1 Universidad de Santiago de Chile, Facultad de Química y Biología, Casilla 40 Correo 33, Estación Central, Santiago, 2 Hospital Félix Bulnes, Servicio de Maternidad, Santiago and 3 Instituto Chileno de Medicina Reproductiva, Santiago, Chile
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Abstract |
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Key words: copper T/gonococcal receptors/Neisseria gonorrhoeae/Norplant®/oviduct
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Introduction |
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On the other hand, because several receptors for N.gonorrhoeae have been identified in cell lines, including CD46, CD66, syndecan and Vß5 integrin (Nassif et al., 1999
; Naumann et al., 1999
), the presence of those receptors in the epithelium of the same oviductal samples was also examined.
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Materials and methods |
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Rabbit anti-gonococcal serum production
Two female rabbits were immunized by s.c. injection (Harlow and Lane, 1988) of heat-cold (180/60°C)-inactivated N.gonorrhoeae suspended in complete Freund's adjuvant (Sigma, St Louis, MO, USA). After 2 weeks, the animals were boosted with the antigen with incomplete Freund's adjuvant (Sigma). Two weeks later the rabbits were killed, their blood collected and titrated for anti-gonococcal activity by enzyme-linked immunosorbent assay (ELISA). Escherichia coli JM109 was used to verify non-specific cross-reaction of the antiserum. For titration, a 96-well ELISA plate was incubated overnight with N.gonorrhoeae or E.coli suspensions, then incubated with the serum. The anti-gonococcal antibodies were detected with alkaline phosphatase-conjugated anti-rabbit immunoglobulin (Ig)G (whole molecule; Sigma). The titration curves with N.gonorrhoeae and E.coli revealed that the anti-gonococcal serum displayed no cross-reactivity with E.coli over a range of dilutions appropriate for the purposes of this study (Figure 1
). Based on these results the 1:1000 dilution was used for the histochemical detection and verified upon smears of bacteria.
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Immunohistochemistry for detection of N.gonorrhoeae
Gonococci attached to, and internalized into, the oviductal epithelium were detected immunohistochemically with rabbit anti-gonococcal serum and alkaline phosphatase (AP) reaction or fluorescein isothiocyanate (FITC)-conjugated antibodies as follows. After overnight incubation in 1% bovine serum albumin (BSA; Sigma)-PBS at 4°C, oviductal slices were incubated during 2 h at room temperature with rabbit anti-gonococcal serum (1:1000). When AP-conjugated antibodies were used, the endogenous AP was inactivated by incubation with 0.074% HCl in ethanol for 10 min at 4°C (Bulman and Heyderman, 1981). Slices were rinsed using PBS and incubated with AP-conjugated anti-rabbit IgG (Sigma; 1:2000) for 1 h at room temperature. After being rinsed, slices were incubated for an additional 1h at room temperature with the AP substrate nitro-blue tetrazolium chloride (NBT; 0.35 mg/ml; Amresco Solon, Ohio, USA)/5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (BCIP; 0.175 mg/ml; Boehringer, Mannheim, Germany). Slices were stained with haematoxylin and mounted with Kaiser's gelatin (Sigma). When FITC-conjugated antibodies were used, slices were first incubated overnight in 1% BSA-PBS, and then incubated for 2 h with the rabbit anti-gonococcal serum followed by 1 h incubation with FITC-conjugated anti-rabbit Ig (gamma and light chains; Biosource, Nivelles, Belgium; 1:1000). All incubations were at room temperature. After being rinsed, slices were stained with propidium iodide (0.5 µg/ml) and mounted in 10% diazabicyclo[2.2.2]octane (DABCO; Sigma) in 90% glycerol (w/v). Gonococcal smears were used as positive controls. Negative controls in which the first antibody was omitted were routinely run in all the experiments.
Inmunohistochemistry for detection of N.gonorrhoeae receptors and quantification of mononuclear cells
The antibodies used were goat anti-human CD46, CD66, or syndecan-1 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-human integrin Vß5 (clone P1F6) from Gibco (1:200). For detection of CD46, CD66 and syndecan-1, 46 µm cryostat sections were placed onto gelatin-coated slides and fixed in acetone at 20°C for 10 min. After blocking with PBS-BSA 1% for 2 h at room temperature, the sections were incubated overnight with the primary antibody at 4°C. After three PBS rinses, the slices were incubated during 1 h at room temperature with FITC-conjugated rabbit anti-goat IgG (1:750; Sigma). After three PBS rinses, samples were counterstained with propidium iodide (1 µg/ml; Sigma). The slices were mounted with Dabco glycerol as indicated above. Detection of
Vß5 integrin was carried out as described by Sülz et al. (1998). Briefly, cryostat sections were fixed in acetone at 20°C for 10 min. After blocking with PBS-BSA 1% for 2 h at room temperature, the sections were incubated overnight at 4°C with the mouse monoclonal antibody anti-human integrin
Vß5. After three PBS rinses the sections were incubated for 30 min at room temperature with biotinylated goat anti-mouse immunoglobulin (1:500). After three rinses, the avidin-FITC complex was incubated on the sections for 1 h at room temperature. Samples were washed with water, counterstained with propidium iodide and mounted in DABCO glycerol.
The tissue sections used to examine the expression of gonococcal receptors allowed the quantification of intraepithelial mononuclear cells, which was done by counting all the cells with a round lymphocyte-like nucleus in an epithelial segment comprising 100200 nuclei. These cells were interpreted as lymphocytes, as reported by others (Wollen et al., 1994). The confocal microscope made it possible to examine the propidium iodide-stained nuclei throughout so that misinterpretation due to inappropriately oriented cells was excluded.
Analysis of gonoccocal attachment and internalization
Slices were analysed by light and confocal microscopy. Attachment and incorporation was quantified by counting the total number of gonococci adhered to, and incorporated into, the oviductal epithelium. Three different slices per time point were examined in each case, and the average of the three measurements was used. Examination was done at x1000 under immersion oil using a BX40 Olympus microscope. The presence of bacteria inside the epithelium (internalization) was always confirmed by confocal microscopy using an Axiovert 100 M Zeiss laser scanning microscope.
Hormone assays
A blood sample was obtained on the day of surgery to measure serum oestradiol and progesterone concentrations by radioimmunoassay using the reagents and methodology of the World Health Organization (WHO) Programme for the Provision of Matched Assay Reagents for the RIA of Hormones in Reproductive Physiology (WHO, 1987). The phase of the menstrual cycle at the time of surgery was determined by the serum hormone concentration according to WHO guidelines (1987) and the clinical records.
Statistical analysis
The number of gonoccoci were compared between groups by analysis of variance (ANOVA) and contingency tables were analysed by the Fisher exact test using the statistical package Quick Statistica (Statsoft Inc., Tulsa, OK, USA).
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Results |
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Discussion |
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Receptors to which N.gonorrhoeae binds to enter the cell have been described in cell lines, but it can at this stage be assumed that, if present in the oviductal epithelium, those molecules will have binding functions toward the gonococcus similar to the functions described in cell lines. So, the presence of CD66 and syndecan-1 in the epithelium of all the cyclic controls examined strongly suggests that these molecules are important mediators of the initial binding of the gonococcus in the human oviduct. The control subjects were three in the follicular and three in the luteal phase, and there was no obvious relationship between the expression of these molecules in the oviductal epithelium and the stage of the menstrual cycle. However, further exploration of this point may be necessary since Vß3 integrin has been found to be expressed in this epithelium only during the implantation window (Sülz et al., 1998
).
As previously reported for the endocytic properties of the epithelium in oviductal explants in vitro (Imarai et al., 1998), in this study there was no association between the internalization of N.gonorrhoeae and the phases of the menstrual cycle. This is to be expected because the entry of the gonococcus into the epithelium is regarded as endocytosis, induced by the binding of pili to gonococcal receptors in the infected cells (reviewed by Cohen and Sparling, 1992; Cooper and Moticka, 1996), in addition to several specific outer membrane proteins (Weel et al., 1991
; Cohen and Sparling, 1992
; Gorby et al., 1994
; Naumann et al., 1999
), and a priori it does not appear probable that a decrease or increase might simultaneously occur in all of those receptors during the menstrual cycle that would lead to differences in the rate of attachment of gonococci. In fact, it has previously been reported that the onset of acute salpingitis, chlamydial or gonococcus-related, is not correlated with the menstrual cycle (Sweet et al., 1986
).
The mechanisms underlying the increased risk of salpingitis associated with copper T IUD have not been disclosed, probably because of the lack of appropriate experimental models. The results of the current study suggest that at least one of those mechanisms is increased adhesiveness and invasiveness of the epithelium by N.gonorrhoeae that is in turn related with changes in the epithelial expression of surface molecules. The OpaA gonococcal proteins are recognized by a heparan sulphate proteoglycan that is a member of the syndecan family (Nassif et al., 1999; Naumann et al., 1999
). So, the disappearance of syndecan-1 in the oviductal epithelium of copper T users is not entirely consistent with the increased attachment of Neisseria in those same organs, but it is evidence of the multiplicity of mechanisms involved in the initial interaction of the gonococcus and the epithelium.
Because of its progestin-like effects, Norplant renders the cervical mucus essentially impenetrable by spermatozoa (Sivin, 1993) and probably also by bacteria ascending towards the upper segments of the reproductive tract. This would explain why Norplant is not associated with higher risk of salpingitis or pelvic inflammatory disease (Sivin, 1993
; Fraser et al., 1998
) despite the findings presented here indicating increased attachment and internalization of bacteria into the oviductal epithelium. The changes found in this study in the pattern of expression of gonococcal receptors in Norplant users can explain the increased attachment because CD46, which appeared in the luminal side of their oviductal epithelium, is a pilus receptor for pathogenic Neisseria (Nassif et al., 1999
; Naumann et al., 1999
). The evidence suggests that gonococcal salpingitis is less probable in Norplant users but it might have an aggressive course in the event that gonococci manage to reach the upper segments of the reproductive tract.
Because, in our population, histological evidence of salpingitis is usually found in ~40% of oviducts from asymptomatic cyclic women requesting sterilization, and the copper T IUD is associated with salpingitis, we decided to look at the number of intraepithelial lymphocytes to verify the presence of inflammation. As expected, a lower percentage of intraepithelial lymphocytes were found in the control group than in users of the copper T IUD group. There are previous reports of intraepithelial lymphocytes in the human oviduct (Cardenas et al., 1998), and the data presented here are similar to those reported by VanBogaert et al. (VanBogaert et al., 1978
). One explanation for the presence of intraepithelial lymphocytes in the human oviduct is chemotaxis by Neisseria, as demonstrated in organ culture by Cooper et al. (Cooper et al., 1987
), but the occurrence of sexually transmitted diseases during the last year was an exclusion criterion, and samples were carefully checked and no evidence was found of N.gonorrhoeae in the oviductal samples not incubated with bacteria.
Totally unexpected was the increased lymphocyte infiltration of the oviductal epithelium of Norplant users. Interestingly, CD46, also known as membrane cofactor protein, that appeared in the oviductal epithelium of Norplant users, has been reported to be associated with glomerular infiltration by immune cells in immunoglobulin A nephropathy (Ootaka et al., 1996). The relationship between the increased gonococcal attachment and internalization, the increased CD46 expression and lymphocyte infiltration in this group is intriguing and merits further experimental studies.
In summary, this paper reports several novel aspects of the initial interaction between N.gonorrhoeae and the epithelium of the human oviduct. First, internalization of the gonococcus into the epithelial cells occurs during the first hours of exposure, which is possible because the oviductal epithelium appears to constitutively express at least two known ligands for the gonococcus, CD66 and syndecan-1. Second, this interaction is modified by chronic treatment with copper T IUD and Norplant subdermal implants. Both contraceptive methods increase the attachment and internalization of gonococci but through different modifications of surface molecules known to act as gonococcal receptors. Copper T leads to disappearance of syndecan-1 and Norplant to appearance of CD46, both in the luminal border of the epithelium. This effect of Norplant is associated with epithelial lymphocyte infiltration which is probably related to the expression of ligands that recruit lymphocytes into the epithelium and lumen of the organ.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed at: Universidad de Santiago de Chile, Facultad de Química y Biología,Casilla 40 Correo 33, Estación Central, Santiago, Chile. E-mail: hcardena{at}lauca.usach.cl
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References |
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Submitted on June 2, 2000; accepted on November 24, 2000.