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Keywords:

  • cell-mediated immunity;
  • chlamydial heat shock protein 60;
  • Chlamydia trachomatis;
  • tubal infertility

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Chlamydia trachomatis-associated tubal factor infertility (TFI) involves enhanced humoral and cell-mediated immune response to the chlamydial 60 kDa heat shock protein (CHSP60). We evaluated the role of CHSP60-induced immune response in TFI by studying lymphocyte proliferation and cytokine (interferon (IFN)-γ, interleukin (IL)-12 and IL-10) secretion in response to C. trachomatis elementary body (EB) and CHSP60 antigens in 57 women with TFI and in 76 women with other causes of infertility. Positive proliferative response of PBMC to CHSP60 was more common in the TFI group (20/57; 36%) than in the other groups (17/76; 22%) although the frequency or the median responses did not differ significantly (1·6, range 0·2–22·1 versus 1·4; 0·2–24·4). C. trachomatis EB induced significantly higher IFN-γ and lower IL-10 secretion in the TFI group compared to the other groups. The EB and CHSP60 induced IL-12 secretion was similar in all study groups and correlated with IFN-γ secretion in the other but not in the TFI group. The lack of correlation between EB-induced IL-12 and IFN-γ production and simultaneously found prominent IL-10 secretion in response to CHSP60 in the TFI group suggests that the CHSP60 may have a specific role in regulating the immune reactions during chlamydial infection and may consequently contribute to the immunopathogenesis of TFI.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Tubal factor infertility (TFI) is a major consequence of pelvic inflammatory disease (PID) [1]. Approximately 25% of women with pelvic inflammatory disease (PID) become infertile due to occlusion of fallopian tubes [2]. TFI is associated strongly with recurrent or prolonged C. trachomatis infection, which results in chronic salpingeal inflammatory responses [3] maintained by mononuclear lymphocytes [4]. The prognosis of in vitro fertilization (IVF) is worse in patients with TFI than in other infertile women [5]. In addition to appropriate antimicrobial chemotherapy, successful eradication of C. trachomatis infection is dependent on T cell-mediated immune mechanisms and secretion of interferon-γ (IFN-γ) [6].

Elevated serum antibody levels to chlamydial heat shock protein 60 (CHSP60) correlate with chronic chlamydial infection, PID and TFI [7,8], and lymphocyte-proliferative responses to the CHSP60 occur more often in PID patients than in healthy controls [9]. A specific role for CHSP60 in the pathogenesis of salpingitis has been suggested by an experimental monkey model of infection [10]. CHSP60 inoculation into subcutaneous pockets of previously C. trachomatis-sensitized monkeys elicits an intense inflammatory response similar to that seen in humans with chlamydial salpingitis. We have shown recently that C. trachomatis-specific T cells are present in the fallopian tubes of women with TFI [11], and that a considerable proportion of these T cells are targeted to CHSP60 antigen [12].

Proinflammatory IFN-γ may modulate immune responses to the CHSP60. Increased expression of CHSP60 in the presence of low IFN-γ levels in vitro[13] and accelerated immune responses to CHSP60 during persistent C. trachomatis infection [14] has led to speculation that these events are also important in vivo. On the other hand, susceptibility to chronic chlamydial infection in mice is associated with a switch from IFN dominating response to interleukin (IL)-10 domination [15,16]. CHSP60 activates inflammatory responses in murine macrophages [17,18]. On the other hand, CHSP60 has been shown to induce both Th1 and Th2 type responses in human lymphocytes [19], suggesting that CHSP60 has the capacity to influence the immune response by stimulating the production of IL-10 or other anti-inflammatory cytokines during the course of prolonged C. trachomatis infection.

To evaluate the role of CHSP60 immune response in TFI, we measured proliferative responses and secretion of IFN-γ, IL-12 and IL-10 of the circulating mononuclear lymphocytes in response to CHSP60 and C. trachomatis EB antigens in TFI patients and in women with other cause of infertility.

PATIENTS AND METHODS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

A total of 133 consecutive women of infertile couples attending the Infertility Clinic of the Department of Obstetrics and Gynecology, University of Helsinki, during May 1999–April 2000 were enrolled in the study. Peripheral blood samples (20 ml) were drawn from the subjects and transferred to the laboratory at room temperature. The study protocol was approved by the Ethics Committee of the Department of Obstetrics and Gynecology, University Hospital, Helsinki.

Measurement of serum antibodies

C. trachomatis-specific IgG serum antibodies were studied using a commercially available EIA kit (Labsystems, Helsinki, Finland) according to the manufacturer's instructions. Specific IgG antibodies present in the serum specimens (diluted 1 : 10) are attached to C. trachomatis peptides bound to the polystyrene surface of the Microstrip® wells and detected using horseradish peroxidase conjugated antihuman IgG. Results are obtained as a mean absorbance of duplicated samples at 450 nm and O.D. > 1 is a positive cut-off level as recommended by the manufacturer.

Chlamydia strains

C. trachomatis serovars E (ATCC VR-348B) and F (ATCC VR-346) were propagated in McCoy cells and elementary bodies (EB) purified using conventional Urografin (Schering AG, Berlin, Germany) density gradient centrifugation. Purified EBs were suspended in sucrose–phosphate–glutamic acid (SPG; 0·2 m sucrose, 3·8 mm KH2PO4, 6·7 mm Na2HPO4 × 2H2O, 5 mm glutamic acid; pH 7·4) buffer and stored in small stock aliquots at − 70°C until used. The number of chlamydial inclusion-forming units (IFU) was determined by infecting McCoy cells with serially diluted EB stock suspension. Chlamydial inclusions were stained with chlamydial genus-specific FITC-conjugated antibody (Sanofi Pasteur Redmond, WA, USA) at 72 h of incubation and counted using a fluorescent microscope.

Lymphocyte cultures

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Ficoll-Paque® (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation, washed three times with Hanks's balanced salt solution (Sigma, St Louis, MO, USA) and suspended in RPMI-1640 medium (Sigma) containing 10% heat-inactivated human AB serum (Finnish Red Cross, Helsinki, Finland) for the PBMC proliferative assay, or 5% fetal calf serum (FCS) for cytokine analyses.

PBMC reactivity was studied in vitro by culture stimulation in round-bottomed 96-well plates (5 × 104 cells/well) with or without antigen in a total volume of 200 µl. Cultures were incubated in humidified 5% CO2 at 37°C for 6 days as described previously by Surcel et al. [20]. Purified, formalin-killed C. trachomatis serovars E and F EB were used as antigens at a protein concentration of 3 µg/ml. Recombinant CHSP60 [21] was used at a protein concentration of 3 µg/ml and contained < 0·03 ng/ml of endotoxin as determined by Limulus assay. Pokeweed mitogen (PWM, Gibco, Paisley, UK; 12·5 µg/ml) was used as a positive control mitogen in each experiment. Optimum concentrations of antigens and mitogen were determined in preliminary experiments as minimum concentrations giving maximal proliferation. [Methyl-3H] thymidine (0·2 µCi per well; Amersham Life Science, Buckhinghamshire, UK) was added to the cultures for the last 18 h of incubation. The PBMC responses were measured as counts per minute (cpm) of incorporated radioactivity using a liquid scintillation counter (Wallac, Turku, Finland). The results were expressed as stimulation indices (SI = mean cpm in the presence of the antigen divided by mean cpm in its absence) of triplicated cultures. A SI > 2·5 was considered a positive response. Viability and reactivity of the cultured PBMC were controlled by SI > 10 in response to the PWM mitogen.

Cytokine analyses

Cytokine analyses were performed on supernatants from antigen stimulated or unstimulated cultures using commercially available enzyme-linked immunosorbent assay kits (ELISA; DuoSet® human IFN-γ, IL-10 and IL-12p40; R&D Systems, Minneapolis, USA) according to the manufacturer's instructions. Briefly, 1·4 × 106 PBMC cells/ml were incubated with or without infectious C. trachomatis EBs (1000 IFU/ml) or with CHSP60 (3 µg/ml). After 5 days of incubation (found in preliminary experiments to be the best-suited time-point to measure all three cytokines from a limited amount of antigen-stimulated PBMC cultures), supernatants were collected by centrifugation and stored at − 70°C until analysis.

Statistical analyses

Statistical analysis was performed using SPSS for Windows 9·1 statistical software (SPSS Inc., Chicago, IL, USA). Kruskal–Wallis non-parametric test was used to compare continuous variables in all four groups and Mann–Whitney U-test to compare two independent groups. Categorical variables were compared with Person's χ2 test. Correlation was tested with Spearman's correlation coefficient.

RESULTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Fifty-seven of 133 IVF patients (43%) had TFI (tubal occlusion with or without severe tubo-ovarian adhesion) verified by laparoscopy or sonosalpingography (Table 1). Twenty-eight women (21%) had other infertility factors, such as endometriosis or ovulation disorder, 23 women (17%) had unexplained infertility and 25 women were partners of couples with male factor infertility. The age range was from 22 to 41 years. There were no differences in mean ages of different patient groups (Table 1). C. trachomatis-specific IgG antibodies were found more frequently in the TFI group than in the other groups (Table 1).

Table 1.  Selected characteristics of the study population of 133 females of infertile couples
Infertility groupnMean age years (range)Positivea antichlamydial IgG titre no. (%)Positive LPb response to CTR EB no. (%)CHSP60 no. (%)
  1. Statistical comparison between TFI and other groups: *P < 0·001. aO.D. value at 450 nm ≥1. bStimulation index> 2·5. TFI: tubal factor infertility; CTR EB: Chlamydia trachomatis elementary bodies.

TFI5734 (22–41)31/56 (55%)*49/57 (86%)20/57 (36%)
Other female factor2833 (24–37) 8/27 (30%)23/28 (82%) 9/28 (32%)
Unexplained2334 (25–40) 2/23 (9%)15/23 (65%) 4/23 (17%)
Male factor2532 (23–39) 8/24 (33%)17/25 (68%) 4/24 (17%)

Lymphocyte proliferative response to EB antigens

Cell-mediated immune responses were assessed by stimulating PBMC with different chlamydial antigens. PBMC proliferative responses to C. trachomatis serovar E EBs (median SI 6·8; range 0·2–247·6) and serovar F EBs (median SI 4·2; range 0·2–247·6) were comparable (a correlation of rs 0·94; P < 0·001). Therefore, the results presented are those obtained with C. trachomatis serovar E antigen only. Positive responses (SI > 2·5) to C. trachomatis EB were more common in the TFI group (49/57, Table 1) than in the other groups but the difference was not statistically significant. Median SI to C. trachomatis EB was slightly higher in the TFI group (SI 11·5 range 0·4–247·6) than in other female-factor groups (SI 6·9, 1·1–64·7) or the unexplained infertility group (SI 7·7, 0·3–59·8) and significantly higher in the male factor group (SI 3·9, range 0·2–74·6; P < 0·005). No correlation was found between the presence of C. trachomatis specific IgG antibodies and PBMC proliferative response.

Lymphocyte proliferative response to CHSP60 antigen

PBMC proliferative responses to CHSP60 were highly variable and did not differ significantly in the study groups (in the TFI group median SI 1·6; range 0·2–22·1; in the other groups 1·4; range 0·2–24·4, Fig. 1). Positive responders (SI > 2·5) were found more often in the TFI group (36%) than in the other groups (Table 1), although the difference was not statistically significant. All the patients who had a positive response to CHSP60 were found with a positive response to chlamydial EB antigen as well.

image

Figure 1. Proliferative responses (stimulation index) of peripheral blood mononuclear cell to chlamydial HSP60 in subjects with different reasons for infertility. The horizontal line in the middle of the box is the median value of the responses and the lower (upper) boundary indicates the 25th (75th) percentile. TFI: tubal factor infertility; NUD: unexplained infertility.

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Cytokine analyses

Secretion of proinflammatory (IFN-γ and IL-12) and anti-inflammatory (IL-10) cytokines was studied from culture supernatants of C. trachomatis EB-stimulated or CHSP60-stimulated PBMC (Fig. 2). The EB antigen induced significantly greater IFN-γ (median 280 pg/ml; range < 10–8914) and weaker IL-10 secretion in the TFI group (138; < 3–6498) than in the other groups (46; < 10–1026 for IFN-γ, P < 0·02 and 871; < 3–6870 for IL-10, P < 0·001). CHSP60 induced significantly greater IFN-γ and greater IL-10 secretion in the TFI group compared to the other groups (32; < 10–7483 versus < 10; < 10–1026 for IFN-γ, P < 0·001 and 1764; < 3–7245 versus 200; < 3–3247 for IL-10, P < 0·001). As shown in Fig. 2, IL-12 secretion was clearly greater in response to CHSP60 than to EB antigen but the secretion did not differ statistically between the groups after stimulation with EB or with CHSP60 antigen. In general, the IL-12 secretion showed a significant correlation with the IL-10 secretion in all groups after stimulation with EB or with CHSP60 antigen (correlation coefficients varied from 0·52 to 0·85; P-values < 0·003). On the other hand, EB-induced IL-12 secretion did not correlate with IFN-γ secretion in the TFI group (r = −0·12) but showed a clear correlation in the other groups (r = 0,58, P < 0·001).

image

Figure 2. Production of IFN-γ, IL-10 and IL-12 (pg/ml) by PBMC stimulated with with C. trachomatis elementary bodies (EB) or CHSP60 antigen in TFI group (1), in other female factor group (2), in unexplained infertility group (3) or in male factor group (4). Statistical comparison between TFI and the other groups; *P < 0·01; **P < 0·001.

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DISCUSSION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

C. trachomatis infection is a common cause of occlusive fallopian tube disease leading to infertility [1]. Our results show that many women with TFI had humoral and cell-mediated immune responses suggestive of past C. trachomatis infection, which is an indirect marker of the association between C. trachomatis infection and fallopian tube disease. In addition, C. trachomatis EB induced a PBMC proliferative response in the TFI patients associated with a prominent secretion of proinflammatory IFN-γ. Surprisingly, IL-12 secretion did not correlate with IFN-γ secretion in the TFI group although a clear correlation was observed in the other subjects. Although more basic research is needed to know the significance of our cytokine data, it is tempting to speculate that the mutual interplay of IL-12 and IFN-γ that is needed for development of protective immunity and for clearance of chlamydial infection [6,22] may in some conditions be interfered and then related with the development of TFI. According to our results, CHSP60-induced IL-10 secretion was clearly stronger in the TFI group than in the other groups. Whether the sources of CHSP60-induced IL-10 secretion are the CHSP60-specific T cells [11,12] or antigen-stimulated monocytes [17], a presence of IL-10 in the cause of immune response to Chlamydia has been shown to be unfavourable for the protective immunity and control of Chlamydia infection [22–23]. Collectively, these results suggest that the immunopathogenesis of TFI is linked with the balance of Th1–Th2 activating cytokines and CHSP60 may have a specific role in regulating the characteristics of these reactions during chlamydial infection.

Although PBL from most subjects secreted IFN-γ when stimulated with EBs, the greatest variation in IFN-γ production, like that in the EB-induced PBMC proliferative responses, occurred in the TFI group. We cannot draw conclusions regarding the IFN-γ-secreting capacity of the chlamydial EB-activated lymphocytes in vivo but the variation in the in vitro IFN-γ secretion is comparable with the results of Arno et al. [24]. They found IFN-γ in endocervical secretion of C. trachomatis-infected women, but the level of IFN-γ varied considerably among individuals (from high to undetectable levels). It is also noteworthy that the link between IFN-γ concentration and eradication of C. trachomatis cannot be judged simply by measuring cytokine concentrations in clinical specimens. This is because C. trachomatis strains differ in sensitivity to inhibition by IFN-γ, which in turn may influence the clinical outcome of infection [25]. However, the possibility remains that in the presence of enhanced IL-10 secretion IFN-γ production may be inadequate to eradicate C. trachomatis.

The enhanced recognition of CHSP60 by circulating lymphocytes occurs in repeated and severe C. trachomatis infections in women [9,26]. According to our results PBMCs from women with TFI responded more often to CHSP60 antigen compared to controls, although the difference did not reach statistical significance. Together with our earlier studies with T cells derived from obstructed fallopian tubes [11,12], these findings seem to reflect a central role of the CHSP60 in the immunopathogenic processes taking place in the genital tract, although the functional significance of the CHSP60 responding cells remains unclear. The possibility that CHSP60 responding T cells down-regulate the IFN-γ-dominant Th1 response by secreting anti-inflammatory cytokines was suggested first in the context of scarring trachoma [19]. Kotake et al. [27] detected both IL-10 and IFN-γ-producing cells in the synovial tissue of patients with C. trachomatis-associated arthritis, and suggest that excessive IL-10 production facilitates persistent chlamydial infection by antagonizing IFN-γ-dependent mechanisms. These findings are supported by the mouse pneumonitis model, where the predominant production of IL-10 over IFN-γ was shown to delay resolution of C. trachomatis mouse pneumonitis infection [23,28].

Acute chlamydial PID progresses to TFI in only some patients suggesting that genetic background, HLA antigens [29–31] and cytokine gene polymorphism [31,32] may be involved. In experimental C. trachomatis mouse pneumonitis infection, secretion of IL-10 appears following lymphocyte activation with both self-HSP60 and CHSP60 antigens [33]. Studying lymphocyte responses in patients with salpingitis, Witkin et al. [26] demonstrated that CHSP60-activated proliferation is directed partly to epitopes that share homology with human HSP60. The authors suggested that autoimmune mechanisms may be involved in the immunopathogenesis of chlamydial genital tract disease. The immunoregulative role of CHSP60 during salpingitis might thus be comparable with the immune processes during Chlamydia-induced arthritis, where HSP60-responding T cells are present in the inflamed joints and produce anti-inflammatory cytokines, e.g. IL-10 [34].

In conclusion, our study shows preliminarily that CHSP60 stimulation of PBMCs from women with TFI produces IL-10 responses specifically, whereas stimulation with EBs enhances the proinflammatory response. These data suggest that CHSP60 may play a role as a central regulator of the immune responses seen in chronic C. trachomatis infection. Specific cytokine balance present at the site of inflammation may influence the tissue damage leading to TFI and further failure of IVF treatment.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Financial support of the Helsinki University Hospital Research Funds (TYH0015) and the Paulo Foundation is gratefully acknowledged. Special thanks are due to Mrs Marja Siitonen, Mrs Marja Suorsa and Mrs Pirkko Timonen for excellent technical assistance.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
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