Expression of the Peptidase "Fibroblast Activation Protein" on Decidual Stromal Cells Facilitating Tissue Remodeling.

INTRODUCTION: Expression of fibroblast activation protein (FAP) has been detected in activated fibroblasts participating in injury response, fibrotic and inflammatory conditions, and tumorigenesis. Human endometrium is equally characterized by rapid tissue remodeling events due to the reproductive tasks comprising the activity of proteolytic enzymes. OBJECTIVE: We therefore hypothesized that FAP-positive fibroblasts could also be involved in physiological processes requiring tissue remodeling, such as decidualization during early pregnancy. METHODS/RESULTS: The expression of FAP was analyzed by immunohistochemistry in frozen sections of decidual tissue from early pregnancy (gestational weeks: 6-12). All tissue samples clearly displayed a strong expression of FAP on the surface of stromal fibroblasts. Additionally, the percentage of FAP-positive fibroblasts freshly isolated from the decidua of the corresponding gestational weeks was calculated by applying FACS analysis. Decidual fibroblasts of different gestational weeks showed a significant decrease in FAP expression between the 6th and 7th weeks of gestation, which was followed by a steady slow reconstitution. By analyzing the expression of cytokines, chemokines, and growth factors of isolated FAP-positive decidual fibroblasts, we detected high levels of monocyte-attracting chemokines (growth-related oncogene alpha and monocyte chemoattractant protein-1 and -2), granulocyte-attracting chemokines (e.g., IL-8), proinflammatory factors (IL-1α and tumor necrosis factor alpha), and angiogenic substances (e.g., vascular endothelial growth factor and IL-8), which all promote an optimal microenvironment for implantation and growth of the conceptus. CONCLUSIONS: Our data demonstrate that the healthy early pregnancy decidua is characterized by a general occurrence of FAP-positive fibroblasts possibly participating in active tissue remodeling during implantation.

CD33(+) /HLA-DR(neg) and CD33(+) /HLA-DR(+/-) Cells: Rare Populations in the Human Decidua with Characteristics of MDSC.

PROBLEM: Human pregnancy needs a remarkable local immune tolerance toward the conceptus. Myeloid-derived suppressor cells (MDSC) are important players promoting cancer initiation and progression by suppressing T-cell functions and thus inducing immune tolerance. Therefore, MDSC were expected within decidua. METHODS: Subpopulations of CD33(+) immune cells were isolated from human early pregnancy decidua and characterized phenotypically and functionally by microscopy, FACS analysis, RT-PCR, Western blotting and in the coculture with T cells. RESULTS: Decidua harbors CD33(+) /HLA-DR(neg) and CD33(+) /HLA-DR(+/-) cells which both express arginase, iNOS and IDO and a typical cytokine profile. Both subtypes potently suppress T-cell proliferation and therefore fulfill the criteria of MDSC. CONCLUSION: We characterized a new population of CD33(+) /HLA-DR(neg) and CD33(+) /HLA-DR(+/-) cells in human early pregnancy decidua with properties of classical MDSC and thus potentially being an important player in immune tolerance in pregnancy.

Quantification of the predominant immune cell populations in decidua throughout human pregnancy.

PROBLEM: To date, a multiplicity of factors contributing to the establishment and progression of a successful pregnancy have been postulated. There is emerging evidence that decidual leukocytes could be decisive factors during pregnancy. Despite numerous investigations on immune cells in human early pregnancy decidua, little is known about the physiological composition and proportion of the various immune cell populations during the different phases of pregnancy. In this study, we therefore analyzed the proportion of the dominant decidual leukocytes in human tissue samples derived from all phases of pregnancy. METHODS: Single cell suspensions were prepared from decidual samples from 205 patients at 6-40 weeks of gestation. Cell populations were analyzed by flow cytometry, and immune cell populations were quantified as percentage of decidual CD45(+) cells. RESULTS: There was generally no difference in immune cell counts comparing decidua of healthy gestations and those with systemic inflammation. Overall, the proportion of uNK cells continuously decreased, while the amount of monocytes, immature dendritic cells, and T cells increased until term. Striking modifications in cell counts were seen during the 7th week compared with the 6th and later weeks of gestation. CONCLUSION: Studying the proportion of decidual immune cells during pregnancy, we detected a unique pattern which could be useful to design novel therapies for pathological conditions during pregnancy.

Mechanisms of tumor immune escape in triple-negative breast cancers (TNBC) with and without mutated BRCA 1.

PURPOSE: Triple negative breast cancers (TNBC) are associated with an adverse outcome, although these tumors are sensitive to chemotherapy. In part, this phenomenon could be caused by tumor immune escape. The current study investigates immunogenicity of TNBC cells in vitro and the presence of immunosuppressive factors in the tumor microenvironment (pAKT and B7H1 expression, infiltration with regulatory T cells, [Tregs]). METHODS: Natural killer (NK)-cell induced lysis was evaluated in estrogen receptor (ER) positive MCF 7 breast cancers, in MDA-MB231 and MDA-MB468 and in HCC-1937 (BRCA 1 mutated) and HCC-1806 TNBC cells. Expression of pAKT, B7H1 and infiltration with Tregs were determined by immunohistochemistry in human specimens of benign and malignant breast disease. RESULTS: NK-cell induced lysis was significantly increased (p < 0.05) in four TNBC cell lines compared to ER + MCF 7 cells. Fibroadenomas and mastectomy samples were not infiltrated with Tregs. Infiltration with Tregs was 0.92 ± 0.21 in ER/PR + breast cancers and significantly higher in TNBC without (2.30 ± 0.34) and also significantly higher with mutation of BRCA 1 (2.10 ± 0.34). Expression of pAKT was absent in benign controls and 1.23 ± 0.36 in ER/PR + breast cancers, 1.78 ± 0.40 in TNBC without and 2.40 ± 0.30 with mutated BRCA 1. No significant differences of B7H1 expression occurred among the breast cancer subgroups. CONCLUSION: TNBC cell stimulate the NK-cell immune response significantly stronger than ER positive breast cancer cells. This could explain why infiltration with immunosuppressive Tregs is increased in human specimens of TNBC with and without mutated BRCA 1. Accordingly, immunomodulatory treatment strategies should be further explored in TNBC.

Dendritic cells: elegant arbiters in human reproduction.

The female reproductive tract represents a great challenge to the residing immune cells: Concomitantly, those immune-competent cells have to provide tolerogenic mechanisms favoring the development of a successful pregnancy while permitting protection against harmful pathogens. The predominant cell population facing this "double edged" regulatory capacity within the reproductive tract is that of dendritic cells (DC). There is evidence that DC represent a highly adaptive cell type, which can either be transformed in an immune-stimulatory phenotype after exposure to inflammatory or infectious signals, or in an immune inhibitory phenotype preventing T cell activation when located in an adequate antiinflammatory microenvironment. Thus, this review highlights this two-faced character of DC focusing on their morphology and function within the human reproductive tract and especially during pregnancy.

Human decidua and invasive trophoblasts are rich sources of nearly all human matrix metalloproteinases.

Trophoblast cell (CTB) invasion into the maternal endometrium plays a crucial role during human embryo implantation and placentation. As for all invasive cell types, the ability of CTB to infiltrate the uterine wall is facilitated by the activity of matrix metalloproteinases (MMPs), which is regulated by tissue inhibitors of MMPs (TIMPs). There is evidence for the expression of several MMPs and TIMPs in decidua. However, published data are limited. Therefore, to set a foundation for future research, we screened a panel of healthy human deciduas obtained during first, second and third trimester of pregnancy in addition to isolated decidual cell populations for the expression of all known human MMPs and TIMPs by RT-PCR, western blot and immunohistochemistry. In the decidual samples, we detected almost all MMPs and all four TIMPs at mRNA level. While the expression of proMMP-3 and active MMP-13 and -23 was down-regulated in the course of pregnancy, the pro forms of MMP-8, -19 and -23, active MMP-9, -10, -12, -15, -16, -26 and -28, and pro- and active MMP-14 increased towards the end of gestation. All MMPs and TIMPs were expressed in uterine natural killer cells, decidual fibroblasts and/or trophoblasts, with the exception of MMP-20 and -25. In summary, a remarkably broad spectrum of MMPs was expressed at the human feto-maternal interface, reflecting the highly invasive and remodelling effect on placenta formation. It can be speculated that expression of MMPs correlates with the invasive potential of CTBs together with a crucial role in activation of labour at term.

Pregnancy-triggered antiphospholipid syndrome in a patient with multiple late miscarriages.

Antiphospholipid syndrome (APS) is a multisystemic disorder of coagulation-causing thrombosis in the arterial and venous system as well as pregnancy-related complications such as miscarriage, stillbirth, preterm delivery and pre-eclampsia. The disease is characterized by the autoimmune production of antibodies against phospholipid, a substance found in the cell membrane. We here report the case of a patient with four second trimester miscarriages, who apart from a heterozygous plasminogen activator-inhibitor-1 mutation, had no risk factors explaining her condition. In the subsequent pregnancy she was therefore put on low-molecular-weight heparin, aspirin and granulocyte colony-stimulating factor. Antiphospholipid antibodies (APL), which had been negative before gestation, increased and remained high throughout pregnancy, thus suggesting a pregnancy-induced or -aggravated APS. The patient was kept on the above-mentioned medication and delivered a healthy male baby by Caesarean section after an otherwise uneventful pregnancy. Thus, in order to diagnose and treat pregnancy-triggered APS in patients with unexplained recurrent miscarriage, screening for APL should also be performed at several time points after conception.

Impact of female sex hormones on the maturation and function of human dendritic cells.

PROBLEM: During pregnancy, the immune and the endocrine system cooperate to ensure that the fetal allograft develops without eliciting a maternal immune response. This is presumably in part achieved by dendritic cells (DCs) that play a dominant role in maintaining peripheral tolerance. In this study, we investigated whether female sex hormones, such as human chorionic gonadotropin (hCG), progesterone (Prog), and estradiol (E2), which are highly elevated during pregnancy, induce the differentiation of DCs into a tolerance-inducing phenotype. METHODS/RESULTS: Immature DCs were generated from blood-derived monocytes and differentiated in the presence of hCG, Prog, E2, or Dexamethasone (Dex) as a control. Unlike Dex, female sex hormones did not prevent the upregulation of surface markers characteristic for mature DCs, such as CD40, CD83, and CD86, except for hCG, which inhibited HLA-DR expression. Similarly, hCG, Prog, and E2 had any impact on neither the rearrangement of the F-actin cytoskeleton nor the enhanced chemokine secretion following DC maturation, both of which were strongly altered by Dex. Nevertheless, the T-cell stimulatory capacity of DCs was significantly reduced after hCG and E2 exposure. CONCLUSION: Our findings suggest that the female sex hormones hCG and E2 inhibit the T-cell stimulatory capacity of DCs, which may help in preventing an allogenic T-cell response against the embryo.

The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation.

BACKGROUND: Pregnancy represents an exclusive situation in which the immune and the endocrine system cooperate to prevent rejection of the embryo by the maternal immune system. While immature dendritic cells (iDC) in the early pregnancy decidua presumably contribute to the establishment of peripheral tolerance, glycoprotein-hormones of the transforming growth factor beta (TGF-beta) family including activin A (ActA) and inhibin A (InA) are candidates that could direct the differentiation of DCs into a tolerance-inducing phenotype. METHODS: To test this hypothesis we generated iDCs from peripheral-blood-monocytes and exposed them to TGF-beta1, ActA, as well as InA and Dexamethasone (Dex) as controls. RESULTS: Both glycoprotein-hormones prevented up-regulation of HLA-DR during cytokine-induced DC maturation similar to Dex but did not influence the expression of CD 40, CD 83 and CD 86. Visualization of the F-actin cytoskeleton confirmed that the DCs retained a partially immature phenotype under these conditions. The T-cell stimulatory capacity of DCs was reduced after ActA and InA exposure while the secretion of cytokines and chemokines was unaffected. CONCLUSION: These findings suggest that ActA and InA interfere with selected aspects of DC maturation and may thereby help preventing activation of allogenic T-cells by the embryo. Thus, we have identified two novel members of the TGF-beta superfamily that could promote the generation of tolerance-inducing DCs.