requiring SI >10 in response to pokeweed mitogen (PWM, Gibco, Paisley, UK; 12.5 µg/ml).

Analyses of HLA class II alleles
HLA-DQA1 and HLA-DQB1 genotyping was performed by the PCR–SSP (sequence-specific primer) method (Olerup et al., 1993Go). Genomic DNA was extracted from the peripheral blood leukocytes using proteinase K digestion in 10% sodium dodecyl sulphate and 7.5 mol/l guanidine–HCl and precipitated with ethanol. The primers (sense 5'-TGCCAAGTGGAGCACCCAAA-3' and anti-sense 5'-GCATCTTGCTCTGTGCAGAT-3'), which amplify the third intron of the DRB1 gene, were used as an internal positive control. Approximately 50 ng of DNA from each sample was amplified in the presence of 200 µmol/l dATP, dCTP, dGTP and dTTP (Finnzymes, Espoo, Finland), 1xPCR buffer (1.5 mmol/l MgCl2; Perkin Elmer, Boston, MA, USA), 0.25 µmol/l of allele or group-specific primers and 0.05 µmol/l of control primers and 0.2 U AmpliTaq DNA polymerase (Perkin Elmer). Amplification consisted of initial denaturation for 5 min at 95°C followed by 35 cycles consisting of denaturation for 20 s at 94°C, annealing for 50 s at 65°C and elongation for 20 s at 72°C. The products were separated on a 2% agarose gel and visualized with ethidium bromide stain under UV light illumination. The results were documented photographically.

Detection of interleukin-10 promoter polymorphism
IL-10 promoter gene polymorphism of a single nucleotide at position –1082 (A/G) was determined using sequence-specific oligonucleotide primers in a bidirectional PCR amplification (Karhukorpi and Karttunen, 2001Go). The primers fpena and revp1 amplify the sequence of 141 bp for the A allele, and the primers afor3 and rpeng amplify the sequence of 200 bp for the G allele. The outermost primers afor3 and revp1 amplify a sequence of 293 bp, which served as an internal control (Table IGo). The PCR reactions were performed in a total volume of 10 µl using ~50 ng of sample DNA, 1x PCR buffer, with 1.5 mmol/l MgCl2 (Perkin Elmer) 10 µmol/l each of the dNTP (Finnzymes), 0.8 IU AmpliTaq DNA polymerase (Perkin Elmer) and 0.5 µmol/l of each primer. After initial denaturation at 95°C for 5 min, 35 cycles, consisting of denaturation for 30 s at 95°C, annealing for 30 s at 63°C and final elongation for 5 min at 72°C, were run. The PCR products were separated on a 2% agarose gel and visualized under UV light illumination with ethidium bromide stain. The results were documented photographically.


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Table I. Nucleotide sequences of the primers used for identifying interleukin-10 promoter gene polymorphisms (Karhukorpi and Karttunen, 2001Go)
 
Statistical analyses
The statistical analyses were performed with SPSS for Windows 9.1 software (SPSS Inc., Chicago, Illinois, USA). The {chi}2-test or Fisher’s exact test was used to compare proportions between groups depending on the numbers involved. Confidence intervals were calculated using the CIA program (BMJ Publishing Group, London, UK). The significance of the HLA association results was corrected using the Bonferroni correction multiplying the P value by the factor 22 (eight tests for HLA DQA1 and 14 tests for DQB1). Spearman correlation coefficient was used to test the association between lymphocyte responses to different antigens.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
C. trachomatis-specific immune responses
C. trachomatis-specific IgG antibodies were present in 23 [49%, 95% confidence interval (CI) 34–64] of the 47 TFI cases for whom serum samples were available and IgA antibodies in 17 (36%, 95% CI 23–52). Cell-mediated immune responses, assessed by stimulating PBL with C. trachomatis serovar E EB (median SI 8; range 0–93) and serovar F EB (median SI 4; range 0.8–105), were comparable (a correlation coefficient rs 0.94; P < 0.001). A positive lymphocyte proliferative (LP) response to chlamydial EB was found in 40 of the 52 TFI cases (77%, 95% CI 63–88). Twenty-four cases (46%, 95% CI 32–61) of the 52 TFI patients also showed cell-mediated reactivity to CHSP60. All the TFI patients who did not respond to EB antigen also had negative LP response to CHSP60.

HLA-DQA1 and HLA-DQB1 alleles
A total of eight HLA-DQA1 alleles and 14 HLA-DQB1 alleles were found among the 52 cases and 61 control subjects, the frequencies being comparable between the groups (Table IIGo). The frequencies of the HLA-DQA1*0102 and HLA-DQB*0602 alleles were nevertheless slightly higher among the cases (0.31 and 0.23) than among the controls (0.16 and 0.10, uncorrected P = 0.01 and P = 0.007 respectively; corrected P = 0.22 and 0.154, not significant). The gene frequency of HLA DQB1*0602 was significantly higher in the TFI cases (22/52) than in the controls (10/61, uncorreced P = 0.002; corrected P = 0.04).


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Table II. Distribution of HLA-DQA1 and HLA-DQB1 alleles in 52 women with tubal factor infertility (TFI) and in 61 control subjects
 
IL-10 promoter gene polymorphism and HLA-DQ alleles
The allele frequency of –1082A was 0.58 in the cases and 0.51 in the controls, and the frequency of the –1082G allele was 0.42 for TFI cases and 0.49 for controls. The heterozygote IL-10 –1082AG genotype was common in both groups (41% in the TFI cases and 52% in the controls; Figure 1AGo), while IL-10 –1082AA homozygotes were found slightly more often among the cases (37%) than among the controls (25%, not significant).



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Figure 1. Distribution of IL-10 –1082 AA, AG and GG genotypes (A) in the 61 control subjects and in the 51 tubal factor infertility (TFI) cases, and in TFI subgroups with cases who are negative or positive in terms of (B) cell-mediated response to C. trachomatis EB antigen (SI-Ctr neg and SI-Ctr pos) or chlamydial heat shock protein 60 (SI-CHsp60 neg and SI-CHsp60 pos), or TFI cases who are (C) positive or negative in terms of C. trachomatis specific IgG or IgA antibodies. P = 0.046 when comparing the number of IL-10 AA genotypes between TFI cases with positive or negative SI-CHsp60. Numbers of subjects shown above bars.

 
Clear associations were found between the IL-10 –1082 AA genotype and both the HLA-DQA1 *0102 and HLA-DQB1* 0602 alleles among the cases. DQA1*0102 and DQB1*0602 alleles together with IL-10 –1082 AA genotype were found significantly more frequently in the TFI cases than in the controls (0.18 and 0.02 respectively; P = 0.005).

C. trachomatis-specific immune response, HLA-DQ alleles and IL-10 promoter gene polymorphism
The possible association of humoral and/or cell-mediated immune responses to C. trachomatis and HLA-DQA1 and HLA-DQB1 alleles was analysed among the TFI cases. For these analyses we chose the ten most frequently found genotypes (DQA1* 0101, 0102, 0103, 0301, 0501 and DQB1* 0201, 0302, 0501, 0602, 0603; Table IIGo). The distribution of the HLA alleles did not differ between the IgG seropositive and seronegative patients (data not shown). When LP response to C. trachomatis EB or CHSP60 was evaluated together with the HLA-DQ genotype frequencies, no significant associations were observed (data not shown).

Finally, we analysed IL-10 –1082 promoter gene polymorphism in association with C. trachomatis-specific cell-mediated immune response among the TFI cases. The IL-10 –1082A allele frequency was significantly higher among the TFI cases who had a positive LP response to CHSP60 than among those cases with a negative response (0.72 versus 0.46, P = 0.01). Accordingly, IL-10 –1082 AA homozygotes were detected more frequently in the CHSP60-responsive TFI cases than in the non-responsive cases (52 versus 25%, P = 0.046; Figure 1BGo). Furthermore, five (22%) of the 23 cases with positive LP response to CHSP60 were positive also for IL-10 –1082AA and for the HLA-DQA1*0102 and HLA-DQB1*0602 genotypes (Table IIIGo), and statistically significantly compared with healthy controls (one out of 61, 2% P = 0.005).


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Table III. Number of tubal factor infertility (TFI) patients and controls with (Positive) or without (Negative) HLA-DQA1*0102, DQB1*0602 alleles and IL-10 –1082AA genotypes
 
No significant associations between the IL-10 polymorphism and C. trachomatis antibody response or with the LP response against chlamydial EB were found (Figure 1B and CGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Immune response to CHSP60 is known to play a central role in PID and C. trachomatis–associated infertility (Neuer et al., 2000Go; Witkin et al., 2000Go), but the immunopathogenic mechanisms are not known. We have previously shown that a considerable proportion of C. trachomatis-specific T cells derived from inflamed Fallopian tubes (Kinnunen et al., 2000Go) are targeted to CHSP60 and produce IL-10 (Kinnunen et al., 2002Go). Women with chlamydial TFI now showed LP responses to CHSP60 antigen, which were associated with the IL-10 –1082A and the HLA-DQA1*0102 and -DQB1*0602 alleles. Although chlamydial infertility probably consists of multifactorial mechanisms, our results are in accordance with the reported association of HLA class II DQ alleles and CHSP60 antibodies (Gaur et al., 1999Go), suggesting that the CHSP60-associated immune response is linked with the host’s genetic factors.

Although no direct comparison was made between IL-10 promoter gene polymorphism and IL-10 secretion, it has been shown by others that the IL-10 –1082 gene polymorphism plays a role in the in-vivo production of IL-10. Turner et al. suggest that the IL-10 –1082 AA genotype is associated with a low production of IL-10 protein compared with the –1082 GG genotype (Turner et al., 1997Go). However, we found in our earlier studies that CHSP60 reactivity is linked with enhanced IL-10 secretion in TFI patients (Kinnunen et al., 2002Go). However, another study (Nieters et al., 2001Go) found no association between cytokine release and lymphocyte stimulation in vitro. Thus, the relationship between in-vivo production of a cytokine and single nucleotide polymorphism is ambiguous, but may involve interactions at several points of a single nucleotide polymorphism (Helminen et al., 1999Go; Gibson et al., 2001Go) and may differ between ethnic groups (Mozzato-Chamay et al., 2000Go). One study of genetic risk factors for C. trachomatis-induced scarring trachoma (Mozzato-Chamay et al., 2000Go) found an association between the disease and the IL-10 –1082GG genotype in only one out of five ethnic groups.

According to our results, TFI was somewhat associated with HLA DQB1*0602 and DQA1*0102 alleles, although the allele frequencies did not differ significantly between TFI cases and controls after multiple comparison. The possible association of these two alleles and TFI might also reflect an association with TFI and other genes, such as HLA DRB1*1501, which are in linkage disequilibrium with HLA DQB1*0602 and DQA1*0102 alleles. Together these three alleles represent a subtype of DR2 antigen. Interestingly, a significant association with HLA DR16, another subtype of DR2 antigen, and scarring trachoma has previously been reported (White et al., 1997Go). More research is needed on the possible role of DR2 antigen in the development of chronic Chlamydia infection and its relationship with damage of the inflamed tissue.

The HLA systems control immune responses by presenting antigenic epitopes to immune T cells. HLA molecules restrict and regulate the range of immune responses to different antigens and mediate susceptibility or resistance to infecting micro-organisms. The linkage between disease susceptibility and HLA molecules is ambiguous, however, and can vary due to the distribution of HLA antigens in different populations. Thus the risk of C. trachomatis-induced scarring trachoma is enhanced in subjects with HLA class I subtype A*0602 in Gambia (Conway et al., 1996Go) or with HLA class II subtype DR16 in Oman (White et al., 1997Go). The possible linkage between TFI and HLA-DQA1*0102 and -DQB1*0602 alleles in Finland found in this study differs from the association between C. trachomatis seropositive TFI and HLA DQA*0101 and DQB*0501 alleles reported in Nairobi (Cohen et al., 2000Go). One possible explanation for the discrepancies between HLA associations found for immunopathologically similar conditions in different populations may be related to the permissive characteristics of HLA molecules in binding processed epitopes that are available from a certain antigen in the phagosomal lysosomes. Moreover, the criteria for case identification may also explain the discrepancies between HLA studies performed on the same geographical populations. Examples of the latter include studies where C. trachomatis-associated PID has been linked with HLA A31 (Kimani et al., 1996Go), whereas C. trachomatis-associated TFI, the severe disease form of PID, has been linked with HLA-DQA*0101 and -DQB*0501 alleles in women in Nairobi (Cohen et al., 2000Go).

We conclude that the characteristics of the cell-mediated response to CHSP60 involve genetic regulation and probably a collaboration between HLA and IL-10 genes. Further research with larger study population and normal fertile women is in progress to determine whether HLA and IL-10 genes can be used as markers of the risk of developing TFI.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Special thanks are due to Mrs Marja Siitonen, Mrs Marja Suorsa and Mrs Pirkko Timonen for their excellent technical assistance, to Helena Kivelä for technical realization of the genetic analysis, and to Pentti Koskela who was responsible for the serological analysis. Financial support from the Helsinki University Hospital Research Funds (TYH0015) and the Paulo Foundation is gratefully acknowledged.


    Notes
 
7 To whom correspondence should be addressed. E-mail: Helja-Marja.Surcel{at}ktl.fi Back


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 Introduction
 Materials and methods
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 Discussion
 Acknowledgements
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Submitted on December 18, 2001; resubmitted on February 2, 2002; accepted on April 5, 2002.