Editor: Willem van Eden
Primary and secondary immune responses of mucosal and peripheral lymphocytes during Chlamydia trachomatis infection
Article first published online: 25 JAN 2007
FEMS Immunology & Medical Microbiology
Volume 49, Issue 2, pages 280–287, March 2007
How to Cite
Vats, V., Agrawal, T., Salhan, S. and Mittal, A. (2007), Primary and secondary immune responses of mucosal and peripheral lymphocytes during Chlamydia trachomatis infection. FEMS Immunology & Medical Microbiology, 49: 280–287. doi: 10.1111/j.1574-695X.2006.00196.x
- Issue published online: 25 JAN 2007
- Article first published online: 25 JAN 2007
- Received 22 August 2006; revised 11 October 2006; accepted 6 November 2006.First published online 25 January 2007.
- proliferative response;
- MOMP antigen;
- Chlamydia trachomatis
Chlamydia trachomatis infection is followed by the development of antigen-specific cell-mediated immunity, which is detectable as a positive lymphocyte proliferation response to the chlamydial major outer membrane protein (MOMP) antigen. To date, however, there have been no studies on the mucosal immune responses to chlamydial antigens. This study aimed to study the primary and secondary immune responses of cervical lymphocytes in response to the chlamydial antigen. Median proliferative responses were found to be significantly (P<0.05) higher in patients with chlamydial infections than in controls. The chlamydial MOMP induced significantly higher IL-6 and IL-10 and lower interferon-gamma (IFN-γ) secretion in cervical lymphocytes of Chlamydia-positive women, resulting in a T helper 2 response. On stimulation of peripheral blood mononuclear cells (PBMC) obtained from Chlamydia-positive women with the chlamydial antigen, the median levels of IL-10, IL-12 and IFN-γ were higher than in controls, but the differences were not significant. Our study suggests that the mucosal immune responses towards Chlamydia trachomatis are different from those of PBMCs and are more helpful in understanding the cytokine responses in the female genital tract during chlamydial infection.
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects the epithelial cells of the genital tract and conjunctivae and can cause diseases that range from salpingitis, conjunctivitis, pelvic inflammatory disease and blinding trachoma to the systemic infection Lymphogranuloma venereum (Grayston & Wang, 1975). A high prevalence of genital C. trachomatis infection has been reported in India (Mittal et al., 1996).
Epithelial cells are the main targets during chlamydial infection, and the receptors through which chlamydiae enter these cells are largely unknown; however, in the case of the L2 serovar, entry is initiated by the attachment of bacteria to heparan sulfates on the cell surface (Zhang & Stephens, 1992). After initial infection with a serovar, most reinfection is caused by a different serovar, suggesting that immunity to C. trachomatis is serovar-specific (Brunham et al., 1996). This is particularly apparent for antibody responses, which predominantly target the major outer membrane protein (MOMP) and are thus serovar-specific (Kinnunen et al., 2001).
Experimental studies, both in rodents and primates, provide strong evidence that cell-mediated immune (CMI) responses play a major role in the clearance and resolution of chlamydial infection. The recruitment of inflammatory cells, as macrophages, to the site of infection and the subsequent release of proinflammatory cytokines appear to be crucial for innate resistance to Chlamydia. It was found in a murine model that Chlamydia-specific T cell lines and clones can transfer protection, which is mediated by the production of interferon gamma (IFN-γ) (Igietseme et al., 1994; Starnbach et al., 1994). T cells of both the CD4+ and CD8+ subsets can adoptively transfer protection (Williams et al., 1984; Buzoni-Gatel et al., 1992). Chlamydial infection generates and produces a variety of cytokine responses, both by direct infection of the epithelial cells lining the mucosal surfaces of the body and by interaction with cells of the immune system. T helper 1 (TH1) cytokines play a major role in polarizing the immune response to Chlamydia spp. towards a protective TH1 type (Johnson, 2004). In contrast, cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-6 and IL-10 might be involved in the pathology associated with infection with Chlamydia spp. (Darville et al., 2003).
Genital infection with C. trachomatis and the resulting cytokine environment together with the route of antigen presentation largely determine the outcome of infection and disease (Igietseme et al., 1998; Morisset et al., 2006). Ultimately, the outcome of genital chlamydial infection depends on the species, as well as on the immunological responses of the host to infection and the balance between the pathogen-specific TH1 and TH2 cell responses (Kelly et al. (1996). While the critical role of cytokines and lymphocyte subset recruitment in infection has been reported in various animal models, there is little information available regarding humans. Most of the studies to date have demonstrated the proliferative responses and cytokine production in PBMCs stimulated with chlamydial antigens (Witkin et al., 1994; Openshaw et al., 1995); however, no study has been undertaken on the mucosal immune responses to chlamydial antigens.
The aim of this study was to examine the primary and secondary immune responses of cervical lymphocytes in response to the chlamydial antigen. We studied the immune responses in terms of lymphoproliferation and TH1 and TH2 cytokine production in cervical lymphocytes and PBMCs obtained from Chlamydia-infected women for the secondary response, and in controls for the primary response in order to gain a better understanding of the mucosal immune responses in humans during chlamydial infections.
Materials and methods
After obtaining informed written consent, 185 symptomatic patients of reproductive age attending the Gynecology Out-Patient Department of Safdarjung Hospital, New Delhi, India who had signs and symptoms of cervicitis were enrolled for the study. All women underwent a pelvic examination for the presence of mucopurulent discharge and friability of the cervix. Sixty-four healthy age-matched controls attending the family-planning department for birth-control measures and with no previous history of any sexually transmitted disease (STD) were also enrolled. Patients with a positive urine pregnancy test or recent antibiotic therapy were excluded from the study. The study received approval from the hospital's ethics review committee.
Collection of samples
The vulva was examined for lesions and the cervix was examined for warts, ulcers, ectopy, erythema and discharge, if any. After the endocervix had been cleaned with a cotton swab (HiMedia, Mumbai, India), endocervical swabs were collected from patients and controls for diagnosis of C. trachomatis and other STD pathogens. Samples were obtained using sterile cotton-tipped swabs for the screening of C. trachomatis. Sterile cotton swabs were collected in sterile vials containing 1.0 mL of phosphate-buffered saline (PBS). An additional endocervical or vaginal cotton swab was collected for making smears on slides for the screening of STD pathogens, namely Candida spp. and Trichomonas vaginalis.
The cervical canal was wiped clean, and a cytobrush was placed within the endocervical canal so that cells from the endocervical region and the zone between the endocervical and ectocervical regions (transformation zone) could be obtained. The cytobrush was then held in a sterile centrifuge tube of sterile PBS (pH 7.2) supplemented with 100 U penicillin mL−1, 100 μg streptomycin mL−1, and 100 μg glutamine mL−1. All cytobrush samples had negative results for blood contamination. Ten milliliters of venous blood was collected into heparinized vials for the isolation and culture of lymphocytes.
Spots were made on clean glass slides using endocervical swabs and were stained with flourescein isothiocyanate-conjugated monoclonal antibodies to C. trachomatis major outer membrane protein using a C. trachomatis direct specimen test kit (Microtrak, Syva Corporation, Palo Alto, CA) according to the manufacturer's instructions. A sample was considered positive if at least 10 elementary bodies (EBs) were detected. Samples with fewer than 10 EBs were confirmed for positivity by PCR analysis using a primer specific for the 517-bp plasmid of C. trachomatis.
Gram-stained cervical smears were examined for the presence of yeast cells (candidiasis) and vaginal smears for clue cells, for diagnosis of bacterial vaginosis. Gram stains showing a predominance of the Lactobacillus morphotype were interpreted as normal. Those showing the Gardenella morphotype or mixed flora were interpreted as consistent with bacterial vaginosis. Wet-mount microscopy was performed for diagnosis of Trichomonas vaginalis. For detection of Neisseria gonorrhoeae, cervical specimens were incubated at 35°C in a humidified CO2 incubator for 48 h on Thayer Martin medium. Colony growth was noted and N. gonorrhoeae was identified on the basis of gram-stained smears. Pleuropneumonia-like organism (PPLO) broth was used for the identification of Mycobacterium hominis and Ureaplasma urealyticum by diluting the cervical samples in arginine-containing and urea-containing liquid media, respectively, and thereafter, incubating the media at 37°C until there was change in colour after 5–7 days for M. hominis or 2 days for U. urealyticum.
Sera from patients and controls were assayed for IgG antibodies to C. trachomatis surface components using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Ridascreen, R-Biopharm AG, Germany) according to the manufacturer's instructions. Results were expressed as mean absorbance at 450 nm of duplicated samples. An OD>1.1 was considered as positive.
Quantification of T-cell subsets was by means of standard flow cytometric technology. Cervical cells were stained with fluorescin isothiocyanate (FITC)-conjugated anti-CD4 and anti-CD8 antibodies (Becton Dickinson, San Jose) for 25 min. Preparations were washed with stain buffer containing 0.1% NaN3 and 2% fetal bovine serum (FBS), and acquired using a fluorescent activated cell sorter (FACS) Calibur (BD Biosciences, San Jose). A total of 10 000 events were acquired. Appropriate isotype-matched control antibodies were also used to rule out nonspecific fluorescence.
Cervical specimens were vortexed before the removal of cytobrush. They were filtered through a 70-mm nylon cell strainer (Becton Dickinson) and centrifuged at 200 g for 10 min, the resulting pellet yielding endocervical lymphocytes. The lymphocytes were washed three times with Hank's balanced salt solution (Sigma, St Louis, MO) and suspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma) supplemented with 10% heat-inactivated human AB serum for lymphoproliferation assay. Briefly, endocervical lymphocytes were cultured in triplicate in round-bottomed 96-well plates (5 × 104 cells well−1) with or without antigen in a total volume of 200 μL. Cultures were incubated in humidified 5% CO2 at 37°C for 6 days.
Synthetic biotinylated peptides corresponding to epitopes of the MOMP antigen of C. trachomatis (VLGTSMAEFISTNVIS) (Techno concept, India) were used as antigens at a protein concentration of 2 μg mL−1. Phytohaematogluttinin (Sigma, 1 μg mL−1) was used as a positive-control mitogen in each experiment. Optimum concentrations of antigens and mitogen were determined in preliminary experiments as minimum concentrations giving maximum proliferations. Tritiated thymidine [3H] (Bhabha Atomic Research Centre, Mumbai, India) was added to the cultures for final 18 h of incubation. Proliferative responses were measured as counts per minute (cpm) of incorporated radioactivity using a liquid scintillation counter (Packard Biosciences, Downers Grove). Results were expressed as stimulation indices (SI=mean cpm in the presence of antigen divided by the mean cpm in its absence). A SI of >2 was considered positive.
Cytokine production assay in supernatants of stimulated lymphocytes
PBMCs were prepared by ficoll-Hypaque density gradient centrifugation. The PBMCs were washed three times with Hank's balanced salt solution (Sigma) and suspended in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated human AB serum for cytokine production. Briefly, endocervical lymphocytes and PBMCs were cultured in triplicate in round-bottomed 96-well plates (5 × 104 cells well−1) with or without antigen in a total volume of 200 μL. Cultures were incubated in humidified 5% CO2 at 37°C for 2 days. The cultures were stimulated with synthetic biotinylated peptides corresponding to MOMP antigen or 1 μg mL−1 phytohemagglutinin (PHA). The cell-free supernatant was collected from each well and stored at −80°C before being evaluated for various cytokines.
Measurement of secreted cytokines
Concentrations of IL-2, IL-4, IL-6, IL-10 and IFN-γ cytokines in serum and cervical washes were detected simultaneously using a human Th1/Th2 cytokine cytometric bead array (CBA) kit II (BD PharMingen, San Diego, CA). Briefly, 50 μL of each sample was mixed with 50 μL of mixed capture beads and incubated, and then washed before adding 50 μL of phycoerythrin (PE)-conjugated anti-human IL-2, IL-4, IL-6, IL-10 and IFN-γ. The samples were incubated at room temperature for 3 h in the dark. After incubation with the PE detection reagent, samples were washed once, and resuspended in 300 μL of wash buffer before acquisition on the FACSCalibur (BD Biosciences, Sunnyvale, CA). Data were analysed using cba software (BD PharMingen). Log–log standard curves were generated for each cytokine using the mixed cytokine standard provided by the kit. The concentration for each cytokine in cell supernatants was determined by interpolation from the corresponding standard curve.
Quantification of IL-8 and IL-12 was performed with ELISA, in accordance with the manufacturer's instructions. Briefly, 96-well ELISA plates were coated overnight with anti-human IL-8 and IL-12 capture antibodies (eBiosciences, SanDiego, CA). Unbound coating antibody was removed and nonspecific protein binding sites were blocked with assay diluent according to the manufacturer's instructions. Duplicate serial dilutions of test samples or controls were incubated for 2 h at room temperature. Detector antibodies to IL-8 and IL-12 were added and incubated for 1 h, followed by incubation with peroxidase-conjugated anti-mouse IgG (eBiosciences) for 30 min. For colour development tetramethylene benzidine was added, and then stopped after 15 min by the addition of 1 N sulfuric acid. Absorbance was read at 450 nm. Log-log standard curves were generated and unknowns were interpolated.
The Mann–Whitney U test was used for comparing any two groups. Categorical variables were compared using the χ2 test.
Chlamydia trachomatis infection was diagnosed by direct fluorescent assay (DFA) and PCR in 79 patients. Of these, 8 women were found to be infected with Candida spp., bacteria vaginosis, T. vaginalis, M. hominis, U. urealyticum or N. gonorrhoeae and were excluded from the study. Four Chlamydia-positive patients and two controls were excluded, as the lymphocyte population in the cervical cells was not homogenous. All healthy controls tested negative for a current C. trachomatis infection. Six healthy controls, who were positive for C. trachomatis IgG antibodies with no apparent chlamydial infection, were excluded from the study. The median ages of Chlamydia-positive women and controls were comparable (29 and 30 years, respectively).
T lymphocyte subsets in the cervix of women
Following microscopic examination of cervical cells, only those patients who had homogenous population of lymphocytes in the cervical cells were included in the study. The range of CD4 and CD8 lymphocytes in patients who were included in the study was between 59% and 86%.
Lymphocyte Proliferative response by cervical lymphocytes and PBMCs
The proliferative response of cervical lymphocytes in response to MOMP antigen was studied in Chlamydia-positive women and controls. The median SI was significantly (P<0.05) higher in patients with a chlamydial infection (SI=2.15, range 0.62−5.55) than in controls (SI=1.23, range 0.24−3.13) (Fig. 1). Proliferative responses of PBMCs to chlamydial MOMP antigen were higher in Chlamydia-positive women than in controls but the difference was not significant (SI=1.57; range 0.32–5.62; controls SI=1.08; range 0.34–2.74) (Fig. 1). The proliferative response of cervical lymphocytes in Chlamydia-positive women was significantly higher (P<0.05) than the proliferative responses of PBMCs in these patients.
Cytokine production by stimulated cervical lymphocytes and PBMCs
The levels of various cytokines were quantified in the culture supernatant of stimulated cervical lymphocytes and PBMCs obtained from Chlamydia-positive women and controls. On stimulation of cervical lymphocytes no significant difference was observed in the levels of IL-2, IL-8 and IL-12 in supernatants of cells obtained from Chlamydia-positive women as compared with controls (IL-2: 58.1 vs. 64.9 pg mL−1; IL-8: 195.6 vs. 218.0 pg mL−1; IL-12: 83.7 vs. 72.9 pg mL−1, respectively). The median levels of IL-6 and IL-10 were significantly (P<0.05) higher and that of IFN-γ was significantly lower in the case of Chlamydia-positive women as compared with controls (IL-6: 174.3 vs. 72.5 pg mL−1; IL-10: 247.6 vs. 126.9 pg mL−1; IFN-γ: 43.4 vs. 135.8 pg mL−1, respectively) (Fig. 2). On stimulation of PBMCs no significant difference was observed in the median levels of IL-2, IL-6, IL-8, IL-10, IL-12 and IFN-γ in supernatants obtained from Chlamydia-positive women as compared with controls (IL-2: 57.3 vs. 65.9 pg mL−1; IL-6: 281.4 vs. 238.7 pg mL−1; IL-8: 274.7 vs. 194.9 pg mL−1; IL-10: 231.5 vs. 211.7 pg mL−1; IL-12: 101.6 vs. 82.5 pg mL−1; IFN-γ: 75.2 vs. 63.9 pg mL−1, respectively) (Fig. 2). IL-4 was below the detection limit in all of the culture supernatants.
In healthy controls, median levels of IL-6 and IL-10 were significantly higher and IFN-γ levels were significantly lower in supernatants of stimulated PBMCs as compared with cervical lymphocytes. In the case of Chlamydia-positive women, median levels of IL-6 and IL-8 were significantly higher in PBMCs compared with cervical lymphocytes.
The immune response to genital chlamydial infection is very complex; it clears infection and confers short-term protection but at the same time sensitizes the host for the development of immunopathological changes (Lehtinen & Paavonen, 1994; Rietmeijer et al., 2002). In women, chlamydial infections are often asymptomatic, and subsequent reinfections lead to inflammatory responses with pathological sequelae (Bailey et al., 1995). We enrolled healthy controls and Chlamydia-positive women, as the cells obtained from controls act as naïve cells mimicking the primary immune response, and cells from Chlamydia-positive women mimic the secondary immune response as they have already been exposed to the pathogen.
There is evidence to implicate the MOMP antigen as the critical one responsible for stimulating immune-mediated inflammation. Lymphocyte proliferation to chlamydial antigens, including MOMP and HSP 60, is enhanced in individuals who spontaneously resolve trachoma infection as compared with those with persistent infections (Holland et al., 1993). Chlamydial infection generates and produces a variety of cytokine responses, and previous reports have demonstrated a relative depression in lymphocyte proliferation in response to C. trachomatis antigens in subjects with persistent clinical signs of inflammatory trachoma and in subjects with severe trachomatous scarring (Holland et al., 1996). Our results suggest that exposure to chlamydial infection could significantly affect mucosal immune function by modifying the release of cytokines.
As an in vitro model of C. trachomatis infection, our study showed variation in the production of various cytokines by cervical cells and PBMCs. Because all studies to date have been performed with PBMCs, we also looked for differences in cytokine production between cervical lymphocytes and PBMCs. Cervical cells are the actual cells encountering the pathogen, and knowledge of their responses would help in gaining a much better understanding of the immunopathogenesis of chlamydial disease. IFN-γ induction was significantly down-regulated in stimulated cervical cells obtained from Chlamydia-positive women when compared with controls. When cytokine levels in cells obtained from controls were compared with those of PBMCs it was found that IFN-γ secretion was significantly higher in cervical cells on primary induction. Previous studies on scarring trachoma have shown down-regulation of TH1 activity and IFN-γ response (Hernandez-Pando & Rook, 1994). IL-12, which is derived from dendritic cells and monocytes, induces TH1 differentiation and induction of IFN-γ (Tripp et al., 1993). Studies have revealed that IL-12 along with TNF-α is a costimulator of IFN-γ production by natural killer cells (Williams et al., 1998). Our study demonstrated that levels of IL-12 were higher in cells obtained from Chlamydia-infected women, but not significantly. These results suggest that the decrease in levels of IFN-γ during the secondary response is not the result of a decrease in IL-12 levels but of some other mechanism. Our study also showed a significantly higher production of IL-6 by cervical cells from Chlamydia-positive women as compared with controls. IL-6 was also higher in PBMCs of Chlamydia-positive women, but the difference was not significant. Although not as protective as IFN-γ, the proinflammatory cytokine IL-6, generated either by epithelial cells or by the interaction of chlamydiae with T lymphocytes of the cell-mediated immune system, is probably important, together with IL-12, for sustaining the protective TH1 cell-mediated immune response (Yu et al., 2003). The increase in levels of IL-6 along with the decrease in IFN-γ levels may suggest that during a secondary immune response the protective role of IFN-γ is taken over by IL-6.
The significant differences between IL-6 and IL-8 levels produced by cervical lymphocytes and PBMCs from Chlamydia-positive women confirms that the response of cervical lymphocytes is much more important than that of PBMCs in understanding chlamydial infection. The level of the TH2 cytokine IL-10 was found to be up-regulated in both cervical cells and PBMCs stimulated with chlamydial MOMP antigen in Chlamydia-positive women. IL-10 has been found to be associated with susceptibility to chlamydial infection and typical pathological changes caused by the infection such as granuloma formation and fibrosis (Conti et al., 2003). IL-10 is not always an inflammatory/inhibitory cytokine; rather, high levels of IL-10 probably prevent the pathological effect of inflammatory cytokines such as IL-1β, IFN-γ and TNF-α (Mosmann & Moore, 1991). Our results are in concurrence with other studies and suggest a higher frequency of IL-10 production by T cells and possibly reflect their capacity to reduce the TH1 response and the effect of IFN-γ that is needed to eradicate C. trachomatis infection.
Our study revealed that cervical cells proliferate more rapidly than PBMCs upon secondary infection and mucosal immune responses of cervical lymphocytes are different from those of PBMCs. Our study also revealed that the production pattern of cytokine cervical cells is closer to the in vitro studies performed for understanding cytokine responses during chlamydial infection, and further research should be carried out with cervical lymphocytes rather than with PBMCs. This study has some limitations. First, we could not enroll asymptomatic Chlamydia-positive women, because in countries like India concern for health care is low and women do not come for regular checkups−they only attend the gynecology out-patient department on the appearance of symptoms. There is a need to study asymptomatic women, as this would give a much clearer picture of the immune mechanisms involved in chlamydial infection. Secondly, only one antigen was used to study the immune responses, and previous studies have indicated that the peptide used can be recognized only by certain women, depending on their class II major histocompatibility complex status (Cohen et al., 2000). Specific class II D alleles may exhibit increased risk of infection and disease, and specific HLA types may be linked to increased IL-10 production (Cohen et al., 2005). Keeping the above limitations in mind, more studies are needed to investigate the role of cytokines and their significance in the development of an adverse outcome of the infection.
We thank Mrs Madhu Badhwar, Mrs Asha Rani, Mrs Rosamma Thomas and Yogendra Singh for providing technical assistance. This study was supported by an Indo-US grant (BT/IN/USCRHR/AM/2002) from the Department of Biotechnology, Government of India. The University Grants Commission is acknowledged for providing assistance to T.A. in the form of a fellowship.
- 1995) Subjects recovering from human ocular chlamydial infection have enhance lymphoproliferative responses to chlamydial antigens compared with those of persistently diseased controls. Infect Immunol 63: 389–392. , , & (
- 1996) The epidemiology of Chlamydia trachomatis within a sexually transmitted disease core group. J Infect Dis 173: 950–956. , , , , , , , , & (
- 1992) Protection against Chlamydia psittaci in mice conferred by Lyt-2 T cells. Immunology 77: 284–288. , , , , & (
- 2000) Human leukocyte antigen class II DQ alleles associated with Chlamydia trachomatis tubal infertility. Obstet Gynecol 95: 72–77. , , , , & (
- 2005) Immunoepidemiological profile of Chlamydia trachomatis infection: importance of heat shock protein 60 and interferon-gamma. J Infect Dis 192: 591–599. , , , , , , , , & (
- 2003) IL-10, an inflammatory/inhibitory cytokine but not always. Immunology Lett 86: 123–129. , , , , , , , , & (
- 2003) Toll like receptor-2, but not Toll like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection. J Immunol 171: 6187–6197. , , , , & (
- 1975) New knowledge of Chlamydiae and the disease they cause. J Infect Dis 132: 87–105. & (
- 1994) The role of TNF-alpha in T-cell-mediated inflammation depends on the Th1/Th2 cytokine balance. Immunology 82: 591–595. & (
- 1993) Conjunctival scarring in trachoma is associated with depressed cell mediated immune responses to Chlamydia antigens. J Infect Dis 168: 1528–1531. , , , & (
- 1996) T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring. Clin Exp Immunol 105: 429–435. , , , , , , , , , & (
- 1994) Role for CD8+ T cells in antichlamydial immunity defined by Chlamydia-specific T-lymphocyte clones. Infect Immunol 62: 5195–5197. , , & (
- 1998) Route of infection that induces a high intensity of gamma interferon-secreting T cells in the genital tract produces optimal protection against Chlamydia trachomatis infection in mice. Infect Immun 66: 4030–4035. , , , , , , & (
- 2004) Murine oviduct epithelial cell cytokine responses to Chlamydia muridarum infection include interleukin-12-p70 secretion. Infect Immunol 72: 3951–3960. (
- 1996) Initial route of antigen administration alters the T cell cytokine profile produced in response to the mouse pneumonitis biovar of Chlamydia trachomatis following genital infection. Infect Immunol 64: 4976–4983. , & (
- 2001) Heat shock protein 60 specific T cell response in chlamydial infection. Scand J Immunol 54: 76–81. , & (
- 1994) Heat shock proteins in the immunopathogenesis of chlamydial pelvic inflammatory disease. Proceedings of the Seventh International Symposium on Human Chlamydial Infections (OrfilaJ, ed) pp. 599–610. Editrice Esculapio, Bologna. & (
- 1996) Host immune response in Chlamydial cervicitis. Br J Biomed Scij 53: 941–947. , & (
- 2006) Sustain interleukin-6 and interleukin-8 expression following infection with Chlamydia trachomatis serovar L2 in a HeLa/THP-1 cell co-culture model. Scand J Immunol 63: 199–207. , & (
- 1991) The role of IL-10 in cross-regulation of Th1 and Th2 responses. Immunol Today 12: A49–A53. & (
- 1995) Heterogeneity of intracellular cytokine synthesis at the single cell level in polarized T helper-1 and T helpher-2 populations. J Exp Med 182: 1357–1367. , & (
- 2002) Incidence and increased infection rates of Chlamydia trachomatis among male and female patients in an STD clinic: implications for screening and rescreening. Sex Trans Dis 25: 65–72. , , & (
- 1994) Protective cytotoxic T lymphocyte are induced during murine infection with Chlamydia trachomatis. J Immunol 153: 5183–5189. , & (
- 1993) Interleukin 12 and tumor necrosis factor α are costimulators of interferon γ production by natural killer cells in severe combined immunodeficiency mice with listeriosis and interlukin-10 is a physiologic antagonist. Proc Natl Acad Sci USA 90: 3725–3729. , & (
- 1984) Cellular immunity to the mouse pneumnitis agent. J Infect Dis 149: 630–639. , , & (
- 1998) A role for interlukin-6 in host defense against murine Chlamydia trachomatis infection. Infect Immun 66: 4564–4567. , , , & (
- 1994) Proliferative response to conserved epitopes of the Chlamydia trachomatis and human 60-kilodalton heat-shock proteins by lymphocyte from women with salpingitis. Am J Obstet Gynecol 171: 455–460. , , & (
- 2003) HeLa cells secrete interleukin-8 and interlukin-10 response to Chlamydia trachomatis entry. Hunan Yi Ke Da Xue Xue Bao 2: 174–176. , & (
- 1992) Mechanism of Chlamydia trachomatis attachment to eukaryotic host cells. Cell 69: 861–869. & (