Mechanisms of irregular bleeding with hormone therapy: the role of matrix metalloproteinases and their tissue inhibitors.

CONTEXT: Irregular bleeding is common in users of combined hormone therapy (HT) and often leads to invasive and expensive investigations to exclude underlying pathology. The mechanisms of HT-related bleeding are poorly understood. Endometrial matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to regulate bleeding during the normal menstrual cycle and are known to be altered in breakthrough bleeding with progestogen-only contraception. OBJECTIVE: The aim of this study was to determine how HT exposure alters endometrial production of MMP-1, -3, -9, and -14 and their tissue inhibitors TIMP-1, -2, -3, and -4 and to determine the relationship between MMP and TIMP production and bleeding patterns in HT users. Endometrial leukocytes regulating MMP production and activation were also assessed. DESIGN: A prospective observational study was conducted between 2003 and 2005. SETTING AND PATIENTS: The study occurred at a tertiary referral menopause clinic at King Edward Memorial Hospital, Western Australia, and included 25 postmenopausal women not taking HT and 73 women taking combined HT. INTERVENTIONS: Endometrium was obtained during and outside bleeding episodes. MAIN OUTCOME MEASURES: We assessed production of MMP-1, -3, -9, and -14 and their tissue inhibitors TIMP-1, -2, -3, and -4 and their relationship to bleeding patterns in HT users. RESULTS: All MMPs studied, with the exception of MMP-9, were expressed at low levels in postmenopausal endometrium. Increases in both MMP-3 and -9 localization were seen in association with irregular bleeding, but these did not reach statistical significance. Endometrial production of TIMP-1 was significantly increased in association with bleeding. Endometrial leukocytes were not related to bleeding, with the exception of uterine natural killer cells, which were significantly increased during bleeding, as previously published. CONCLUSIONS: Irregular bleeding in HT users is associated with a distinct pattern of MMP and TIMP production that differs from that seen in normal menstrual bleeding and from that seen in contraceptive-related breakthrough bleeding. This suggests that the endometrial balance between MMP and TIMP contributes to vascular breakdown with HT but by a different mechanism than that seen in normal menstruation or in breakthrough bleeding.

Menopausal hormone therapy and irregular endometrial bleeding: a potential role for uterine natural killer cells?

CONTEXT: Irregular bleeding affects many users of combined menopausal hormone therapy (HT) and commonly leads to invasive and expensive investigations to exclude underlying malignancy. In most cases no abnormality is found. OBJECTIVE: The main objective of this study was to explore the role of uterine natural killer (uNK) cells and their regulatory cytokine IL-15 in irregular bleeding in HT users. DESIGN: This was a prospective observational study conducted between 2002 and 2004. SETTING: The study was conducted in a tertiary referral menopause clinic at King Edward Memorial Hospital, Western Australia. PATIENTS: Patients included 117 postmenopausal women taking combined HT. INTERVENTIONS: Outpatient endometrial biopsies were taken during and outside bleeding episodes. MAIN OUTCOME MEASURES: The relationship between endometrial uNK cells (CD56+) and bleeding patterns was measured. We also addressed the impact of HT exposure on uNK cell populations, the relationship between endometrial IL-15 expression and uNK cell populations, and killer Ig like receptor genotype in subjects with irregular bleeding. RESULTS: Endometrial CD56+ uNK cells were significantly increased in biopsies obtained during bleeding episodes (P < 0.001), compared with HT users with no bleeding. The highest level of IL-15 expression was also seen in biopsies taken during bleeding. No clear relationship between killer Ig like receptor genotype and bleeding on HT was observed. CONCLUSIONS: Little is known about the mechanisms underlying irregular bleeding in HT users. This is the first report of uNK cells and their association with regulating cytokines in postmenopausal endometrium and demonstrates a possible mechanism by which HT may induce irregular bleeding.

Endotoxin-induced uveitis. Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body.

PURPOSE: Footpad injection of bacterial lipopolysaccharide (LPS) causes pronounced anterior uveitis in susceptible species and strains. Recent studies using wholemount techniques have demonstrated the presence of rich networks of major histocompatibility complex (MHC) class II-positive dendritic cells (DC) and resident tissue macrophages in the iris and ciliary body. The aim of this investigation was to determine the immunophenotype and dynamics of the inflammatory cell infiltrate during LPS-induced anterior uveitis using the wholemount method and to examine the response of the resident tissue macrophages and DC to an acute inflammatory episode in the anterior segment. METHODS: Female Lewis rats (8 to 12 weeks old, n = 49) received a single footpad injection of 100 micrograms of LPS and were killed at various times up to 6 weeks after injection. The iris-ciliary body complex from each eye was removed intact and subdivided into segments and immunostained using a panel of monoclonal antibodies to a variety of immune cell types. RESULTS: The wholemount method clearly illustrates that during endotoxin-induced uveitis (EIU), the earliest cellular infiltrate includes small, round ED1+ mononuclear cells marginating in the iris vasculature approximately 2 hours after injection. Marginating Ox42+ polymorphonuclear leukocytes were detectable in the iris vessels approximately 4 to 6 hours after injection and were especially numerous in the ciliary body base approximately 24 hours after injection. The overall density of resident tissue macrophages (ED2+) remained largely unchanged in the course of EIU. In contrast, the total number of MHC class II-bearing (Ox6+) cells (putative dendritic cells) increased 30% in the first 6 hours and 200% by 72 hours. During the acute phase of the inflammatory response (up to 24 hours), the proportion of these cells with a dendritiform morphology decreased (93% to 50%). The number of T cells showed a biphasic response peaking at 4 to 6 hours and again at 24 hours (290 cells/mm2); however, their numbers had resumed normal low density (4 cells/mm2 to 25 cells/mm2) by 6 weeks. CONCLUSIONS: The results suggest that the neutrophilic infiltration in EIU occurs predominantly in the base of the ciliary body, whereas the monocytic and lymphocytic infiltrate occurs in the iris vasculature. Resident tissue macrophages do not undergo marked changes in density or morphology in the early course of the disease. Recruitment of T cells into the anterior segment in EIU may suggest a previously unsuspected role for these cells in the immunopathology of this disease. Changes in density and morphology of MHC class II+ DC in the iris, which persisted for at least 6 weeks, were interpreted as an increase in recruitment and migration of these cells that may serve to enhance the efficiency of immune surveillance in the anterior segment at crucial times of bacterial infection.

Integrity of epithelium and endothelium in organ-cultured human corneas.

PURPOSE: To examine components of the junctional complex and the actin cytoskeleton and the incidence of apoptosis in epithelium and endothelium of organ-cultured human corneas. METHODS: Human corneas, either organ-cultured for 1 to >28 days or excised directly from eyes stored in moist chambers, were stained with antibodies to ZO-1, vinculin, and caspase 3 coupled to FITC-conjugated secondary antibody. These markers were combined with rhodamine-phalloidin staining for F-actin and DAPI labeling for DNA. The corneas were examined by confocal microscopy. RESULTS: The depth of the epithelium was reduced during organ culture, but no changes were observed in the distribution of ZO-1 or vinculin, or in the F-actin cytoskeleton. The appearance of apoptotic epithelial cells positive for caspase 3 or with condensed DNA increased with time after 14 days in organ culture, but there was no correlation with donor age. ZO-1 and F-actin staining patterns in endothelium were similarly undisturbed by organ culture, but apoptotic endothelial cells were only rarely seen and then only after >28 days in organ culture. CONCLUSIONS: Organ culture maintained the integrity of tight junctions and the actin cytoskeleton in epithelial and endothelial cell layers. Apoptosis was evident in epithelium but was observed rarely in the endothelium and then only after extended periods in organ culture.

Immunomorphologic studies of macrophages and MHC class II-positive dendritic cells in the iris and ciliary body of the rat, mouse, and human eye.

PURPOSE: To establish the presence of distinct populations of macrophages and MHC class II (Ia)-positive dendritic cells (DC) in the iris and ciliary body of the rat, mouse, and human eye. METHODS: Iris-ciliary body wholemounts from a variety of rat strains, balb/c mice, and human eyes were investigated by single and double immunohistochemistry, immunoelectron microscopy, and confocal microscopy to determine the phenotype, density, distribution, and location of macrophage and DC populations. RESULTS: Dendritiform and pleiomorphic macrophages were distributed in a regular array within the rat iris and ciliary body stroma (600 to 700 cells/mm2 or 7000 cells per iris). Ia+ DC were distributed in a similar regular network (400 cells/mm2 or 5500 cells per iris) within the iris stroma and ciliary epithelium. In the rat, a strain-dependent variation in the numbers of DC was noted, F344 rats displaying highest numbers of DC (962 +/- 398 cells/mm2) and WAG strain the lowest numbers (285 +/- 218 cells/mm2). Double color immunoperoxidase staining using anti-Ia and anti-pan specific macrophage monoclonal antibodies revealed that macrophages and Ia+ DC are distinct populations with only 5% to 15% overlap. Single immunoperoxidase of mouse iris and ciliary body using anti-pan macrophage and anti-Ia antibodies produced findings identical to those in rat. Preliminary studies of human tissue using confocal microscopy of immunostained whole irides also revealed a regular array of macrophages and MHC class II (HLA-DR)+ dendritiform cells. CONCLUSIONS: The mammalian iris contains rich networks of dendritiform-pleiomorphic macrophages and MHC class II+ DC. These findings suggest that the DC in the tissues lining the anterior chamber represent a rich network of putative antigen presenting cells and are the most likely candidates for transmitting antigen-specific signals from the anterior chamber in vivo and in experimental models such as ACAID: These observations have wide implications for the understanding of the pathogenesis of anterior and posterior uveitis.

Resident and infiltrating cells in the rat iris during the early stages of experimental melanin protein-induced uveitis (EMIU).

Experimental melanin protein-induced uveitis (EMIU) is reported to be a model of anterior uveitis and choroiditis in which the retina is spared. In this study, we chose to compare EMIU with experimental autoimmune uveoretinitis (EAU), a well-recognised model of endogenous posterior uveitis, with regard to the nature and dynamics of the cellular infiltrate in the iris. Female Lewis albino rats were immunised with mixtures of crude retinal extract/complete Freund's adjuvant (CFA) (EAU), phosphate-buffered saline/CFA (controls), or iris melanin/CFA (EMIU) using established protocols. Animals were sacrificed on days 10 and 13 (around disease onset). Following whole body perfusion fixation, irides were dissected from the remainder of the globe. Iris wholemount preparations were then subjected to immunohistochemical analysis in order to investigate both the dynamics of infiltrating leukocytes and the effects of the inflammatory changes on resident immune cells in the iris. The nature of the cellular infiltrate in both EMIU and EAU models was essentially similar, namely there was a rich infiltrate of EDI+ mononuclear cells, Ox42+ neutrophils and T cells. Resident tissue macrophages (ED2+) were slightly below normal densities in the iris of EAU animals and marginally elevated in EMIU animals, MHC class II (Ia) staining, associated in the normal eye with dendritic cells (DC), was considerably elevated in EMIU. It is likely that this was due to both increased DC numbers and an influx of Ia+ exudate macrophages. No striking difference was found in the nature and phenotype of the cellular infiltrate in the iris at the onset of the disease in these two models of uveitis (EAU and EMIU). This suggests that the anterior segment inflammation in both models represents non-specific changes secondary to cytokine release associated with interaction of activated antigen-specific T cells and target antigens, namely retinal photoreceptors in EAU and uveal tract melanin-containing cells in EMIU. Alternatively, it may suggest that antigen-presenting cells resident in the iris and ciliary body in normal eyes have access to ocular antigens on both sides of the blood-ocular barrier and are capable of activating circulating antigen-specific T cells in these models.

Cellular localisation and dynamics of nitric oxide synthase expression in the rat anterior segment during endotoxin-induced uveitis.

The present study examined the temporal pattern and cellular localisation of nitric oxide synthase in Endotoxin-Induced Uveitis (EIU). Lewis rats (n=40) received a single footpad injection of 200 microg of bacterial lipopolysaccharide. Animals were killed at 0, 2, 4, 6, 12, 24, 48 and 72 hr after injection and ocular tissues prepared as iris-ciliary body wholemounts or frozen sections of the anterior segment. The expression of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) was investigated at all time points by immunohistochemistry. A further group of animals (n=6) were killed at the peak of the disease (12 hr) and the cellular co-localisation of iNOS on resident and infiltrating immune cells was investigated by double immunohistochemistry utilising the biotinylated monoclonal antibodies ED1, ED2 and Ox6. Expression of cNOS on iris vessels did not alter during the course of EIU. Quantitative analysis of iris-ciliary body wholemounts revealed the first evidence of iNOS+ at 2 hr which increased dramatically at 4 and 6 hr with a peak at 12 hr. The expression of iNOS in the early phase of the disease (2-6 hr) was associated with small round marginating and newly extravasated cells that on morphological criteria were most likely neutrophils and monocytes. At 12 hr, cells of more mixed morphologies began to express iNOS and double labelling revealed 70% of these cells were also ED1(+) (a lysosomal antigen present in monocytes/macrophages and dendritic cells), 52% were Ox6(+) (MHC class II) (dendritic cells, activated macrophages and some T-cells) and 19% were ED2(+) (pan-specific resident tissue macrophages). Expressed in an alternative manner, 10% of the total ED1(+) cell population, 11% of the ED2(+) cells and 44% of Ox6(+) cells co-expressed iNOS. Expression of iNOS decreased significantly by 24 hr to near baseline levels and was absent by 48 and 72 hr. Within the ciliary processes iNOS+ dendriform cells were noted at 6 hr and accumulations of many small round iNOS+ cells were present at 12 hr. The ciliary epithelium did not at any time express iNOS at the protein level detectable by immunohistochemistry. The results of this study suggest that iNOS expression early in EIU is associated with infiltrating or newly recruited neutrophils and monocytes/macrophages in the iris whereas later in the disease resident tissue macrophages and MHC class II+ cells (activated macrophages and putative dendritic cells) in the iris and ciliary body may synthesise nitric oxide. The role of this late phase of nitric oxide synthesis may include lymphocytostasis and immunosuppression as proposed in other tissue sites. The outcome of the present study may help in planning therapeutic strategies using NOS inhibitors.