Characterization of Toll-like receptors in the female reproductive tract in humans

doi:10.1093/humrep/deh775 Human Reproduction Vol.20, No.5 pp. 1372–1378, 2005 Advance Access publication February 3, 2005 Characterization of Toll-like receptors in the female reproductive tract in humans A.Fazeli1, C.Bruce and D.O.Anumba Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK To whom correspondence should be addressed.E-mail: BACKGROUND: Rapid innate immune defences against infection involve the recognition of invading pathogens by specific pattern recognition receptors recently attributed to the family of Toll-like receptors (TLR). Little is known about the in vivo protein expression or distribution of TLR in the female reproductive tract in humans. It is likely that TLR distribution in the female reproductive tract reflects the immunological tolerance to the commensal organisms in lower parts of the tract (vagina, ectocervix and, partially, endocervix) and the intolerance to commensal microbial flora in the upper tract (the uterus and uterine tubes). METHODS: Using immunohistochemistry techniques, distribution of TLR1 –6 was studied in surgical sections from the vagina, ecto- and endocervix, endometrium and uterine tubes, obtained from patients undergoing abdominal hysterectomy for benign gynaecological conditions. RESULTS: TLR1, 2, 3, 5 and 6 were present in the epithelia of different regions of female reproductive tract. However, TLR4 was only present in the endocervix, endometrium and uterine tubes and absent in vagina and ectocervix. In addition, a secretory form of TLR4 seems to be produced by the endocervical glands. CONCLUSION: TLR4 may play an important role in modulation of immunological tolerance in the lower parts of the female reproductive tract, and in host defence against ascending infection. Key words: female reproductive tract/innate immunity/Toll-like receptors Introduction In health the vagina is colonized by several hundred bacterial commensal species which have a protective function against infection by pathogenic organisms. Organisms such as the Lactobacillus species serve to maintain vaginal acidity by glycogen cleavage in epithelial cells to release lactic acid (Kasprowicz and Bialecka, 1993). Vaginal acidity appears to play a key role in determining the vaginal microbial flora and preventing ascending infection by pathogenic organisms that thrive under more alkaline conditions (Hillier, 1999). The mucosal surface of the reproductive tract provides a physical barrier against infection, and has adapted to a dynamic nonsterile environment challenged by several antigenic and inflammatory stimuli associated with sexual intercourse and the endogenous vaginal microbial flora. In contrast to the lower parts of the female reproductive tract (vagina, ectocervix and to some extent endocervix), the upper parts of the female reproductive tract, including the uterine cavity and uterine tubes, are virtually free of organisms, with little by way of commensal microbial activity (Heinonen et al., 1985). The mechanisms which account for this contrasting distribution of organisms between the upper and lower parts of the female reproductive tract are not known. It is likely that the divergent immunological tolerance of the tract towards microorganisms is modulated by an alert innate immune system in the upper regions of the female reproductive tract, and a tolerant system in the lower parts of the tract. Rapid innate immune defences against infection usually involve the recognition of invading pathogens by specific pattern recognition receptors recently attributed to the family of Toll-like receptors (TLR). Originally identified in the early Drosophila larvae (Stein et al., 1991), 10 structurally related mammalian receptor proteins have now been identified (Akira, 2003). Consistent with their role in pathogen recognition, TLR family members are expressed by cells involved in the first line of host defence, including neutrophils, macrophages, dendritic cells, dermal endothelial cells and mucosal epithelial cells. Collectively, TLR function to alert the immune system to the presence of microorganisms. The different members of the TLR family are expressed on different cell organelles and appear to mediate signal transduction to different antigenic stimuli by engaging with specific ligands leading to the production of various proinflammatory cytokines, chemokines and effector molecules, depending on the cell type that is activated (Hirschfeld et al., 2001; Jones et al., 2001). The role and signal transduction mechanisms for the various members of the Toll receptor family are increasingly becoming recognized. Although they have been shown to 1372 q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: 1 Toll-like receptors in female reproductive tract Materials and methods Patients and samples Tissue from nine (eight Caucasians and one African) patients undergoing hysterectomy for benign gynaecological conditions were studied. The median age (range) of the women was 45 (33– 56) years. Two of the subjects were post-menopausal, two were in the secretory whilst four were in the proliferative menstrual phase. Menstrual phases were consistent with endometrial histological dating. Women on hormonal therapy at the time of their operation and those with a recent history of genital tract infection were excluded. Written informed consent was obtained prior to the collection of samples, and the study was approved by the local institutional Research Ethics Committee. Full thickness wedge biopsies including epithelial and stromal tissue were freshly taken post-operatively from five sites: uterine tubes, endometrium, endocervix, ectocervix and vagina. Small sections of tissue (5 £ 5 mm) were fixed in 10% formalin. Tissue sections (3 mm) were prepared from these samples. In addition, tissue sections (, 5 £ 5 mm) from the same samples were embedded in optimal cutting temperature compound (VWR, UK) and snap-frozen in hexane (Sigma –Aldrich, UK) suspended in liquid nitrogen. The cryosections (5 mm) were prepared and stored at 280 8C until use. Antibodies and peptides Antibodies and peptides used in the experiments were obtained from Santa Cruz Biotechnology Inc. (USA). These were goat polyclonal antibodies specific for N-terminal domains of TLR1, TLR2, TLR3, TLR5, TLR6 (catalogue nos., sc8687, sc8689, sc8691, sc8695, sc5657 respectively) and goat polyclonal antibody specific for C-terminal domains of TLR4 (catalogue no. sc8694). Blocking peptides specific for the respective antibodies were used to detect non-specific staining. Immunostaining Cryosections were air-dried before use for 30 min, fixed in acetone at 2 20 8C for 10 min, air-dried again for 30 min, and then washed in phosphate-buffered saline (PBS). Formalin-fixed sections were dewaxed (...truncated)