Chlamydia trachomatis reactive T lymphocytes from upper genital tract tissue specimens

A. Kinnunen1, P. Molander3, A. Laurila1, I. Rantala4, R. Morrison5, M. Lehtinen2, R. Karttunen6, A. Tiitinen3, J. Paavonen3 and H.-M. Surcel1,7

1 National Public Health Institute, Aapistie 1, Box 310, 90101 Oulu, Finland and 2 Helsinki, 3 Department of Obstetrics and Gynaecology, University of Helsinki, Helsinki, 4 Pathology Unit, Tampere University Hospital, Tampere, 5 Department of Medical Microbiology, University of Oulu, Finland and 6 Department of Microbiology, Montana State University, Bozeman, MT, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chlamydia trachomatis infection is associated with pelvic inflammatory disease (PID) and tubal factor infertility (TFI). We investigated the role of C. trachomatis as a target antigen of endometrial and salpingeal tissue lymphocytes derived from PID and TFI patients. Antigen specificity of the tissue originated T lymphocyte lines (TLL) was tested against C. trachomatis elementary bodies and chlamydial heat shock protein 60 (CHSP60). C. trachomatis antigen stimulated proliferation in two out of eight endometrial TLL derived from PID patients and three out of four TLL derived from TFI patients. All (n = 4) TLL derived from the salpingeal specimens responded to CHSP60 compared with only one out of 12 TLL derived from the endometrial specimens. In-vivo expression of interferon-gamma (IFN-{gamma}) mRNA revealed that it was present in nine of 13 specimens obtained from PID patients. The dominant activity of type-1 T lymphocytes was confirmed by the in-vitro production of IFN-{gamma} (median 1007 pg/ml) from all (n = 5) C. trachomatis specific TLL while IL-5 secretion was lower (median 779 pg/ml). In conclusion, C. trachomatis reactive TLL were established from in-vivo activated lymphocytes from the upper genital tract tissue of PID and TFI patients. The reactivity of the salpingeal TLL to CHSP60 provided further evidence that immunoreactivity to CHSP60 is a predominant response in patients with tubal damage.

Key words: cell-mediated immunity/heat shock protein 60/IFN-{gamma}/pelvic inflammatory disease/tubal factor infertility


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chlamydia trachomatis is the major cause of sexually transmitted infection in the western world. It has been associated with pelvic inflammatory disease (PID), tubal factor infertility (TFI) and ectopic pregnancy by sero-epidemiology (McCormack, 1994Go; Paavonen and Lehtinen, 1996Go). The presence of C. trachomatis DNA and antigens in Fallopian tubes of women with postinfectious TFI and ectopic pregnancy (Campbell et al., 1993Go; Patton et al., 1994Go; Dieterle et al., 1998Go; Gerard et al., 1998Go) has confirmed that C. trachomatis is associated with silent salpingitis. Chlamydiae may induce inflammatory reaction that provokes tissue injury and results in tubal occlusion. Moreover, a comparable relationship between C. trachomatis infection and salpingitis or TFI has been established in animals repeatedly exposed to chlamydial antigens (Cappuccio et al., 1994Go; Patton et al., 1994Go; van Voorhis et al., 1997Go). Enhanced humoral (Toye et al., 1993Go; Arno et al., 1995Go; Eckert et al., 1997Go; Peeling et al., 1997Go; Domeika et al., 1998Go) and cell-mediated responses (Witkin et al., 1993Go, 1994aGo) to chlamydial heat shock protein (CHSP60) suggests that CHSP60 participates in the immunopathological response. In animal models of chlamydial ocular infection (Watkins et al., 1986Go; Taylor et al., 1987Go; Morrison et al., 1989Go) or salpingitis (Patton et al., 1994Go), purified CHSP60 elicits an immune response comparable to that in chronic chlamydial infection.

T cells are functionally differentiated into Th1 or Th2 subtypes depending on their cytokine secretion profiles. Type 1 cells typically produce gamma-interferon (IFN-{gamma}) and other pro-inflammatory cytokines. Conversely, type 2 cells secrete interleukin (IL)-4, IL-5, IL-10 and inhibit production of IFN-{gamma} (Constant and Bottomly, 1997Go). Based on experimental animal models of C. trachomatis infection, activation of type 1 cells and the presence of IFN-{gamma} are needed for protection against re-infection (Su and Caldwell, 1995Go; Perry et al., 1997Go; van Voorhis et al., 1997Go). However, the impact of the cytokine balance in the context of immunopathogenesis is unresolved.

In this study, we analysed inflammation in the female upper genital tract and characterized in-vivo activated T-cells present in the inflamed tubal tissue of patients with acute PID and TFI. Our purpose was to evaluate further the role of C. trachomatis in the pathogenesis of TFI, to demonstrate the extent of inflammation and the presence of in-vivo activated C. trachomatis specific T cells from endometrium to the tubal tissue. The antigen specificity of the response and cytokine secretion of the responding cells were also analysed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
The original study population consisted of 22 consecutive patients with clinical diagnosis of PID, who attended the Department of Obstetrics and Gynaecology at the University Hospital of Helsinki, and 22 patients who attended the infertility clinic of the same hospital because of tubal factor infertility. The PID patients underwent an operative laparoscopy within 24 h following admission. Laparoscopy confirmed the diagnosis of acute PID in 14 of the 22 patients. The laparoscopic diagnosis of PID was based on commonly accepted visual findings (Tukeva et al., 1999Go). Diagnosis of PID was placed on commonly accepted visual findings (Tukeva et al., 1999Go). Acute salpingitis was diagnosed when a hyperaemic oedematous Fallopian tube was seen with signs of intraluminal pus at the fimbriated end. Pyosalpinx was diagnosed when an enlarged tube with partial or total destruction of the fimbriated end was seen. Tubo–ovarian abscess was diagnosed when the ovary and the Fallopian tube could not be distinguished from each other, forming an adnexal complex with abscess formation. Those 14 cases were then included in the immunological studies. Endometrial biopsy specimens were obtained from the PID (n = 14) and TFI patients (n = 22) for histology, immunohistochemistry, and lymphocyte cultures. In addition, salpingeal tissue specimens were obtained from six infertility clinic patients who underwent salpingectomy because of hydrosalpinx formation.

Specimens
Tissue specimens for morphological studies were fixed in 10% neutral buffered formalin and embedded in paraffin. Specimens for lymphocyte cultures were immediately immersed in tissue culture medium RPMI 1640 (Sigma, St Louis, MO, USA) supplemented with glutamine (2.0 mmol/l), antibiotics (20 µg/ ml streptomycin) and 10% heat-inactivated human AB serum (Finnish Red Cross, Helsinki, Finland). Transportation to the laboratory occurred at room temperature within 24 h. Heparinized blood samples were drawn from all subjects for immunological analysis and kept at room temperature for no longer than 24 h prior to the separation of peripheral blood mononuclear lymphocytes.

Specimens for isolation of RNA for reverse transcription- polymerase chain reaction (RT-PCR) analyses were taken from cell pellets of tubal aspirates and peritoneal aspirates of acute PID patients, and from Fallopian tube tissue of salpingectomy patients. Specimens were immersed in RNase-free buffer and stored at -70°C until analysed.

Immunohistochemistry
For histological examination, 5 µm thick paraffin sections were routinely stained with haematoxylin and eosin. Immunohistochemical staining of T-cell populations was carried out either on paraffin or cryostat sections. The following primary antibodies were used: CD45RO (T memory cells, clone UCHL1; Dako a/s, Glostrup, Denmark), CD4 (T helper cells, clone SK3; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA), CD8 (T suppressor cells, clone SK1; Becton Dickinson), CD15 (monocytes, clone MMA; Becton Dickinson), CD20 (B cells, clone L26; Dako) and CD25 (IL-2 receptor, clone 2A3; Becton Dickinson).

For the staining of CD45RO, CD15 and CD20 antigens, 5 µm thick paraffin sections were cut on ChemMateTM capillary gap microscope slides (Dako). Rehydrated sections were heated in a microwave oven at 850 W for two 7-min cycles using 0.01 mol/l citrate buffer (pH 6.0) as antigen retrieval solution. The immunohistochemical staining was performed using the indirect streptavidin-biotin-peroxidase method in TechMateTM 500 Immunostainer (Dako). The primary antibodies were visualized by ChemMateTM detection kit (Dako) with diaminobenzidine as chromogen and haematoxylin as nuclear stain.

CD4 and CD8 antigens were demonstrated on 5 µm cryostat sections after fixation in acetone for 10 min. Specimens for CD4 and CD8 were additionally fixed in chloroform for 30 min. After fixation the sections were washed in phosphate-buffered saline (pH 7.4) and incubated in primary antibodies for 1 h. The bound antibodies were revealed with avidin-biotin peroxidase technique (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA) using aminoethylcarbazole as chromogen. Endogenous peroxidase activity was inhibited for 30 min after biotinylated secondary antibodies with 0.5% H2O2 in methanol. Counterstaining was performed with haematoxylin. The number of each cell type was counted in 10 representative high power fields (x250) per sample.

The presence of C. trachomatis in genital tract tissues was visualized by direct immunofluoresence microscopy of paraffin sections as earlier described (Rantala and Kivinen, 1998Go). Before immunostaining with the fluorescein-conjugated anti-chlamydial major outer membrane protein monoclonal antibody (clone 512F; Cellabs, Brookvale, NSW, Australia), the sections were treated with chlamydial antigenicity retrieval solution (Cellabs). Evans blue (combined with the antibody solution) was used as a counterstain.

T cell lines
T cells were cultured from the endometrial biopsy and salpingeal tissue specimen as previously described (Halme et al., 1999Go). Briefly, tissues were minced into small pieces with a sterile scalpel and placed on a plastic Petri dish (Corning, New York, USA) in RPMI 1640 supplemented with 10% human AB serum in a humidified 5% CO2 atmosphere at 37°C. In-vivo activated T lymphocytes expressing IL-2 receptor were propagated by adding 10% IL-2 (Biotest, Dreieich, Germany) into the culture medium. Half of the IL-2 containing medium was changed every third day. After 10 days of incubation without antigen, the growing lymphocytes were further augmented by stimulation with formalin-inactivated, purified C. trachomatis elementary bodies (EB) (0.3 µg/ml) antigen, and autologous irradiated peripheral blood mononuclear lymphocytes as the antigen presenting cells (APC) in the presence of IL-2. The expanding T cells were restimulated with C. trachomatis antigen (0.3 µg/ml) twice over a period of 10 days to obtain sufficient cells to perform antigen specificity tests. IL-2 was added no later than 4 days prior to testing.

The antigen specificity of the TLL was tested by culturing 20 000 cells in triplicate in 96-well round bottomed microtitre plates (Sterilin Ltd, Feltham, UK) in the presence of 20 000 irradiated autologous APC and C. trachomatis (3 µg/ml) or the CHSP60 (20 µg/ml) antigen suspended in RPMI 1640 supplemented with 10% AB serum in a total volume of 200 µl. Cultures were incubated in humidified 5% CO2 at 37°C for 72 h as previously described (Halme et al., 1999Go). Methyl-[3H]thymidine (0.2 Ci/well; Amersham Life Science, Amersham, Bucks, UK) was added to the wells for the last 18 h. The cells were harvested from each well on nitrocellulose filters (Wallac, Turku, Finland) using an automated cell harvester (Skatron AS, Lier, Norway) and the lymphocyte proliferation responses were measured in counts per minute (c.p.m.) of radioactivity incorporated into the proliferating cells using a liquid scintillation counter (Wallac). The results are expressed as mean c.p.m. or as stimulation indexes (SI; the ratio of c.p.m. value in the presence of antigen to the c.p.m. in the absence of antigen) calculated from triplicate cultures.

The chlamydial reactivity of genital tract tissue derived in-vivo activated T lymphocytes was compared to peripheral blood derived TLL. Following the protocol described above, peripheral blood derived TLL were established and tested concomitantly with corresponding TLL derived from the tissue specimens.

The TLL were also analysed for their surface antigens by double immunofluorescence flow cytometric analysis. Cultured lymphocytes were stained using phycoerythrin (PE)-conjugated anti-CD4 and fluorescein isothiocyanate (FITC)-conjugated anti-CD8 monoclonal antibodies (Caltag Laboratory, San Francisco, CA, USA) and then analysed by FACScan (Becton Dickinson and Co., Mountain View, CA, USA).

Cytokine secretion of TLL
Cytokine production was induced by incubating the TLL (106 cells/ml) with or without PHA (2 µg/ml) in the presence of autologous APC cells (106 cells/ml). After 48 h of incubation as above, supernatants were collected by centrifugation and stored at -70°C until analysis. Analysis of IFN-{gamma} and IL-5 production was performed using commercially available enzyme-linked immunosorbent assay (ELISA; DuoSetTM Human IFN-{gamma}; Genzyme Diagnostics, Cambridge, MA, USA and Pharmingen QuantigenTM Human IL-5 Set; San Diego, CA, USA) according to the manufacturer's instructions.

Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis
In-vivo expression of IFN-{gamma} and IL-4 specific mRNA in cell pellets of acute PID patient specimens and in the salpingeal tissue specimens from TFI patients were analysed by RT-PCR as previously described (Halme et al., 1997Go). Commercially available kits were used for total RNA isolation (RNeasy Midi Isolation Kit; Quiagen, Crawley, West Sussex, UK) and RT-PCR analyses (GeneAmpR RNA PCR Core Kit; Perkin Elmer, Branchburg, NJ, USA).

Statistical analyses
The statistical analysis was performed by SPSS software (SPSS Inc. Chicago, IL, US) with Mann–Whitney U-test and with Fisher's {chi}2 test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Histopathology and immunohistochemistry
Mean age of both PID and TFI patients was 36 years (range 15–57). Endometrium samples from 13 out of the 14 PID patients and from all 22 TFI patients were available for histopathologic examination. Routine histology showed endometritis with plasma cell infiltrations in 54% (7/13) of the PID patients and in 18% (4/22) of the TFI patients. The total number of lymphocytes varied widely; in cases with plasma cell endometritis, the median number of lymphocytes/10 HPF was clearly higher than in non-endometritis cases (612.5; range 137–2220 versus 91; 27–890; P < 0.001). Chronic salpingitis with mucosal atrophy was detected in five of six salpingeal samples.

Immunohistology revealed that the majority of the endometrial lymphocytes were CD45RO+ T cells in the PID group, but these were found less often in the TFI group (Table IGo). Most of the CD45RO+ were CD4+ T cells as they outnumbered CD8+ T cells in all tissues. The CD4/CD8 ratio was greater in the four PID patients compared to the two TFI patients (median 7 versus 2 respectively). The CD4/CD8 ratio in TFI patients was equal to that normally found in blood lymphocytes. Monocytes and B cells were also present but were consistently fewer than T cells.


View this table:
[in this window]
[in a new window]
 
Table I. Mononuclear lymphocyte subsets (number of positive staining cells in 10 high power fields) in the female upper genital tract tissue specimen from acute pelvic inflammatory disease (PID) patients and patients undergoing IVF for tubal factor infertility (TFI)
 
C. trachomatis EB were detected by direct immunofluorescence staining in 33% (4/12) of the patients with acute PID, and in 22% (4/18) of the endometrial and 20% (1/5) of the salpingeal specimens of the TFI patients. However, C. trachomatis positivity was not associated with the number of lymphocytes or plasma cells found in tissue samples.

IL-2 supported T cell growth
The IL-2 containing medium without antigen supported T cell growth more frequently from endometrial specimens of PID patients (57%; 8/14) than TFI patients (18%; 4/22; P < 0.05). In addition, IL-2 supported T cell growth in 67% (4/6) of salpingeal tissue specimens obtained from TFI patients. The majority of the growing lymphocytes were CD4 positive (the range of the CD4/CD8 ratio was 16–42).

C. trachomatis EB antigen supported proliferative responses (SI >3) in 25% (two out of eight) of the TLL established from PID patients (Figure 1Go), but more frequently of TLL established from four endometrial (75%, three out of four) or four salpingeal (100%, four out of four) tissue specimens from TFI patients (Figure 1Go). Moreover, the proliferative responses of the TLL originating from salpingeal specimens were greater (P < 0.001; median 16 822 c.p.m., range 11 668–24 187) than those originating from the endometrial specimens (1502; 194–2035). Background TLL proliferation in the absence of antigen ranged from 31 to 608 c.p.m.



View larger version (39K):
[in this window]
[in a new window]
 
Figure 1. Percentage of T lymphocyte lines (TLL) showing a positive proliferative response (stimulation index >3) toC. trachomatis elementary bodies (dark bar) or chlamydial heat shock protein 60 (open bar) antigen. TLL were derived from endometrial or salpingeal tissue of patients with pelvic inflammatory disease (PID) or tubal factor infertility (TFI).

 
T cell reactivity to C. trachomatis and to chlamydial HSP60
Antigen reactivity of the TLL was further analysed with CHSP60. CHSP60 was recognized only by TLL derived from TFI patients as all four salpingeal and one endometrial TLL derived from TFI patients responded to CHSP60, and none of the TLL derived from PID patients responded (Figure 1Go).

To obtain information about the original location of the activated C. trachomatis specific T cells, TLL from peripheral blood lymphocytes (PBL) of the same patients were isolated and evaluated. One PID patient and one patient with TFI had C. trachomatis EB responding TLL (SI >3) both in tissue and peripheral blood. The PBL TLL did not respond to CHSP60.

Cytokine analysis
Five representative C. trachomatis specific TLL were analysed for the cytokine production profile. After mitogenic stimulation, all TLL secreted high quantities of IFN-{gamma} (median 1007 pg/ml, range 765–1080 pg/ml), whereas IL-5 production varied (median 779 pg/ml, range 91–1034 pg/ml). When IFN-{gamma} production was compared with IL-5 production, IFN-{gamma} was prominent in two TLL and comparable to IL-5 secretion in three TLL (Figure 2Go), indicating both type 1 and type 0 T cell reactivity, an intermediate type of type 1 and type 2 T cells.



View larger version (44K):
[in this window]
[in a new window]
 
Figure 2. Secretion of gamma interferon (IFN-{gamma}) (dark bar) and interleukin (IL)-5 (open bar) from five representative T lymphocyte lines (TLL) obtained from tissue specimens of patients with pelvic inflammatory disease (PID) (no. 1) or tubal factor infertility (TFI) (nos 2–5). Positive reactivity (stimulation index >3) of the TLL to C. trachomatis (CTR) and to chlamydial heat shock protein 60 (CHSP60) is shown below.

 
We found in-vivo expression of IFN-{gamma} mRNA in nine (69%) of 13 PID patients from whom the cell pellets of the peritoneal fluid or tubal aspirate specimens were available. In the salpingectomy patients, IFN-{gamma} mRNA was found in three of four (75%) salpingeal specimens.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We were able to establish TLL from the endometrium of 57% of PID patients and 18% of TFI patients. Since the lymphocyte propagation was supported by IL-2 in the absence of exogenous antigen, the cells have arisen from in-vivo activated T-cells that are probably actively participating in the inflammatory process in the salpingeal tissue (Stamm, 1999Go). Although the number of salpingeal tissue specimens from the TFI patients was limited, establishing T cells from four out of six specimens is in accordance with the hypothesis that tissue inflammation is playing a central role in the development of tissue damage and eventual tubal obstruction (Paavonen and Lehtinen, 1996Go; Stamm, 1999Go). C. trachomatis was recognized as a specific target antigen by 25% of the TLL obtained from the PID patients and by 75% of the TLL obtained from the TFI patients. That finding provides evidence supporting the premise that C. trachomatis is important in maintaining tissue inflammation in tubal infertility (Paavonen and Lehtinen, 1996Go; Stamm, 1999Go). Moreover, C. trachomatis was a target antigen for each TLL derived from the obstructed salpingeal tissue.

The high frequency of positive immune response to the CHSP60 in women with TFI (Witkin et al., 1993Go; McCormack, 1994Go; Witkin et al., 1994aGo; Paavonen and Lehtinen, 1996Go) suggests that CHSP60 may be involved in the immunopathological events following chronic C. trachomatis infection. In our study, the TLL originated from the TFI patients recognized the CHSP60 more frequently than those from the PID patients. Although functional analysis (i.e. cytokine profile and cytotoxic capacity) of the T cells responding to CHSP60 are needed to know precisely the role of CHSP60 in the pathological events, our results are in accordance with previous studies showing that blood lymphocytes from patients with laparoscopically verified salpingitis react to CHSP60 (Witkin et al., 1994aGo). Thus this antigen, either alone or together with other antigens, activates tissue lymphocytes in chlamydial disease.

The numbers of lymphocytes and subsets of lymphocytes in the upper genital tract is known to vary during the menstrual cycle (Klentzeris et al., 1992Go), which makes it difficult to compare relative proportions. However, we found that Chlamydia reactive TLL were more frequently established from tissue specimens with a high number of mononuclear cells, which is indicative of an ongoing immunological reaction. The endometrial specimens from PID patients contained more mononuclear cells than corresponding specimens from TFI patients. In the latter group, the inflamed tissue was limited to the obstructed Fallopian tube where the number of mononuclear cells was comparable to that in endometrial tissue specimens from PID patients.

Cell-mediated immune response to C.trachomatis typically involves secretion of IFN-{gamma}, which contributes to the resolution of the infection (Williams et al., 1997Go) and protection against re-infection (Perry et al., 1997Go). It has been reported (Arno et al., 1990Go) that women who are culture positive for C. trachomatis have higher concentrations of IFN-{gamma} in endocervical secretions than culture negative women. In our study, IFN-{gamma} mRNA was found in the Fallopian tube specimens and peritoneal cavity specimens of PID patients. Also, Chlamydia reactive TLL secreted IFN-{gamma}, suggesting that the progression of chlamydial infection into PID may not be related to a shift from a pro-inflammatory type 1 (IFN-{gamma}) cytokine response to an anti-inflammatory type 2 (IL-5) cytokine response. Presence of the type 2 cytokines in the tissues of TFI patients and their in-vitro production by CHSP60 reactive TLL suggests that the pathological mechanisms may in some cases, however, involve impaired type 1 cell function as has been reported in chlamydial trachoma (Holland et al., 1993Go).

Chronic Chlamydia infection has been linked with impaired embryo implantation (Witkin et al., 1994bGo). For some years it has been known that patients with severe tubal damage have a poor prognosis in IVF and embryo transfer programmes (Csemiczky et al., 1996Go). In particular, hydrosalpinges present during IVF–embryo transfer have negative consequences on the pregnancy rates (Camus et al., 1999Go). A recent prospective, randomized study showed that preventive salpingectomy increased pregnancy and delivery rates in patients with bilateral hydrosalpinges (Strandell et al., 1999Go). Although the mechanism for the negative association of hydrosalpinges with pregnancy outcome is not known, the hydrosalpinx fluid has been shown to impair the blastocyst development in animal studies (Mukherjee et al., 1996Go; Beyler et al., 1997Go), but not in human studies (Granot et al., 1998Go). However, it is possible that hydrosalpinx fluid contains immunological mediators that interfere in vivo with the regulation of fertilization and implantation outcome. Based on our study, IFN-{gamma} may be continuously present in local genital tract tissue due to chronic inflammation and chlamydial infection. IFN-{gamma} is a powerful down-regulator of anti-inflammatory cytokines such as IL-10 (Belardelli, 1995Go) which is increased during pregnancy and is probably needed to prevent rejection of the fetus (Marzi et al., 1996Go; Kelemen et al., 1998Go).

In women, upper genital tract infection with C. trachomatis causes a number of clinically important negative consequences, including tubal infertility. The pathogenesis of tubal infertility is incompletely understood. We have shown that T-lymphocytes derived from salpingeal tissue of TFI patients show specific reactivity with CHSP60 and respond to C. trachomatis by IFN-{gamma} production. This suggests that the specific inflammatory response against C. trachomatis contributes to the pathogenesis of tubal infertility.


    Acknowledgments
 
This study was supported by the Academy of Finland and the University of Helsinki University Hospital Research Funds (TYH0015). Special thanks are due to Marja Siitonen and Pirkko Timonen for technical assistance.


    Notes
 
7 To whom correspondence should be addressed at: National Public Health Institute, Aapistie 1, Box 310, 90101 Oulu, Finland. E-mail: helja_marja.surcel{at}ktl.fi Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arno, J.N., Ricker, V.A., Batteiger, B.E. et al. (1990) Interferon-{gamma} in endocervical secretions of women infected with Chlamydia trachomatis. J. Infect. Dis., 162, 1385–1389.[ISI][Medline]

Arno, J.N., Yuan, Y., Cleary, R.E. et al. (1995) Serologic responses of infertile women to the 60-kD chlamydial heat shock protein (hsp60). Fertil. Steril., 64, 730–735.[ISI][Medline]

Belardelli, F. (1995) Role of interferons and other cytokines in the regulation of the immune response. APMIS, 103, 161–179.[ISI][Medline]

Beyler, S.A., James, K.P., Fritz, M.A. et al. (1997) Hydrosalpingeal fluid inhibits in-vitro embryonic development in a murine model. Hum. Reprod., 12, 2724–2728.[Abstract]

Campbell, L.A., Patton, D.L., Moore, D.E. et al. (1993) Detection of Chlamydia trachomatis deoxyribonucleic acid in women with tubal infertility. Fertil. Steril., 59, 45–50.[ISI][Medline]

Camus, E., Poncelet, C., Goffinet, F. et al. (1999) Pregnancy rates after in-vitro fertilization in cases of tubal infertility with and without hydrosalpinx: a meta-analysis of published comparative studies. Hum. Reprod., 14, 1243–1249.[Abstract/Free Full Text]

Cappuccio, A.L., Patton, D.L., Kuo, C.-C. et al. (1994) Detection of Chlamydia trachomatis deoxyribonucleic acid in monkey models (Macaca nemestrina) of salpingitis by in situ hybridization: implications for pathogenesis. Am. J. Obstet. Gynecol., 171, 102–110.[ISI][Medline]

Constant, S.L. and Bottomly, K. (1997) Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu. Rev. Immunol., 15, 297–322.[ISI][Medline]

Csemiczky, G., Landgren, B.-M., Fried, G. et al. (1996) High tubal damage grade is associated with low pregnancy rate in women undergoing in-vitro fertilization treatment. Hum. Reprod., 11, 2438–2440.[Abstract]

Dieterle, S., Rummel, C., Bader, L.W. et al. (1998) Presence of the major outer-membrane protein of Chlamydia trachomatis in patients with chronic salpingitis and salpingitis isthmica nodosa with tubal occlusion. Fertil. Steril., 70, 774–776.[ISI][Medline]

Domeika, M., Domeika, K., Paavonen, J. et al. (1998) Humoral immune response to conserved epitopes of Chlamydia trachomatis and human 60-kDa heat-shock protein in women with pelvic inflammatory disease. J. Infect. Dis., 177, 714–719.[ISI][Medline]

Eckert, L.O., Hawes, S.E., Wolner-Hanssen, P. et al. (1997) Prevalence and correlates of antibody to chlamydial heat shock protein in women attending sexually transmitted disease clinics and women with confirmed pelvic inflammatory disease. J. Infect. Dis., 175, 1453–1458.[ISI][Medline]

Gerard, H.C., Branigan, P.J., Balsara, G.R. et al. (1998) Viability of Chlamydia trachomatis in Fallopian tubes of patients with ectopic pregnancy. Fertil Steril., 70, 945–948.[ISI][Medline]

Granot, I., Dekel, N., Segal, I. et al. (1998) Is hydrosalpinx fluid cytotoxic? Hum. Reprod., 13, 1620–1624.[Abstract]

Halme, S., Saikku, P. and Surcel, H.-M. (1997) Characterization of Chlamydia pneumoniae antigens using human T cell clones. Scand. J. Immunol., 45, 378–384.[ISI][Medline]

Halme, S., Juvonen, T., Laurila, A. et al. (1999) Chlamydia pneumoniae reactive T lymphocytes in the walls of abdominal aortic aneurysms. Eur. J. Clin. Invest., 29, 546–552.[ISI][Medline]

Holland, M.J., Bailey, R.L., Hayes, L.J. et al. (1993) Conjunctival scarring in trachoma is associated with depressed cell-mediated immune responses to chlamydial antigens. J. Infect. Dis., 168, 1528–1531.[ISI][Medline]

Kelemen, K., Paldi, A., Tinneberg, H. et al. (1998) Early recognition of pregnancy by the maternal immune system. Am. J. Reprod. Immunol., 39, 351–355.[ISI][Medline]

Klentzeris, L.D., Bulmer, J.N., Warren, A. et al. (1992) Endometrial lymphoid tissue in the timed endometrial biopsy: morphometric and immunohistochemical aspects. Am. J. Obstet. Gynecol., 167, 667–674.[ISI][Medline]

Marzi, M., Vigano, A., Trabattoni, D. et al. (1996) Characterization of type 1 and type 2 cytokine production profile in physiologic and pathologic human pregnancy. Clin. Exp. Immunol., 106, 127–133.[ISI][Medline]

McCormack, W.M. (1994) Pelvic inflammatory disease. N. Engl. J. Med., 330, 115–119.[Free Full Text]

Morrison, R.P., Lyng, K. and Caldwell, H.D. (1989) Chlamydial disease pathogenesis. Ocular hypersensitivity elicited by a genus-specific 57-kD protein. J. Exp. Med., 169, 663–675.[Abstract]

Mukherjee, T., Copperman, A.B., McCaffrey, C. et al. (1996) Hydrosalpinx fluid has embryotoxic effects on murine embryogenesis: a case for prophylactic salpingectomy. Fertil. Steril., 66, 851–853.[ISI][Medline]

Paavonen, J. and Lehtinen, M. (1996) Chlamydial pelvic inflammatory disease. Hum. Reprod. Update, 2, 519–529.[Abstract/Free Full Text]

Patton, D.L., Askienazy -Elbhar, M., Henry-Suchet, J. et al. (1994) Detection of Chlamydia trachomatis in Fallopian tube tissue in women with postinfectious tubal infertility. Am. J. Obstet. Gynecol., 171, 95–101.[ISI][Medline]

Peeling, R.W., Kimani, J., Plummer, F. et al. (1997) Antibody to chlamydial hsp60 predicts an increased risk for chlamydial pelvic inflammatory disease. J. Infect. Dis., 175, 1153–1158.[ISI][Medline]

Perry, L.L., Feilzer, K. and Caldwell, H.D. (1997) Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-{gamma}-dependent and -independent pathways. J. Immunol., 158, 3344–3352.[Abstract]

Rantala, I. and Kivinen S. (1998) Demonstration of Chlamydia trachomatis in Papanicolaou-stained gynecological smears. Eur. J. Clin. Microbiol. Infect. Dis., 17, 46–48.[ISI][Medline]

Stamm, W.E. (1999) Chlamydia trachomatis infections: progress and problems. J. Infect. Dis., 179, S380–383.[ISI][Medline]

Strandell, A, Lindhard, A., Waldenström, U. et al. (1999) Hydrosalpinx and IVF outcome: a prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum. Reprod., 14, 2762–2769.[Abstract/Free Full Text]

Su, H. and Caldwell, H.D. (1995) CD4+ T cells play a significant role in adoptive immunity to Chlamydia trachomatis infection of the mouse genital tract. Infect. Immun., 63, 3302–3308.[Abstract]

Taylor, H.R., Johnson, S.L., Schachter, J. et al. (1987) Pathogenesis of trachoma: the stimulus for inflammation. J. Immunol., 138, 3020–3027.

Toye, B., Laferrière, C., Claman, P. et al. (1993) Association between antibody to the chlamydial heat-shock protein and tubal infertility. J. Infect. Dis., 168, 1236–1240.[ISI][Medline]

Tukeva, T.A., Aronen, H.J., Karjalainen, P.T. et al. (1999) MR imaging in pelvic inflammatory disease: comparison with laparoscospy and US. Radiology, 210, 209–216.[Abstract/Free Full Text]

van Voorhis, W.C., Barrett, L.K., Cosgrove Sweeney, Y.T. et al. (1997) Repeated Chlamydia trachomatis infection of Macaca nemestrina Fallopian tubes produces a Th1-like cytokine response associated with fibrosis and scarring. Infect. Immun., 65, 2175–2182.[Abstract]

Watkins, N.G., Hadlow, W.J., Moos, A.B. et al. (1986) Ocular delayed hypersensitivity: a pathogenetic mechanism of chlamydial conjunctivitis in guinea-pigs. Proc. Natl Acad. Sci. USA, 83, 7480–7484.[Abstract]

Williams, D.M., Grubbs, B.G., Pack, E. et al. (1997) Humoral and cellular immunity in secondary infection due to murine Chlamydia trachomatis. Infect. Immun., 65, 2876–2882.[Abstract]

Witkin, S.S., Jeremias, J., Toth, M. et al. (1993) Cell-mediated immune response to the recombinant 57-kDa heat-shock protein of Chlamydia trachomatis in women with salpingitis. J. Infect. Dis., 167, 1379–1383.[ISI][Medline]

Witkin, S.S., Jeremias, J., Toth, M. et al. (1994a) Proliferative response to conserved epitopes of the Chlamydia trachomatis and human 60-kilodalton heat-shock proteins by lymphocytes from women with salpingitis. Am. J. Obstet. Gynecol., 171, 455–460.[ISI][Medline]

Witkin, S.S., Sultan, K.M., Neal, G.S. et al. (1994b) Unsuspected Chlamydia trachomatis infection and in vitro fertilization outcome. Am. J. Obstet. Gynecol., 171, 1208–1214.[ISI][Medline]

Submitted on January 6, 2000; accepted on April 3, 2000.