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
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Abstract |
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Key words:
cell-mediated immunity/heat shock protein 60/IFN-/pelvic inflammatory disease/tubal factor infertility
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Introduction |
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T cells are functionally differentiated into Th1 or Th2 subtypes depending on their cytokine secretion profiles. Type 1 cells typically produce gamma-interferon (IFN-) and other pro-inflammatory cytokines. Conversely, type 2 cells secrete interleukin (IL)-4, IL-5, IL-10 and inhibit production of IFN-
(Constant and Bottomly, 1997
). Based on experimental animal models of C. trachomatis infection, activation of type 1 cells and the presence of IFN-
are needed for protection against re-infection (Su and Caldwell, 1995
; Perry et al., 1997
; van Voorhis et al., 1997
). 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.
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Materials and methods |
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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, 1998). 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., 1999). 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., 1999). 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- and IL-5 production was performed using commercially available enzyme-linked immunosorbent assay (ELISA; DuoSetTM Human IFN-
; Genzyme Diagnostics, Cambridge, MA, USA and Pharmingen QuantigenTM Human IL-5 Set; San Diego, CA, USA) according to the manufacturer's instructions.
Reverse transcriptasepolymerase chain reaction (RT-PCR) analysis
In-vivo expression of IFN- 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., 1997
). 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 MannWhitney U-test and with Fisher's 2 test.
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Results |
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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 I). 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.
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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 1642).
C. trachomatis EB antigen supported proliferative responses (SI >3) in 25% (two out of eight) of the TLL established from PID patients (Figure 1), 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 1
). Moreover, the proliferative responses of the TLL originating from salpingeal specimens were greater (P < 0.001; median 16 822 c.p.m., range 11 66824 187) than those originating from the endometrial specimens (1502; 1942035). Background TLL proliferation in the absence of antigen ranged from 31 to 608 c.p.m.
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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- (median 1007 pg/ml, range 7651080 pg/ml), whereas IL-5 production varied (median 779 pg/ml, range 911034 pg/ml). When IFN-
production was compared with IL-5 production, IFN-
was prominent in two TLL and comparable to IL-5 secretion in three TLL (Figure 2
), indicating both type 1 and type 0 T cell reactivity, an intermediate type of type 1 and type 2 T cells.
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Discussion |
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The high frequency of positive immune response to the CHSP60 in women with TFI (Witkin et al., 1993; McCormack, 1994
; Witkin et al., 1994a
; Paavonen and Lehtinen, 1996
) 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., 1994a
). 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., 1992), 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-, which contributes to the resolution of the infection (Williams et al., 1997
) and protection against re-infection (Perry et al., 1997
). It has been reported (Arno et al., 1990
) that women who are culture positive for C. trachomatis have higher concentrations of IFN-
in endocervical secretions than culture negative women. In our study, IFN-
mRNA was found in the Fallopian tube specimens and peritoneal cavity specimens of PID patients. Also, Chlamydia reactive TLL secreted IFN-
, suggesting that the progression of chlamydial infection into PID may not be related to a shift from a pro-inflammatory type 1 (IFN-
) 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., 1993
).
Chronic Chlamydia infection has been linked with impaired embryo implantation (Witkin et al., 1994b). 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., 1996
). In particular, hydrosalpinges present during IVFembryo transfer have negative consequences on the pregnancy rates (Camus et al., 1999
). A recent prospective, randomized study showed that preventive salpingectomy increased pregnancy and delivery rates in patients with bilateral hydrosalpinges (Strandell et al., 1999
). 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., 1996
; Beyler et al., 1997
), but not in human studies (Granot et al., 1998
). 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-
may be continuously present in local genital tract tissue due to chronic inflammation and chlamydial infection. IFN-
is a powerful down-regulator of anti-inflammatory cytokines such as IL-10 (Belardelli, 1995
) which is increased during pregnancy and is probably needed to prevent rejection of the fetus (Marzi et al., 1996
; Kelemen et al., 1998
).
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- production. This suggests that the specific inflammatory response against C. trachomatis contributes to the pathogenesis of tubal infertility.
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Acknowledgments |
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Notes |
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References |
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Submitted on January 6, 2000; accepted on April 3, 2000.