Peritoneal macrophages during peritonitis. Phenotypic studies.

The expression of a range of surface molecules/receptors that are important in the host response to infection and foreign antigens was examined using peritoneal macrophages isolated from patients on continuous ambulatory peritoneal dialysis (CAPD) with peritonitis. The macrophage phenotypic profile was compared with that of normal peripheral blood monocytes. Consistently there was increased expression by macrophages of CD14, ICAM-1 (CD54), Fc gamma RI (CD64), Fc gamma RII (CDw32), Fc gamma RIII (CD16), transferrin receptors (CD71) and tissue factor. Increased expression of MHC class II was marginally significant. There was no detectable expression of either the p55 (CD25) or p70 chains of the IL-2 receptor. The expression of the complement receptors, CR1 (CD35) and CR3 (CD11b, CD18), was reduced. The activity of well-known inflammatory cytokines, rather than uraemic molecules, can account for the phenotypic profile of these extravasated peritoneal macrophages. The results of this study indicate that peritoneal macrophages from CAPD patients with peritonitis display a phenotype consistent with them being in vivo-derived inflammatory macrophages, and that they are appropriate for use in studies of anti-inflammatory agents.     

Immunolocalization of the apoptosis regulating proteins Bcl-2 and Bax in human endometrium and isolated peritoneal fluid macrophages in endometriosis

Endometriosis, a debilitating disease associated with infertility, is characterized by the prolonged presence of ectopic endometrial tissue and the involvement of activated peritoneal fluid macrophages. Apoptosis, which occurs in both endometrium and peritoneal fluid macrophages, is controlled in part by members of the Bcl-2/Bax family of proteins. Here, through immunohistochemical staining, we investigated the Bcl-2/Bax status in endometrium and peritoneal fluid macrophages in endometriosis. Bcl-2/Bax immunoreactivity was found predominantly in the glandular epithelial cells, mainly during the proliferative phase of the menstrual cycle for Bcl-2 but throughout the entire menstrual cycle for Bax. Ectopic endometrium contained a population of Bcl-2 positive. Bax negative tissue macrophages. Fluorescence-activated cell sorting of isolated peritoneal fluid macrophages showed that women with endometriosis had a significantly higher proportion of Bcl-2 positive macrophages than the non- endometriotic group. The proportion of Bax positive peritoneal fluid macrophages was significantly elevated in women without endometriosis. The increased proportion of Bcl-2 positive macrophages found in women with endometriosis may predispose these cells to resist apoptosis. The continued survival of these active cells could have important consequences for the survival and proliferation of the ectopic endometrial tissue.      

Endometrial leucocytes: expression of steroid hormone receptors.

BACKGROUND: Stromal leucocyte populations in human endometrium comprise T cells, macrophages, and phenotypically unusual endometrial granulated lymphocytes. Their proportions vary during the menstrual cycle and, in particular, endometrial granulated lymphocytes increase in number in the late secretory phase. The stimulus responsible for these cyclical changes is unknown but it is likely that the steroid hormones oestrogen and progesterone play a role. AIMS: To define further the expression of steroid hormone receptors by leucocytes in non-pregnant and pregnant human endometrium. METHODS: Frozen and paraffin wax embedded sections of endometrium from non-pregnant women and early pregnancy decidua were labelled using single and double immunohistochemical techniques with monoclonal antibodies directed against oestrogen and progesterone receptors and various leucocyte subpopulations. RESULTS: Despite the prominence of CD56 positive endometrial granulated lymphocytes in late secretory phase endometrium and early pregnancy decidua, double immunohistochemical labelling showed no evidence of expression of either progesterone or oestrogen receptors by these cells or other endometrial leucocyte populations. CONCLUSIONS: Rather than acting directly, steroid hormones are likely to influence endometrial leucocyte populations indirectly via products of endometrial stromal or epithelial cells that express steroid hormone receptors.     

小柴胡汤联合丹那唑抑制子宫内膜异位症大鼠血管生成的研究

目的:探讨小柴胡汤、丹那唑及二者联合用药对子宫内膜异位症(EM)模型大鼠血管生成因子和异位内膜微血管密度的影响。方法:EM模型大鼠随机分为不用药组(U)、小柴胡汤(XCH)、丹那唑组(D)和联合用药组(S),设假手术组(C)为对照。四周后观察各组异位子宫内膜体积和形态学的变化,计数腹腔液巨噬细胞,放射免疫法测定各组大鼠外周血、腹腔液及巨噬细胞培养液白细胞介素－8(IL－8)、肿瘤坏死因子(TNF-α)的含量,免疫组化法测定血管内皮生长因子(VEGF)在异位内膜中的表达,并通过CD34标记异位子宫内膜血管,测定其微血管密度(MVD)。结果:XCH组、D组和S组异位内膜呈现不同程度萎缩,腺体明显减少,腹腔液巨噬细胞计数减少,XCH组、D组及S组大鼠外周血、腹腔液及巨噬细胞培养液IL－8、TNF-α含量降低,异位内膜VEGF表达减弱,MVD明显减少,其中以S组最为明显。结论:小柴胡汤和丹那唑可以抑制EM模型大鼠异位内膜的血管生成,使异位内膜萎缩,当二者联合用药时,作用更强。     

Aberrant expression of CD95 and CD69 molecules among CD56 cells in women with endometriosis

PROBLEM: Activated lymphocytes can be eliminated by Fas/Fas ligand (FasL)-induced cell death. Endometrial cells express FasL. The aim of our study was to determine the expression of CD56+ cells (natural killer and natural killer T cells) Fas antigen CD95 and the early activation molecule CD69 in the peritoneal fluid of women with endometriosis. METHOD: Two-colour flow cytometry was used. RESULTS: In the early stages of endometriosis, more CD56+ cells expressed CD69 and CD95 molecules when compared with the control group. However, in case of severe endometriosis the percentage of CD95+CD56+ cells in peritoneal fluid was similar to that of the control group, but the expression of CD69 molecules remained high. CONCLUSION: Because of Fas/FasL mechanisms, in the initial stages of endometriosis the activated peritoneal fluid CD56+ cells can be intensively eliminated, thus providing conditions for the survival of ectopic endometrial cells and the development of the disease.     

Markers of endometrial function in women with unexplained recurrent pregnancy loss: a comparison between morphologically normal and retarded endometrium

BACKGROUND: Endometrial defect, usually described as luteal phase defect (LPD), is associated with recurrent miscarriage. Recurrent miscarriage has also been associated with the abnormal expression of various molecules by endometrial cells. The aim of this study was to determine if any of these molecules or cells could be used to distinguish LPD from in-phase endometrium. METHODS: Immunocytochemistry was used to compare endometrial expression of CD45+, CD56+, CD3+ and CD4+ cells, leukaemia inhibitory factor, interleukin-6 and estrogen and progesterone receptors in precisely timed endometrial biopsies obtained between days LH+6 and LH+11 from recurrent miscarriage women with in-phase and retarded endometrium. RESULTS: In all samples there was a positive correlation between the number of CD45+ cells and LH day and a negative correlation between progesterone receptor and LIF expression and LH day. A significantly lower number (P<0.05) of CD56+ cells in peri-implantation endometrium and a decreased mid-cycle estrogen level (P<0.05) was seen in women with LPD compared to in-phase endometrium when single analysis was carried out. However, these differences were not significant after application of the Bonferroni correction for multiple analysis. CONCLUSIONS: The results are in line with previous associations observed between estrogen levels and LPD and suggest that the number of CD56+ cells is different in LPD and in-phase endometrium, although this could be due to delayed endometrial development in women with LPD. Interpretation must be cautious because these differences could have arisen by chance.     

Regulation of numbers of macrophages in the endometrium of the sheep by systemic effects of pregnancy, local presence of the conceptus, and progesterone

Many species exhibiting hemochorial placentation experience an accumulation of macrophages in the endometrium during pregnancy. For the present study, it was tested whether macrophages also accumulate in the endometrium of the sheep, which is a species undergoing an epitheliochorial placentation. An additional objective was to determine whether regulation of endometrial macrophage number occurs via systemic or local signals and whether progesterone is one of these signals. The approach was to evaluate presence of macrophages immunohistochemically using antibodies to CD68 and CD14. Tissues examined were from cyclic ewes in the luteal phase of the estrous cycle, unilaterally-pregnant ewes at day 140 of pregnancy in which pregnancy was surgically confined to one uterine horn, ovariectomized ewes, and ovariectomized ewes treated with progesterone for 44 days. Macrophages were localized predominately to the stromal compartment of the stratum compactum region of the endometrium. In non-pregnant ewes, macrophages were not abundant regardless of physiological status. Increased numbers of endometrial macrophages were seen for both the pregnant and non-pregnant uterine horns of unilaterally pregnant ewes. Numbers of macrophages were higher in the endometrium from the pregnant uterine horn than from endometrium from the non-pregnant uterine horn. Results indicate that macrophages accumulate in the endometrium by day 140 of pregnancy in the sheep and that this induction is because of both systemic and local signals. Progesterone appears not to be an important regulator of numbers of endometrial macrophages.     

Effects of granulocyte/macrophage colony-stimulating factor on the development and differentiation of CD5-positive macrophages and their potential derivation from a CD5-positive B-cell lineage in mice.

In co-cultures of either the murine pre-B cell line J13, fetal liver cells, or adult peritoneal or bone marrow cells with ST2 mouse bone marrow stromal cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), the development of CD5+ macrophages was demonstrated by immunohistochemical staining and flow cytometry. Although CD5+ macrophages were not present in the peritoneal cavities of normal mice, approximately 30% of the peritoneal macrophages in viable motheaten (mev/mev) mice, deficient in SHP-1 protein tyrosine phosphatase, expressed cell surface CD5 and B220, markers for B cells. In the mev/mev mice, GM-CSF level in peritoneal fluid was increased significantly. At 5 days after daily intravenous injection with GM-CSF, many CD5+ macrophages appeared in the peritoneal cavity and in omental milky spots of normal mice but fewer in osteopetrosis (op) mutant mice, deficient in macrophage (M)-CSF. These results indicate that GM-CSF, in combination with M-CSF, induces the development and differentiation of CD5+ macrophages in the peritoneal cavity, particularly in the omental milky spots of mice. In the peritoneal cavity of GM-CSF-treated mice, the percentages of hematopoietic progenitor cells doubly positive for CD5 and CD34 or c-kit and of macrophage precursor cells doubly positive for CD5 and ER-MP58 or ER-MP20 were increased significantly during the development of CD5+ macrophages and CD5 B cells, suggesting that CD5+ macrophages and B cells may share a bipotential progenitor in vivo.     

Phenotypic and functional studies of leukocytes in human endometrium and endometriosis

The aetiology of endometriosis, a common and disabling disorder, is presently unknown, although immune dysfunction could allow ectopic endometrial fragments to survive outside the uterine cavity. These studies investigate the relationship between leukocyte populations, steroid hormone receptor expression, proliferative activity, bcl-2 expression and apoptosis in eutopic and ectopic endometrium from women with endometriosis or adenomyosis at different phases of the menstrual cycle. Significantly increased oestrogen receptor expression, bcl-2 expression and numbers of CD8+ leukocytes were found in ectopic compared with eutopic endometrium in endometriosis, and CD56+ endometrial granulated lymphocytes (eGLs) were significantly reduced in ectopic endometrium. Apoptotic cells were rarely found in control and subject endometria. In contrast with endometriosis, adenomyotic lesions showed identical steroid hormone receptor expression, proliferative activity, bcl-2 expression and leukocyte subpopulations to eutopic endometrium, indicating different aetiologies for these disorders. The unusual CD56+ CD16- eGLs present in large numbers in late secretory phase eutopic endometrium were highly purified (>98%) by immunomagnetic separation. Except for a negligible cytotoxic activity of eGLs from early proliferative samples, cytotoxic activity of eGLs from non-pregnant endometrium during the menstrual cycle was comparable with those in peripheral blood, predominantly CD56+ CD16+ natural killer cells. eGLs from non-pregnant endometrium and early pregnancy showed a variable proliferative response to 5 and 100 U/ml interleukin-2 over 48-h and 120-h time courses. eGLs are evidently functionally important in the eutopic endometrium. Their absence in endometriotic lesions together with increased CD+8 T-cell numbers and increased oestrogen receptor and bcl-2 expression may have significant effects on the development and progression of endometriosis.     

Decreased expression of killer cell inhibitory receptors on natural killer cells in eutopic endometrium in women with adenomyosis

BACKGROUND: Increased expression of killer cell inhibitory receptors (KIRs) has been found on natural killer (NK) cells in peritoneal fluid in women with endometriosis. In this study, we tried to measure the expression of KIRs on NK and T cells in women with adenomyosis, in an attempt to find the possible role of KIRs in the development of adenomyosis. METHODS: A total of 10 women with adenomyosis (study group) and 12 women with uterine myoma (control group) were included in this study. The expression of KIRs, including NKB1, GL183, EB6 and CD94, on NK and T cells in myometrium and endometrium was examined by flow cytometry. RESULTS: There was a decreased expression of NKB1 and GL183 on NK cells in the endometrium, but not in the myometrium, in women with adenomyosis. However, the expression of KIRs on T cells, either CD4+ or CD8+, was not different in either myometrium or endometrium between women with and without adenomyosis. CONCLUSIONS: The expression of KIRs on NK cells was decreased in eutopic endometrium in women with adenomyosis. It may be a compensatory effect in which the NK cytotoxicity is activated in order to eradicate the abnormal endometrial cells that might exit of the eutopic site of the endometrium.     

Phenotypic Characterization of Macrophages in the Endometrium of the Pregnant Cow

Problem  Macrophages are recruited in large number to the interplacentomal endometrium of the cow during pregnancy. We evaluated whether endometrial macrophages also accumulate in placentomal regions of endometrium during pregnancy and whether endometrial macrophages are regionally differentiated.
Method of study  Interplacentomal endometrium and placentomes were subjected to dual-color immunofluorescence using CD68 as a pan-macrophage marker.
Results  CD68⁺ cells were abundant in stroma of the interplacentomal endometrium and caruncular septa of the placentomes. CD68⁺ cells were not present in fetal villi of the placentomes or in the interplacentomal chorion. Regardless of location, the majority of CD68⁺ cells also expressed CD14. In interplacentomal endometrium, CD68⁺CD11b⁺ cells were present in deeper areas of the stroma but not in shallow endometrial stroma. In caruncular septa of the placentome, CD68⁺ cells were negative for CD11b. CD68⁺ cells in the interplacentomal endometrium were negative for MHC class II while most CD68⁺ cells in caruncular septa were positive for MHC class II.
Conclusion  CD68⁺CD14⁺ macrophages present in the stroma of the interplacentomal endometrium and caruncular septa of the placentome are regionally differentiated with regard to expression of CD11b and MHC class II.     

Immunohistochemical Localization of C9 Neoantigen and the Terminal Complement Inhibitory Protein CD59 in Human Endometrium

PROBLEM : Human endometrium expresses complement components, receptors, and regulatory proteins, many of which appear to be expressed in a hormone-dependent manner. Whether terminal complement components are also present in the endometrium is unknown. CD59, a broadly expressed protein that blocks association of C9 with C8 in the membrane attack complex, is localized in reproductive tissue to human spermatozoa, seminal plasma, amniotic fluid, and placenta. The present study examines human endometrium for the presence of CD59 and terminal complement proteins. METHOD : Endometrial biopsies were obtained from six normal women from various phases of the menstrual cycle and analyzed by immunohistochemistry, using MEM-43 anti-human CD59 and anti-human SC5b-9 murine monoclonal antibodies and the immunoperoxidase technique. RESULTS : Both CD59 protein and SC5b-9 (C9 neoantigen) were demonstrated to be present in endometrial glandular epithelium throughout the menstrual cycle. No specific staining was demonstrated in the stromal compartment. CONCLUSION : CD59 protein and terminal complement proteins are expressed in glandular epithelial cells of normal human endometrium, in both proliferative and luteal phases, suggesting that expression is not hormonally dependent. These analyses further support the presence of a functionally active complement system in normal human endometrium.     

Effects of depot-medroxyprogesterone acetate on the immune microenvironment of the human cervix and endometrium: implications for HIV susceptibility

Depot-medroxyprogesterone acetate is a commonly used injectable contraceptive that has been associated with an increased risk of HIV acquisition. This study compares effects of depot-medroxyprogesterone acetate on immune parameters from several upper reproductive tract compartments relevant to HIV-1 susceptibility in repetitive samples from 15 depot-medroxyprogesterone acetate users and 27 women not on hormonal contraceptives. Compared with samples from unexposed women in the mid-luteal phase, depot-medroxyprogesterone acetate use was associated with: increased endocervical concentrations of MCP1 and IFNalpha2; decreased endocervical concentrations of IL1beta and IL6; increased proportions of endometrial CD4+ and CD8+ cells expressing the activation marker HLADR; increased density of endometrial macrophages; and decreased density of endometrial regulatory T cells. Unlike previous reports with samples from the vagina, we did not observe increased expression of the HIV co-receptor CCR5 on CD4+ T cells in the endocervix or endometrium. Our results indicate important differences in anatomic compartments regarding mechanisms by which depot-medroxyprogesterone acetate could be associated with increased risk of HIV acquisition, including increased recruitment of macrophages to the endometrium, decreased levels of pro-inflammatory cytokines in the endocervix possibly leading to enhanced susceptibility to viral infection, and activation of endometrial T cells.     

Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle

Relaxin (RLX) is a pregnancy-associated polypeptide hormone. In non-pregnant women, the peak of circulating relaxin coincides with the window of endometrial receptivity and both in vivo and in vitro experiments showed that it plays a role in the decidualization process. Recently, two receptors, LGR7 and LGR8, have been identified as high affinity receptors for relaxin. Here we describe LGR7 mRNA and protein expression in human endometrium using semi-quantitative and quantitative fluorescent PCR (Q-PCR) and immunohistochemical analyses. Three different experimental designs were used. First, endometrial biopsies from five different phases of the menstrual cycle were analysed. Secondly, we assessed the early luteal phase in more detail. Finally we analysed the expression at LH+2 (2 days after the natural LH surge, pre-receptive endometrium) versus LH+7 (receptive endometrium) within the same menstrual cycle from the same patient to avoid inter-cycle or inter-person variations in gene expression. Our results indicate that there is no consistent regulation of LGR7 mRNA expression, neither during the menstrual cycle nor during the early-mid-luteal phase. In general, we observed a large degree of variation in LGR7 mRNA expression levels between patients. LGR7 immunoreactive protein was identified in all stages of the menstrual cycle. LGR7 protein was localized in both the epithelial and the stromal compartments, except for the mid-luteal phase when the expression was restricted to the endometrial epithelium. We conclude that no consistent regulation of LGR7 mRNA expression can be detected in human endometrium during the menstrual cycle.     

Human Peritoneal Macrophage and T Lymphocyte Populations in Mild and Severe Endometriosis

PROBLEM : The aim of this study was to characterize the phenotype of peritoneal lymphocyte and macrophage populations in mild versus severe endometriosis. METHOD : Using dual staining, antigen expression on peritoneal leukocytes from 24 women with endometriosis and 21 control patients was analyzed by flow cytometry. RESULTS : All groups had CD4:CD8 ratios of 0.6, with subpopulations of CD8+ cells expressing cytotoxic marker S6F1. Mild and severe endometriosis patients had increased CD3/ DR+ cells, relative to controls. Two populations of macrophages were identified by size in all groups. Mild endometriosis patients had increased percentages of small macrophages expressing CD14 and HLA DQ, compared to controls and severe disease patients. In severe disease patients, antigen expression on small macrophages did not differ from controls, but decreased percentages of large macrophages expressed CD14 relative to controls and mild disease patients. CONCLUSION : All women with endometriosis exhibit activated peritoneal lymphocytes, whereas macrophage expression of CD 14 is differentially expressed as a function of disease stage. Alterations in the functional capacity of these cells may contribute to the pathophysiology of this disease.     

Differential Expression of VLAβ1 (CD29) on Monocytes From Patients With Endometriosis

PROBLEM : Previous studies have established that in vitro proliferation of endometrial cells is enhanced by peripheral blood monocytes (PBM) and suppressed by peritoneal macrophages (PM) from patients with endometriosis but only suppressed by PBM and PM obtained from normal subjects. The functional activity of PBM and PM is influenced by the engagement of numerous cell surface receptors with their respective physiological ligands. METHOD : In this study, PBM and PM from fertile women (Group 1), women with unexplained infertility (Group 2), and women with limited (Group 3) or severe (Group 4) endometriosis were isolated in order to analyze these cells for the expression of CD54, CD58 and HLA-DR (immunoglobulin supergene antigens) CD18 and CD29 (integrins) and CD44 (an addresin). These cell surface antigens are involved in monocyte/macrophage trafficking, activation, signal transduction and/or adhesion. RESULTS : No differences were detected in the percentage of PBM expressing CD18, CD44, CD54, CD58, or HLA-DR among the four groups of subjects. Furthermore, the density of these antigens expressed on PBM was identical in patients and control subjects. In contrast, the percentage of PBM expressing CD29 (also known as VLAβ1) and the density of CD29 expressed per cell were significantly reduced (P < 0.01) in patients with limited endometriosis compared to controls and patients with severe disease. Interestingly, although the percentage of CD29+ PBM from women with severe endometriosis was not statistically different from the percentage of CD29+ PBM from controls, the density of CD29 expressed per cell was significantly elevated among patients with severe disease. Analysis of PM from the four subject groups revealed no differences in CD29 expression or density. However, the percentage of PM expressing CD18 was significantly decreased in patients with limited (but not severe) endometriosis. CONCLUSION : Since both CD18 and CD29 play a role in cell trafficking and/or adhesion, alterations in their expression among patients with endometriosis suggest that these integrin β chains may play a role in the pathogenesis of the disease.     

Increased expression of human macrophage metalloelastase (MMP-12) is associated with the invasion of endometrial adenocarcinoma

To evaluate the association between the expression of human macrophage metalloelastase (matrix metalloproteinase-12, MMP-12) with cancer invasion and differentiation of endometrial adenocarcinoma, specimens from endometrial adenocarcinoma (n=61) of diverse stages and histologic types were collected from patients having undergone hysterectomy, and specimens from normal endometrium (n=38) were obtained from patients with benign diseases. The expression of MMP-12 was analyzed immunohistochemically, by Western blot, and RT-PCR. The positive rate of MMP-12 was significantly increased in endometrial adenocarcinoma (81.97%) as compared with that in normal endometrium (13.16%). The results showed that expression of MMP-12 correlated with stage (p=0.022) and grade (p=0.018) of endometrial cancer. MMP-12 immunoreactive proteins were found mainly on the glandular epithelial cells of endometrial adenocarcinoma. The macrophage infiltration detected by CD68 immunohistochemical staining in endometrial adenocarcinoma was also higher than that in normal endometrium. In this study, we show that in addition to macrophages, endometrial adenocarcinoma cells are able to express MMP-12. Increased MMP-12 expression tended to be associated with the extent of adenocarcinoma invasion accompanied by marked macrophage infiltration. Our results suggest that MMP-12 is an important oncogene in high-stage and high-grade endometrial adenocarcinoma.     

A defective expression of ICAM-1 (CD54) on secretory endometrial cells is associated with endometriosis.

A defect in the cytolytic activity against autologous endometrial cells refluxed in the peritoneal cavity has been hypothesized as being involved in the etiology of endometriosis, although its causes have not been definitively identified. Cell adhesion molecules are necessary not only for cell-to-cell contact but also for the binding of immune effectors to their targets. In this study, the expression of CD54, CD58 and CD106, three adhesion molecules with a crucial role in cytotoxic mechanisms, was quantitatively studied on fresh endometrial cells by immunofluorescence and flow cytometry. Samples were collected from endometriosis patients (n=10) and controls (n=12), either during the follicular or luteal phase of the cycle. While no significant differences were observed for CD58 and CD106, a significantly reduced expression of CD54 in the secretory endometrial cells of women with endometriosis was observed (-75% with respect to apparently healthy controls). These findings could account for an apparently cyclic defect in the expression of CD54 that could result in poor binding of immune effectors to secretory endometrial cells in vivo. The defective recognition and removal of refluxed endometrial cells could, at least in part, be involved in the pathogenesis of endometriosis.