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
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Abstract |
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Key words: Chlamydia trachomatis/CHSP60/HLA alleles/infertility/interleukin-10
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Introduction |
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Protective immunity and eradication of chlamydial infection is dependent on cell-mediated immunity and the presence of interferon- (IFN-
), a typical product of Th1-type immune response (Brunham, 1999
). 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., 1999
; Yang et al., 1999
) thus giving more room to a Th2-type of immune response. This suggests that the balance between IFN-
and IL-10 regulates the final course of chlamydial infection (Arno et al., 1990
). Cytokine gene polymorphisms and secreted levels of IFN-
and IL-10 can be highly variable between individuals (Turner et al., 1997
; Gibson et al., 2001
). It is also possible that the balance between the Th1 and Th2 types of responses, characterized by IFN-
or IL-10 secretion respectively (Surcel et al., 1994
), 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., 2000; Witkin et al., 2000
). Furthermore, an association between CHSP60 antibodies and HLA class II alleles has been described in humans (Gaur et al., 1999
). The CHSP60-induced immune response is associated with the levels of both IFN-
and IL-10 in mice (Yi et al., 1997
). We have recently found that IL-10 is frequently secreted by the CHSP60-specific T cells in women with TFI (Kinnunen et al., 2002
). CHSP60-induced IL-10 secretion is more vigorous in women with TFI than in controls without TFI (A.H.Kinnunen et al., unpublished data).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.
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Materials and methods |
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Serology
C. trachomatis-specific IgG and IgA serum antibodies were determined using commercially available enzyme immunoassay kits (Labsystems, Helsinki, Finland) (Närvänen et al., 1997) according to the manufacturers 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., 2000) 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., 1993). 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 PCRSSP (sequence-specific primer) method (Olerup et al., 1993). Genomic DNA was extracted from the peripheral blood leukocytes using proteinase K digestion in 10% sodium dodecyl sulphate and 7.5 mol/l guanidineHCl 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, 2001). 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 I
). 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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Results |
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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 II). 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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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 II). 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 1B). 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 III
), and statistically significantly compared with healthy controls (one out of 61, 2% P = 0.005).
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Discussion |
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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., 1997). However, we found in our earlier studies that CHSP60 reactivity is linked with enhanced IL-10 secretion in TFI patients (Kinnunen et al., 2002
). However, another study (Nieters et al., 2001
) 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., 1999
; Gibson et al., 2001
) and may differ between ethnic groups (Mozzato-Chamay et al., 2000
). One study of genetic risk factors for C. trachomatis-induced scarring trachoma (Mozzato-Chamay et al., 2000
) 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., 1997). 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., 1996) or with HLA class II subtype DR16 in Oman (White et al., 1997
). 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., 2000
). 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., 1996
), 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., 2000
).
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.
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Acknowledgements |
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Notes |
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References |
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Submitted on December 18, 2001; resubmitted on February 2, 2002; accepted on April 5, 2002.