Defensins are a family of small cationic proteins that have been shown to confer the host with anti-bacterial, anti-fungal and anti-viral protection. Defensins have six cysteine residues that form three disulphide bonds and they are divided into two main groups on the basis of the position of these bonds: α-defensins are found in neutrophils (human neutrophil peptides (HNP)1-4) and at epithelial sites (human defensin (HD) 5 and 6) while the β-defensins (human β defensins (HBD)) are found mainly at epithelial surfaces e.g. gut, lung 34. It has recently been reported that there are 28 defensin-like sequences in the human genome 5 but currently only HBD1-6 have been characterized 67891011. The β-defensins further subdivide into those that are constitutively expressed (e.g. HBD1) and those that are induced upon challenge with inflammatory or pathogen-derived stimuli 910121314. In addition to their role as anti-microbials, the β-defensins are chemoattractants allowing an interaction between the innate and the acquired immune systems 8915. The expression, regulation and role of the β-defensins at non-reproductive sites has been reviewed elsewhere 161718.

Proteins containing the WAP motif have a 50 amino acid domain, which forms a tight core containing four disulphide bonds 19. This family includes secretory leukocyte protease inhibitor (SLPI), elafin, eppin and HE4 20. SLPI and elafin are the most widely studied WAP proteins. These two molecules share 40% homology, with eight identical cysteines and five identical prolines, and both are expressed at mucosal surfaces where they have anti-protease and anti-microbial actions 212223242526. Both proteins inhibit serine proteases with SLPI inhibiting several proteases including neutrophil elastase and cathepsin G 27 while elafin regulates only neutrophil elastase and proteinase 3 282930. These actions combined with their anti-microbial activity allow SLPI and elafin to protect epithelial surfaces during infection and inflammation. The similarity between SLPI, elafin and other proteins containing the WAP motif suggests that they may also have anti-protease and anti-microbial actions at epithelial surfaces 20.

As at other mucosal surfaces the epithelial cell mediates the initial response of the endometrium to infection 31. In 1997 and 1998 the first reports of defensin expression in the human endometrium were published. These described the expression of the α-defensin, HD5, and the β-defensin, HBD1, in the endometrial epithelium 323334. Subsequently, messenger RNA (mRNA) for HBD1-4 has been detected in endometrium and it has been shown that each defensin has a unique temporal expression profile 3536. HBD1 and 3 are expressed at highest levels during the secretory phase 3536. A similar pattern of expression has been reported for HD5 33. In contrast, HBD2 mRNA expression shows a dramatic peak during menstruation while HBD4 is expressed mainly in the proliferative phase 3536. These data are summarised in Figure 1. The reasons for these differential patterns of expression have not been determined and may relate to differences in anti-microbial activity of members of the defensin family or to their other activities such as chemotaxis. Acquisition of sexually transmitted infections is reported to be influenced by oestradiol and progesterone 37 and changes to the expression of innate immune molecules such as defensins may contribute to this. As in other epithelial cells, expression of both HBD2 and 3 in endometrial epithelial cells can be upregulated in response to inflammatory stimuli suggesting that these defensins will be induced in response to infection 3638.

The WAP protein, SLPI, has also been detected in the endometrial epithelium with peak expression in the mid-late secretory phase (Figure 2a) 39. This protein may have important actions during the window of implantation. SLPI has several anti-inflammmatory actions including inhibition of the proinflammatory transcription factor, nuclear factor κB 40, and this combined with its anti-protease activity may prevent an excessive inflammatory response from occurring in the uterus at the time of implantation and during early pregnancy. In addition, SLPI has recently been suggested to have an effect on epithelial growth. Proepithelin (PEPI) is an epithelial growth factor that can be converted to a growth inhibitor, epithelin (EPI), by elastase. SLPI can form a complex with PEPI preventing its cleavage by elastase and hence, promote epithelial growth 41. In vitro studies using the Ishikawa epithelial cell line (derived from an endometrial adenocarcinoma) have shown that SLPI can also increase expression of the cyclin D1 gene while inhibiting expression of anti-proliferative genes such as insulin-like growth factor binding protein-3 and lysyl oxidase 42. The anti-microbial actions of SLPI are likely to be particularly important in the protection of the uterus from infection during implantation and early pregnancy. Indeed, it has been shown that the antibacterial activity of apical secretions from polarized endometrial epithelial cells can be reduced by incubation with an anti-SLPI antibody confirming that SLPI contributes to the antibacterial defences at the epithelial surface at least in vitro 43. SLPI concentrations and the ratio of SLPI to elastase have also been found to increase in the peritoneal fluid of women suffering from endometriosis. Local peritoneal inflammation is one of the factors thought to be responsible for the symptoms of this disease and it has been suggested that SLPI concentrations are increased in response to this and that the anti-inflammatory and anti-protease actions of SLPI may limit the inflammatory process 44.

It is likely that the endometrial epithelium will also express additional natural anti-microbials. For example, the iron-binding protein lactoferrin is expressed by the endometrial epithelium 45. Lactoferrin has anti-microbial actions 46 and has been shown to be oestrogen regulated with maximal expression in the proliferative phase 47. Lactoferrin has been shown to act in synergy with SLPI 48 and it is likely that interactions between natural anti-microbials in endometrium will also be important.

Leukocyte populations are an important component of the endometrium, constituting 8.2% of the stroma during the proliferative phase with this figure increasing to 31.7% in first trimester decidua 49. The presence of several leukocyte populations in human endometrium is cycle dependent. The populations of neutrophils, uterine NK cells (uNK), macrophages and mast cells are relevant to endometrial anti-microbial expression. uNK cells are present in endometrium in the mid-late secretory phase of the menstrual cycle and are the major leukocyte population in first trimester decidua accounting for 70% of the leukocytes present at this time 50. They are believed to have a role in implantation, decidualization and placentation 51. uNK cells differ from the main population of NK cells in peripheral blood as they are CD56^bright 51. The presence of these cells in endometrium is dependent on progesterone although this may be an indirect action, as uNK cells do not express the nuclear progesterone receptor 52. These cells express receptors for the glucocorticoid receptor and oestrogen receptor β suggesting that they do respond to these steroid hormones 53. Macrophages are present throughout the cycle with a small increase in number premenstrually 5054. There are also increased numbers present in early pregnancy with a high concentration found at the implantation site 55. Immediately prior to menstruation there is an influx of neutrophils into the endometrium 56. Leukoctyes are involved in the tissue breakdown and repair that occurs at this time 57. Infiltration of endometrium with both neutrophils and macrophages around the time of menstruation is coincident with falling progesterone concentrations. The effects of progesterone withdrawal on these leukocyte populations is believed to be mediated by increased expression of inflammatory mediators such as interleukin-8 and monocyte chemoattractant protein-1 in the endometrium premenstrually 585960. Mast cells are also present throughout the menstrual cycle, although in smaller numbers, and are activated at menstruation 61.

Neutrophils are a rich source of natural anti-microbial molecules (Figure 3). The infiltration of the endometrium by neutrophils during menstruation will enhance natural anti-microbial protection at a time when the epithelial barrier is disrupted. In addition, neutrophils will rapidly infiltrate during infection, again increasing the innate defences at an appropriate time. Peripheral blood neutrophils have been shown to express α-defensins, the cathelicidin, human cationic anti-microbial peptide 18 (hCAP-18), lactoferrin and lysozyme 626364. It is likely that endometrial neutrophils will also express these molecules although this has not yet been documented. Moreover, it is possible that the cytokine environment in the uterus may, at certain stages of the menstrual cycle, modulate neutrophil granule release. The WAP protein, elafin, has been detected in endometrial neutrophils (Figure 2b) 65. Elafin mRNA and protein expression rises dramatically around the time of menstruation and immunohistochemical studies have determined that the cellular source of endometrial elafin is the neutrophil. The differing expression profiles of the two WAP proteins, SLPI and elafin (Figures 1 and 2), in endometrium suggest that they have independent roles. The anti-protease actions of elafin may aid tissue repair during menstruation while the protein's anti-microbial activity will increase innate immune defences at this time.

Granulysin is an anti-microbial molecule that is stored in the granules of cytolytic T cells and peripheral blood NK cells 66. Granulysin mRNA has been found to be maximally expressed in endometrium from the late secretory phase suggesting that the uNK cell is also a source of this anti-microbial 35. Expression of granulysin during the late secretory phase may provide additional protection around the time of implantation or in its absence, menstruation.

It is also likely that endometrial macrophages and mast cells will express natural anti-microbial peptides. There are reports that HBD1 and 2 are expressed by blood monocytes and alveolar macrophages 67 and that hCAP-18 is expressed by human skin mast cells 68. However, localization of these peptides to endometrial leukocytes has not yet been described.