Mol. Hum. Reprod. Advance Access originally published online on October 15, 2004
Molecular Human Reproduction 2004 10(12):861-870; doi:10.1093/molehr/gah117
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Surfactant protein D is present in the human female reproductive tract and inhibits Chlamydia trachomatis infection
Departments of 1Anatomy and Cell Biology and 2Obstetrics and Gynecology, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 and 3Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA
4 To whom correspondence should be addressed at: Department of Anatomy and Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, IA 52242, USA. Email: jeanne-snyder{at}uiowa.edu
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Abstract |
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Surfactant protein D (SP-D) is a lung collectin involved in innate host defence mechanisms in the lung. SP-D is also expressed at other mucosal sites throughout the human body. In the present study, we show that SP-D mRNA and protein are expressed in the human female reproductive tract. SP-D protein was localized in the apical portion of the reproductive epithelial cells. We also demonstrate that endometrial and endocervical cell lines and primary endocervical cells in culture produce SP-D mRNA and protein. Chlamydia trachomatis is an intracellular pathogen that infects the female reproductive tract, primarily the cervix, and is responsible for the most prevalent infectious disease in the USA. Untreated chlamydial infections of the female reproductive tract often result in sterility of the infected woman. Since SP-D protein is produced in cervical glands, we examined the effect of SP-D on chlamydial infection of cervical epithelial cells in vitro. We found that SP-D protein inhibits the infection of HeLa cells (an endocervical epithelial cell line) by C. trachomatis in a dose-dependent manner. We further demonstrate that the SP-D lectin-binding domain is involved in inhibiting infection of HeLa cells by Chlamydia. In conclusion, we detected SP-D in the female reproductive tract and determined that one of the functions of the SP-D protein may be to protect cervical epithelial cells from infection by C. trachomatis.
Key words: human/surfactant protein D/Chlamydia trachomatis/innate host defence/reproductive tract
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Introduction |
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Surfactant protein D (SP-D) is a pulmonary surfactant-associated protein that is involved in innate host defence mechanisms in the lung (Wright, 1997
). SP-D is a member of the collectin family of proteins, i.e. a calcium-dependent lectin with a collagen-like domain (Wright, 1997). SP-D can function as an opsonin by binding to carbohydrates present on bacterial surfaces (Wright, 1997). For example, SP-D enhances the phagocytosis and killing of Pseudomonas aeruginosa and Klebsiella pneumoniae in vitro (Restrepo et al., 1999; Ofek et al., 2001). SP-D enhances the in vivo clearance of influenza A virus, respiratory syncytial virus and Asperigillus fumigatus from the lung (Hickling et al., 1999; LeVine et al., 2001; Madan et al., 2001). SP-D has also been shown to inhibit the uptake of Mycobacterium tuberculosis and Candida albicans by macrophages (van Rozendaal et al., 2000; Ferguson et al., 2002). Recently, it was shown that SP-D inhibits the growth of several strains of Escherichia coli, K. pneumoniae, and Enterobacter aerogenes by increasing membrane permeability (Wu et al., 2003). SP-D has also been shown to modulate the adaptive immune response in the lung (Borron et al., 2002).
SP-D mRNA and protein have been detected at several sites throughout the body, usually at mucosal surfaces, i.e. the salivary gland, the gastrointestinal tract, the prostate, the kidney, and the pancreas (Madsen et al., 2000; Stahlman et al., 2002). SP-D has also been detected by immunostaining in the female reproductive tract including the uterus, the endocervical glands, the ovary, and the placenta (Madsen et al., 2000; Stahlman et al., 2002; Leth-Larsen et al., 2004). However, the function of SP-D in these extra-pulmonary sites remains unclear.
Little is known about innate host defence mechanisms in the female reproductive tract (Cohen, 1999). The lower portion of the reproductive tract, i.e. the vagina and the ectocervix, contributes to antimicrobial host defence (Cohen, 1999). For example, it is known that mucus from the cervical glands contains antimicrobial factors such as lactoferrin, lysozyme, and secretory leukoprotease inhibitor (Cohen, 1999). In addition, the epithelial cells that line the female reproductive tract secrete defensin 5, an antimicrobial peptide that disrupts pathogen membranes (Quayle et al., 1998).
Chlamydia trachomatis is a pathogen that infects the female reproductive tract with high propensity, resulting in the most commonly reported infectious disease in the USA (Washington et al., 1987; Schachter, 1999). Although C. trachomatis infects both women and men, women are infected at a higher rate and these infections result in more serious problems (Harrison et al., 1983). Women infected with Chlamydia can develop pelvic inflammatory disease and scarring of the oviduct, which in turn can lead to infertility or ectopic pregnancies (Harrison et al., 1983). Women who are pregnant while infected with C. trachomatis are at risk of premature delivery and babies born to women infected with Chlamydia can acquire the infection during parturition (Harrison et al., 1983).
Chlamydia trachomatis is an obligate intracellular pathogen. The infectious form of Chlamydia (the elementary body) becomes internalized by the host cell, then avoids the endosomal pathway—the fate of most phagocytosed bacteria—by creating an intracellular inclusion in the host cell (Hackstadt, 1999). The internalized Chlamydia grow and replicate inside the intracellular inclusion (Hackstadt, 1999). Unlike the phagosome/lysosome compartments found in most cells, chlamydial inclusions never acidify (Hackstadt, 1999). About 48 h post-infection, the bacteria complete replication and begin to be released from the host cell and can infect new host cells (Hatch, 1999). By replicating within a host cell, the bacteria evade many immunological defence mechanisms, which makes chlamydial infections difficult to treat (Hackstadt, 1999).
Chlamydia trachomatis elementary bodies use a protein embedded in their outer membrane, called the major outer membrane protein (MOMP), to attach to host cells (Kuo et al., 1996). Interestingly, the MOMP is heavily glycosylated with high mannose carbohydrate modifications (Kosma, 1999). In previous studies, it was shown that another collectin, mannose-binding protein (MBP), binds to carbohydrate residues present on the chlamydial MOMP and can inhibit the infection of HeLa cells in vitro (Swanson et al., 1998). However, MBP is not produced in the female reproductive tract, but is synthesized in the liver and secreted into the blood (Swanson et al., 1998).
In the present study, we demonstrate, utilizing a variety of methods, that SP-D mRNA and protein are present throughout the human female reproductive tract. Because SP-D is involved in the innate host defence mechanisms in the lung, we hypothesized that the SP-D protein in the female reproductive tract might be involved in innate immunity. In this report, we also show that SP-D protein inhibits the infection of cervical epithelial cells by C. trachomatis in vitro. This is the first study to demonstrate that SP-D plays a role in the innate immune system of the female reproductive tract. These findings are exciting because SP-D protein may protect the female genital tract from other bacteria and viruses that infect the female reproductive tract.
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Human reproductive tissue
The experimental protocol for this study was approved by the Human Subjects Review Committee at the University of Iowa, Iowa City, IA. Portions of adult cervical tissues were obtained from the Cooperative Human Tissue Network (Columbus, OH). Some cervical tissues were obtained via biopsies and others were obtained through autopsy 1–8 h after death. The cervical tissue donors were females aged between 42 and 65 years. Sections of paraffin-embedded cervix, oviduct, and endometrium tissues were also obtained from the archives of the Pathology Department at the University of Iowa.
Cell lines
HeLa cells were grown in Eagle's minimal essential medium [American Type Culture Collection, ATCC # CCL-2.1, minimum essential medium (MEM), GIBCO, Grand Island, NY] supplemented with sodium bicarbonate (26 mM), fetal calf serum (10%), non-essential amino acids (10 mM), L-glutamine (200 µM), gentamicin/vancomycin (10 µg/ml), and sodium hydroxide (0.2%). Cells were maintained at 37°C in the presence of 5% CO2 and passed 1:4 approximately every other day (Yee et al., 1985). Hec1b cells (ATCC # HTB-113) were grown in 1:1 Dulbecco's modified Eagle's medium (DMEM)/F-12 media (GIBCO) supplemented with newborn calf serum (10%), penicillin/streptomycin (10 units/ml, 10 mg/ml), gentamicin/vancomycin (both 50 mg/ml), and Fungizone (2.5 mg/ml) (Kuramoto et al., 1972). Hec1b cells were maintained at 37°C in the presence of 5% CO2 and cells were passed 1:10 approximately once a week (Myers and Clements, 2001). KLE cells (ATCC # CRL-1622) were grown in 1:1 DMEM/F-12 media (GIBCO) supplemented with fetal bovine serum (10%), penicillin/streptomycin (10 units/ml, 10 mg/ml), gentamicin/vancomycin (both 50 mg/ml), and Fungizone (2.5 mg/ml) (Hendricks et al., 1997). KLE cells were incubated at 37°C in the presence of 5% CO2 and cells were passed 1:10 approximately once a week (Myers and Clements, 2001).
Endocervical primary cell isolation and maintenance
Biopsy punches (obtained from the Department of Pathology at the University of Iowa) taken from the mucus portion of human cervix tissue were placed epithelial side down on the bottom of a well in a 6-well tissue culture plate as previously described in Edwards et al. (2000). Briefly, once endocervical cells were observed growing from the tissue fragment, the tissue was removed and transplanted to a new well to collect more endocervical cells. The endocervical cells were trypsinized and grown in 60-mm dishes to confluency (Edwards et al., 2000). The epithelial cells express a cytokeratin profile similar to endocervical cells in situ (Edwards et al., 2000).
Human fetal lung explants
The experimental protocol for this portion of the study was approved by the Human Subjects Review Committee at the University of Iowa, Iowa City, IA. Human fetal lung tissue was obtained from mid-trimester abortuses and minced into 1-mm3 pieces. Explants were placed on a grid in a 35-mm culture dish supplemented with serum-free Waymouth's MB medium and allowed to grow for 6 days, as described (Patel et al., 2000). It has been previously established that the epithelial cells in the explants differentiate into alveolar type II cells that produce SP-D (Dulkerian et al., 1996).
Production of recombinant rat SP-D protein
Recombinant rat SP-D protein was stably expressed in CHO-K1 cells as previously described by Crouch et al. (1994). The SP-D dodecamers were isolated by sequential maltosyl-agarose and gel filtration chromatography. The recombinant SP-D protein was stored in a buffer containing EDTA and recalcified before use to a final concentration of 2 mM free calcium. Endotoxin contamination was routinely monitored using a chromogenic assay (Cambrex, East Rutherford, NJ), and the final endotoxin concentrations of the diluted protein were less than approximately 0.7 pg/µg SP-D.
Production of C. trachomatis elementary bodies
Chlamydia trachomatis serovar E was propagated in HeLa cells grown in monolayer culture, and the elementary bodies were isolated and quantified as previously described (Beswick et al., 2003). Briefly, HeLa cell monolayers were treated with DEAE-dextran (30 µg/ml) for 20 min and then infected with C. trachomatis. At 48 h post-infection, the inoculum was removed from the tissue culture flasks and cold HEPES (10 mM)–sucrose (0.2 mM)–calcium (0.15 µM) (HSC) was added along with 3-mm glass beads. The flasks were shaken to dislodge the cells; the cells were then sonicated to release the Chlamydia elementary bodies. The cell sonicate was centrifuged at 482 g for 10 min. The supernatant was removed and then centrifuged for an additional 30 min at 30 877 g. The elementary bodies were then resuspended in the HSC buffer.
Immunostaining for SP-D in human tissue
Paraffin-embedded tissue was obtained from the Pathology Department at the University of Iowa. Each type of tissue, i.e. cervix, oviduct, and endometrium, was obtained from five different individuals. The tissues were deparaffinized and rehydrated serially in 100, 95, 75, and 50% EtOH. The slides were then boiled in 0.1 M sodium citrate, pH 6.0 for 10 min and cooled for 20 min. The slides were rinsed in phophate-buffered saline (PBS) and endogenous peroxidase activity was quenched by incubating in 0.3% H2O2 in methanol for 30 min. Slides were rinsed with PBS and incubated in goat normal serum (1.5%) for 20 min. The slides were then incubated in SP-D polyclonal primary antibody (rabbit anti-mouse, which cross-reacts with human SP-D, Chemicon, Temecula, CA, 1:1000) at 4°C overnight. The slides were rinsed in PBS and incubated in secondary antibody [anti-rabbit immunoglobulin G (IgG) conjugated to biotin, Cappel, Aurora, OH] for 30 min at room temperature. The slides were rinsed and incubated in Vectastain ABC reagent (Vector Labs, Burlingame, CA) for 1 h at room temperature. The slides were rinsed in PBS and incubated in diaminobenzadine (0.7 mg/ml) as substrate. The slides were then rinsed with PBS followed by H2O, counterstained with haematoxylin, dehydrated, and mounted using Permount.
In situ hybridization of SP-D mRNA
Frozen sections of cervical tissues were cut at –20°C and mounted on glass slides (Superfrost Plus, Fischer, Chicago, IL). Sections from several individuals were mounted on slides to facilitate comparison. Sense and antisense [35S]-SP-D cRNA probes (
3 x 105 cpm) were applied to each slide. The methods used for in situ hybridization have been previously described (Wohlford-Lenane and Snyder, 1992). The developed slides were photographed under brightfield and darkfield conditions using a Nikon photomicroscope.
RT–PCR for SP-D mRNA
RNA was extracted from 100-mm plates of confluent HeLa, Hec1b, KLE, or primary endocervical cells, by scraping cell layers into 1 ml of Trizol (Invitrogen, Carlsbad, CA). In some cases, cervical tissue (obtained from the Cooperative Human Tissue Network) was homogenized in Trizol. RNA was extracted using chloroform and precipitated with ice-cold isopropanol. An RT reaction was performed using an SP-D oligomer: TCA GAA CTC GCA GAC CA as a primer and the isolated RNA as a template to produce cDNA for each of the cell lines or tissues. PCR for SP-D was then performed, using the RT reaction as a template, with a forward primer ATG TTG CTT CTC TGA GG and the reverse primer TCA GAA CTC GCA GAC CAC AAG (Lu et al., 1992). The template DNA was initially denatured at 94°C for 3 min followed by 35 cycles with denaturation at 94°C for 30 s, annealing at 62°C for 1 min, and extension at 72°C for 2 min. The amplified 431 bp PCR product was visualized on a 1% agarose gel.
Sequencing of PCR product
The 431 bp fragment, obtained by RT–PCR of RNA extracted from human cervical tissue, was excised and the DNA extracted using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The DNA (2.5 ng/100 bp) was sequenced at the DNA Core Facility at the University of Iowa.
Chlamydia trachomatis infection of HeLa cells
A HeLa cell monolayer (grown in either a 6-well plate or chamber slide) was incubated with C. trachomatis [MOI (multiplicity of infection) of 1 or 10] in the presence or absence of SP-D (1 µg/ml) for 3 h at 37°C. After incubation, the media were removed and fresh media MEM with no antibiotics were added. The cells were then allowed to grow for an additional 48 h. HeLa cells that were grown on chamber slides were immunostained for MOMP (as described below). HeLa cells grown in 6-well plates were harvested for MOMP immunoblot analysis (as described below).
Immunostaining of HeLa cells
HeLa cells were seeded in 2-well chamber slides and were either uninfected or exposed to C. trachomatis (MOI of 1) in the presence or absence of SP-D protein (1 µg/ml). After 48 h of growth, the cells were fixed in ice-cold 50% acetone/50% methanol for 10 min. The cells were then immunostained using the protocol described above with a C. trachomatis MOMP primary antibody (goat anti-MOMP, United States Biological, 1:200) followed by an incubation in secondary antibody (1:10 000, anti-goat IgG conjugated to biotin, Cappel).
Immunostaining quantification
Ten random cell counts were conducted for each condition [chlamydial-infected HeLa cells versus chlamydial-infected HeLa cells in the presence of SP-D (1 µg/ml)] for three independent experiments. Total cell numbers and the number of infected cells were counted for each random field. A percentage of infected cells was calculated for each field and the average of the ten fields was then calculated for each condition. Each condition was counted by three different individuals.
Immunoblot analysis
Media from HeLa cells, primary endocervical cells, and from HeLa cells infected with Chlamydia in the presence or absence of SP-D were removed and lysis buffer [10 mM Tris–HCL, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 50 mM NaF, 5 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 10% glycerol, 1% Triton X-100, 0.5% NP-40, 1 mM phenylmethylsulphonyl fluoride (PMSF)] was added to the cells and incubated for 30 min at room temperature. The lysed cells were scraped off the plate, collected, vortexed, and then centrifuged at 1000 g to pellet cellular debris. The cervical tissue was homogenized in 1 mM PMSF, centrifuged at 1000 g and the supernatant collected. Bradford analysis for protein content was performed on every sample. Equal amounts of supernatant homogenate protein obtained from the cultured cells or cervical tissues was separated by gel electrophoresis on a 10% Tris–HCl polyacrylamide gel. The cervical tissues were electrophoresed in either the presence or absence of ß-mercaptoethanol. The proteins were then electrophoretically transferred to nitrocellulose at 100 V for 1 h and placed in 7% non-fat dry milk diluted 0.1% TNT (0.02 M Tris, 0.15 M NaCl, 0.1% Tween 20) overnight to block non-specific binding. The blots were then incubated with either a rabbit anti-SP-D primary antibody (Chemicon) at a dilution of 1:1000 or MOMP primary antibody (United States Biological) at a dilution of 1:200 for 1 h at room temperature, then washed in 0.1% TNT, three times at 15 min per wash. The blots were incubated with a secondary antibody [anti-rabbit IgG conjugated to horse radish peroxidase (HRP), 1:10 000 (Cappel) or anti-goat IgG conjugated to HRP, 1:10 000 (Cappel)] for 45 min at room temperature, then washed in 0.1% TNT three times at 15 min per wash, and finally incubated with enhanced chemiluminescence solution (Amersham, Buckinghamshire, UK) followed by exposure to X-ray film. Densitometric analysis was performed on the films to determine the relative amount of immunoreactive protein present in each sample.
SP-D binding assay to Chlamydia or HeLa cells
Chlamydial elementary bodies or HeLa cells were sonicated, washed in PBS and resuspended in coating buffer (0.05 M sodium carbonate/bicarbonate buffer, pH 9.6). The chlamydial or HeLa proteins (100 µl, 2 µg/well) were placed on the bottom of a 96-well plate that was centrifuged for 10 min at 350 g. Ethanol (200 µl) was added above the supernatant of each well, this mixture was immediately discarded and methanol (100 µl) was then added. After a 5 min incubation, methanol was discarded and the plates were dried at 37°C, as previously described (Barka et al., 1985). The plate was then blocked in 1% bovine serum albumin (BSA)/calcium–magnesium–barbital (CMB) buffer, pH 7.3 (1.45 sodium chloride, 20 mM barbital, 8 mM magnesium chloride, and 27 mM calcium chloride) for 1 h at room temperature. SP-D protein (4 µg/ml) was diluted in 1% BSA/CMB buffer and incubated on the plate for 2 h at room temperature. In some cases, maltose (100 mM) or EDTA (10 mM) was added to the buffer along with the SP-D protein. The plate was washed three times with CMB buffer and then incubated with rabbit-anti-SP-D protein antibodies (1:10 000, Chemicon) for 1 h at room temperature. The plate was washed three times with CMB buffer and then incubated in secondary anti-rabbit IgG conjugated to HRP (1:10 000, Cappel) for 45 min at room temperature. The plate was then washed seven times with CMB buffer and incubated with tetramethlylbenzidine substrate for 30 min at room temperature in the dark. The reaction was stopped with 2 N H2SO4 and the plate was read at 450 nm on a spectrophotometer plate reader.
Statistics
All data were derived from at least three experiments. The data were analysed by one-way analysis of variance followed by a paired Student's t-test.
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SP-D mRNA is present in human cervical tissue
PCR using SP-D-specific primers (35 cycles) was performed using cDNA from human cervical tissue as a template (Figure 1). A band of 431 bp corresponding to SP-D mRNA was present in the human cervical tissue as well as in the lung (Figure 1). A sample that did not contain template was also run as a negative control. The 431 bp PCR fragment was excised from the gel and the DNA extracted and sequenced. The cervical and lung PCR sequences were identical and in turn identical to the human SP-D cDNA sequence previously reported (data not shown) (Lu et al., 1992
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In situ hybridization for SP-D mRNA was performed on human cervix tissue using sense and antisense SP-D [35S] cRNA probes. The SP-D mRNA-specific autoradiographic grains were localized over epithelial cells that line cervical glands (Figure 2A and B). As a control, cervical tissue was probed with a sense cRNA probe and the darkfield image revealed only background audiogradiographic grains (Figure 2C and D).
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SP-D protein in cervical tissues
SP-D protein was immunolocalized to the apical aspect of the epithelial cells that line cervical glands as well as the surface epithelium that lines the lumen of the cervix (Figure 3A and B). SP-D staining was also present in infected cervical tissue (cervicitis sample, Figure 3C). No specific staining was detected in cervical tissue when the primary antibody was replaced with PBS as a staining control (Figure 3D). SP-D protein was detected in the endometrial lining epithelium as well as in the epithelium of endometrial glands (Figure 4A and B). SP-D protein was also detected in the oviduct where it was localized to the apical aspect of the epithelial cells (Figure 4C). Staining controls were negative (Figure 4D).
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SP-D immunoblot analysis revealed an immunoreactive band of 43 kDa in all cervical samples examined as well as in the lung tissue (human fetal lung explants), which was the positive control for SP-D protein (Figure 5A). Similar amounts of homogenate protein were loaded on the gels; thus, it appears that the SP-D protein content of the cervical samples is much greater than the lung sample. To further confirm that the 43 kDa immunoreactive band is SP-D protein, the cervical proteins were electrophoresed under non-reducing conditions (Figure 5B). As predicted, SP-D monomers (43 kDa), dimers (86 kDa), and trimers (
200 kDa) were detected in the cervical samples (Figure 5B). SP-D trimers usually migrate at a higher apparent molecular weight (200 kDa) due to disulphide bonds crosslinking the collagenous domains together (Figure 5B).
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SP-D mRNA is present in female reproductive system epithelial cell lines
An RT reaction was performed on RNA extracted from HeLa cells (an endocervical cell line), Hec1b, KLE cells (both endometrial cell lines), and primary endocervical cells (Myers and Clements, 2001
; Edwards et al., 2002). SP-D PCR was then performed for the different cell preparations (Figure 6A and B). The SP-D PCR product 431 bp was present in the HeLa, Hec1b, and KLE cell lines as well as in the primary endocervical cells (Figure 6A and B), thus demonstrating the presence of SP-D mRNA in these cells.
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SP-D protein detected in female reproductive system epithelial cell lines
SP-D immunoblot analysis was performed on HeLa and primary endocervical cell homogenates (Figure 7A and B). An immunoreactive SP-D protein band (
43 kDa) was detected in both the HeLa cells and the primary endocervical cells (Figure 7A and B).
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SP-D protein inhibits infection by C. trachomatis
Immunostaining for MOMP was performed in HeLa cells, a cervical epithelial cell line, infected with C. trachomatis (MOI of 1) in the presence or absence of SP-D (Figure 8). There was no staining in uninfected cells (Figure 8A). Many HeLa cells contained C. trachomatis inclusions when infected in the absence of SP-D (Figure 8B, as shown by the arrows). However, when infected in the presence of SP-D, the number of HeLa cells that contained C. trachomatis inclusions was decreased (Figure 8C, arrows). These results were quantified by determining the percentage of cells infected for each condition (Figure 8D). There was a significant decrease in the number of infected cells (cells staining positively for MOMP) in the presence of SP-D protein (Figure 8D).
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We have recently shown that the amount of immunoreactive MOMP present within chlamydial infected cells is directly correlated with the number of infectious chlamydial elementary bodies inside the cells (Oberley et al., 2004
). Thus, for these experiments, immunoreactive MOMP was assayed as a measure of the number of infectious Chlamydia present within infected cells. C. trachomatis MOMP immunoblot analysis was performed on HeLa cells infected with C. trachomatis in the presence or absence of SP-D protein (1 µg/ml, Figure 9A). Uninfected HeLa cells did not contain MOMP (Figure 9A, first and fourth lanes). HeLa cells infected with C. trachomatis (MOI of 10 or 1), in the absence of SP-D, contained MOMP (Figure 9A, second and fifth lanes). HeLa cells infected with C. trachomatis (MOI of 10 or 1), in the presence of SP-D (1 µg/ml), contained less MOMP than their respective controls (HeLa cells infected with Chlamydia alone) (Figure 9A, third and sixth lanes). Densitometric analysis of the three experiments revealed a statistically significant decrease in MOMP levels in HeLa cells infected with bacteria in the presence of SP-D when compared to cells infected with Chlamydia without SP-D (Figure 9B). HeLa cells infected with C. trachomatis (MOI of 10), in the presence of SP-D, had an 25% reduction of MOMP as compared to HeLa cells infected with Chlamydia alone (Figure 9B), while there was an 50% reduction in MOMP present in HeLa cells infected with C. trachomatis (MOI of 1), in the presence of SP-D (Figure 9B). Also, with increasing amounts of SP-D protein (0–4 µg/ml) the amount of C. trachomatis MOMP present in HeLa cells was decreased (Figure 10A). Densitometric analysis revealed a significant, dose-dependent decrease in MOMP levels in HeLa cells infected with bacteria in the presence of increasing amounts of SP-D (1, 2, and 4 µg/ml; Figure 10B). Finally, we determined that SP-D protein binds to chlamydial proteins in a solid phase bioassay (Figure 11). The binding of SP-D to chlamydial proteins was significantly inhibited by either EDTA or maltose (Figure 11). These data are suggestive that the SP-D carbohydrate-binding domain is involved in the binding of SP-D to Chlamydia (Figure 11). Since the Chlamydia elementary bodies were propagated in HeLa cells, we wanted to confirm that SP-D was binding to elementary body components and not to an elementary body contaminate from the host HeLa cells. SP-D protein binding to HeLa cell proteins was significantly less than binding to the elementary bodies (40.0±4.8% of the level of SP-D binding to elementary bodies, P<0.05). Thus, these data are suggestive that SP-D is binding to Chlamydia elementary body components and not to a contaminate from the host cells used to propagate the bacteria.
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In this study, we show that SP-D mRNA and protein are present in epithelial cells of human cervical tissue. SP-D protein was also detected in the epithelial cells of the endometrium and oviduct. SP-D mRNA was detected in HeLa cells (an endocervical cell line) as well as in Hec1b and KLE cells (endometrial cell lines). Primary endocervical cells obtained from three different individuals also contained SP-D mRNA and protein. These findings are consistent with previous reports indicating that SP-D mRNA and protein are present in the female reproductive tract (Madsen et al., 2000
; Stahlman et al., 2002; Leth-Larsen et al., 2004).
After documenting that SP-D protein and mRNA are present in cervical glands, we next wanted to assess the hypothesis that local production of SP-D could contribute to host defence mechanisms in the female reproductive tract. As reported previously, MBP, a member of the collectin family of proteins, binds to the MOMP present in the membrane of C. trachomatis and inhibits the ability of C. trachomatis to bind to and infect HeLa cells (Swanson et al., 1998). However, MBP is not produced locally in the female reproductive tract (Swanson et al., 1998). SP-D has a similar structure to MBP and, as we and others have shown, is present in the female reproductive tract (Wright, 1997; Madsen et al., 2000; Stahlman et al., 2002; Leth-Larsen et al., 2004). Therefore, we investigated the effects of SP-D on the infection of HeLa cells by C. trachomatis. We found that SP-D significantly inhibited chlamydial infection of the HeLa cells in a dose-dependent manner.
SP-D may inhibit infection in the same way MBP inhibits chlamydial infection (Swanson et al., 1998). We showed that SP-D protein binds to C. trachomatis via its carbohydrate-binding domain. We speculate that SP-D protein may bind to carbohydrates associated with the chlamydial MOMP and that this binding physically blocks C. trachomatis from interacting with surface receptors present in HeLa cells, thus, inhibiting HeLa cell infection by C. trachomatis. SP-D (at 1 µg/ml) blocks chlamydial infection more effectively at a lower (MOI of 1) than at a higher bacterial concentration (MOI of 10). At an MOI of 1, the SP-D may bind proportionally more bacteria and, thus, inhibit more bacteria from infecting the HeLa cells than at a higher multiplicity of infection. The mannose-binding protein inhibited infection of HeLa cells by C. trachomatis (MOI 1) by 20% at 6.25 µg/ml, by 53% at 25 µg/ml, and at the highest concentration tested, 100 µg/ml, the MBP inhibited infection by 76% (Swanson et al., 1998). Thus, in the present study, SP-D significantly inhibited infection of HeLa cells by Chlamydia at much lower concentrations than those described for mannose-binding protein.
Recently, Leth-Larsen et al. (2004) have reported that SP-D protein is present in the uterus, in the placenta, and in the amniotic fluid; however, the function of the SP-D protein in these sites was not explored. From our studies, it appears that SP-D may play a critical role in inhibiting infection of the female genital tract by bacteria. Thus, we speculate that the SP-D present in the placenta and amniotic fluid may also prevent infection of these tissues and the developing embryo.
Since SP-D protein has been shown to protect the lung from a variety of pathogens, i.e. yeast, fungus, viruses, and bacteria (Wright, 1997), we speculate that SP-D may protect the female reproductive tract from a wide variety of infections in addition to Chlamydia. Our findings may prove to be relevant in devising new methods for treating and preventing sexually transmitted diseases.
In conclusion, we have shown that SP-D, a lung collectin, is present in the human female reproductive tract. In particular, SP-D protein appears to be made in the cervical glands in relatively high amounts. Since the cervix is a target for C. trachomatis infection, we then investigated the effects of SP-D on chlamydial infections of the reproductive tract epithelial cells. We showed that SP-D blocks the infection of HeLa cells by C. trachomatis in a dose-dependent manner, probably via binding of its lectin domain. Thus, we have described a function of SP-D protein in the female reproductive tract, one that may lead to novel approaches to prevent chlamydial infections.
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Acknowledgements |
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This research was funded by the National Institutes of Health (NIH), R01 grant HL-50050 (J.S.), a NIH Training Grant Award # 2 T32 HL07638-16 (R.O.), the Diabetes and Endocrine Research Center, DK-25295, and an American Heart Association Predoctoral Fellowship 0215282Z (R.O.). Some experiments were further supported by NIH R01 grants HL-29594 and HL-44015 (E.C.). The authors would like to thank Paul Reimann and Dennis Dunnwald for their help in preparing the photomicrographs for this manuscript. The authors would also like to thank the Cooperative Human Tissue Network for the human tissue used to conduct experiments presented in this manuscript.
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Submitted on August 13, 2004; resubmitted on September 13, 2004; accepted on September 19, 2004.
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