- Email: [email protected]
Macrophages are crucial for embryo implantation
Abstract / Journal of Reproductive Immunology 101–102 (2014) 18–39
33
Results: Compared with the value of the mid-luteal phase before pregnancy, the ratio of TNF-␣/IL-10, NK cytotoxicity, and the absolute count of CD3+ T cells and NK cells were significantly decreased at both week 12 and week 24 of the pregnancy. The TNF-␣ cytokine and absolute count of CD4+ T cells were significantly decreased at week 12 of the pregnancy; the IFN-␥ cytokine, the ratio of IFN-␥/IL-10, the percentage of NK cells and the absolute count of B cells were significantly decreased whereas the percentages of CD3 + T cells, CD4+ T cells, CD69+ T cells, and CD69+ NK cells were significantly increased at week 24 of the pregnancy. Conclusions: The decrease in TNF-␣/IL-10 ratio, NK cytotoxicity, NK cell and T cell counts probably contribute to the immune tolerance to the fetus and successful pregnancy in patients with a history of unexplained recurrent miscarriage. Furthermore, these parameters could be the potential markers for immunotherapy for unexplained recurrent miscarriage.
acteristic cytokines. Th 1 dominance points to elevated cellular immune response while Th 2 dominance represents humoral immune response and suppressive immunoregulation. Analysis of natural killer (NK) cells: If elevated NK cell numbers are detected in the blood and/or their reactivity is elevated indicates a generally upregulated immune reactivity of the patient. In the present work we summarize our experience with the 104 RSA patients investigated so far in our laboratory. We analyze the efficiency of the different measurements and emphasize the importance of the integrated evaluation of the results. Most informative results were concluded from the MLC measurement, where 32% of the patients showed hyper-reactivity against the partner and 77% of patients’ serum contained factors that enhanced the reactivity. In contrast, only 21 patients had components in their serum that blocked the reaction against the partner cells.
http://dx.doi.org/10.1016/j.jri.2013.12.105
http://dx.doi.org/10.1016/j.jri.2013.12.106
O31
O32
Immunological methods to investigate alloimmune habitual abortion
Macrophages are crucial for embryo implantation
˝ o˝ Petrányi 1 , Ádám David Fekete 1,∗ , Eszter Ujhelyi 1 , Gyoz 2 3 2 Galamb , András Széll , Attila Pajor , István Vályi-Nagy 1 1 United St. István and St. László Hospital and Outpatient Clinic, Budapest, Hungary 2 Semmelweis University, 2nd Department of Obstetrics and Gynaecology, Budapest, Hungary 3 G1 Laboratory, Budapest, Hungary
Recurrent spontaneous abortion (RSA) is repeated pregnancy loss at 6–12th week of gestation, despite normal implantation and normal embryonic development. In several cases where no anatomical, genetic, hematological, endocrine or infectious cause can be identified we speak of RSA of unknown cause. These cases can occur against a background of autoimmune/alloimmune conditions or disorders of immune regulation. Clinical observations suggest that immunotherapies used to treat RSA are effective only in those cases, where the immunological cause of RSA is accurately assessed. For this reason a national RSA committee was founded in 2013 at the 2nd Department of Obstetrics and Gynaecology. In cooperation with this committee we introduced a complex protocol for the investigation of immunological RSA. The protocol consists of the following: Flowcytometry cross-match: Detection of IgG molecules in the patient serum that react with the T or B lymphocytes of the partner showing HLA sensitization or immune regulation. Mixed leukocyte culture (MLC) and blocking antibody analysis: With this method we can determine the cellular reactivity of the patient against the partner. We can also measure whether the patients’ serum contains any factor that modifies this reactivity. Determination of Th 1–Th 2 ratio: The determination of Th 1 vs. Th 2 cell ratio is based on measuring their char-
Kenneth D. Beaman ∗ , Mukesh K. Jaiswal Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Among immune cells, the macrophages have the most potential as regulators of tissue homeostasis under various inflammation-associated conditions. During pregnancy proper, immune response is crucial to support trophoblast proliferation and differentiation for successful placental development. Our data show that semen initiates a receptive immune/inflammatory reaction in a maternal environment that will be vital for successful implantation. The sperm’s expression of vacuolar ATPase, particularly the a2 isoform, plays an important role in commencing progressive changes in a composition of immunocompetent cells at the endometrial mucosa, such as at the levels and in the phenotypes of endometrial macrophages. Once initiated, these changes result in establishment of the critical balance of inflammation at the maternal–fetal interface. This balance also allows the angiogenic portion of the inflammatory reaction to prevail, while limiting the destructive aspects of the immune response. An alteration of vacuolar ATPase expression in the male mouse via siRNA knock down or by monoclonal antibody administration results in changes related to the functional characteristics of testicular macrophages, namely modified cytokine production. These experiments mimic our earlier findings using the classic CBA/DBA model in a role of cytokine profile (systemic or testicular?) in male mouse determinative between success and failure in this recurrent miscarriage model and not an assumed adaptive immune difference. Perhaps the most interesting fact is that the alteration of cytokine production by testicular macrophages manifests in poor pregnancy outcome despite viable blastocysts being implanted.
34
Abstract / Journal of Reproductive Immunology 101–102 (2014) 18–39
This supports the idea that macrophages on both sides, maternal as well as paternal, are the chief immune cells that regulate the pregnancy outcome by controlling the inflammatory process. Moreover, paternal macrophages could be the key players in the pathogenesis of recurrent miscarriage that was previously thought to be caused only by female immune problems. http://dx.doi.org/10.1016/j.jri.2013.12.107 O33 Expression of granzyme B by non-cytotoxic cells in the decidual region of the placenta—a novel immunoregulatory mechanism during pregnancy? Christoph Scholz 2,∗ , Arthur Krause 1 , Thamara Beyer 1 , Anke Faul 2 , Emanuel Bauer 2 , Frank Reister 2 , Lisa Wiesmüller 2 , Bernd Jahrsdörfer 1 1
Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden-Württemberg-Hessen and Institute of Transfusion Medicine, Ulm University, Ulm, Germany 2 Department of Gynecology and Obstetrics, Ulm University, Ulm, Germany Certain lymphocyte subpopulations such as regulatory T cells can potently suppress T cell expansion in a granzyme B (GrB)-dependent manner. Recently, we found that plasmacytoid dendritic cells (pDC) and B cells (BC) can also secrete the cytotoxic serine protease GrB and potently suppress T cell proliferation by GrB-dependent degradation of the T cell receptor zeta chain. The fetomaternal interface constitutes a physiological immunoprivileged environment that allows the fetus to grow in spite of its semi-allogeneic character with respect to the maternal immune system. Here we hypothesized that non-cytotoxic cells in the decidual region of the placenta might be able to contribute to this immunoprivileged environment by expressing the immunoregulatory molecule GrB. Using flow cytometry, we were able to demonstrate that various non-cytotoxic cell subsets of the decidua express substantial intracellular levels of GrB. These cell subsets, which are primarily associated with antigen-presenting capacity or the capacity to differentiate into such cells included monocytes, pDC and BC. Importantly, GrB levels were significantly higher in the decidual region than in the villous stroma. Additionally, short term incubation (12 h) with various activating agents including specific cytokines (IL-3, IL-10, and IL-21), TLR agonists (CpG, LPS) or CD40 ligand resulted in consistent expression of GrB in decidual monocytes, pDC or B cells and in CD5+ cord blood B cells. Our results show that GrB-expressing non-cytotoxic cells, most of them with antigen-presenting capacity, are present at the fetomaternal interface, and may therefore play an important role in the induction of materno-fetal immunoprotection. http://dx.doi.org/10.1016/j.jri.2013.12.108
O34 Immunomodulatory pretreatment improves the success rate of the IVF-ET method in women suffering from immunologically induced infertility ˇ Jindrich Madar ∗ , Jitka Rezᡠcová, Andrea Kestlerová, Lucie Melicharová, Jaroslav Feyereisl Institute for the Care of Mother and Child, Prague, Czech Republic Problem: The in vitro fertilization-embryo transfer (IVF-ET) technique is a potent tool for the treatment of fertility disorders, but its success rate is limited by the failure of implantation. Nowadays, there is valid evidence that in many cases a discrepancy between the embryo and the mother’s immunity disturbs the implantation. In this presentation, we analyze the possibility of pre-treating the mother’s adverse immunity before the transfer of the embryo (ET). Methods: In this particular presentation we identified the “adverse immunity” as an increase in specific cell-mediated immunity (CMI) measured by the method of migration inhibition index (MII, Dimitrov et al., J. Immunol. Methods 154:147–153, 1992, slightly modified). Briefly, leukocytes separated from the woman’s blood were allowed to migrate either alone, or in the presence of antigens (sperm, trophoblast) under the agarose layer. After a 16-h incubation (37 ◦ C, 5% CO2 ), the zones of migration were measured by image analysis. When the migration in the presence of antigens was 65% or less of the control zones, the samples were considered to be CMI-positive. Immunomodulatory pretreatment (IP): At least 6 weeks prior to the ET, the couple used condom occlusion; the female partner was advised to allow any gynecological infections to heal and took a low dose of corticoids (5 mg prednisolone daily). Patients: A pool of 387 women treated by IVF-ET in the Department of Assisted Reproduction of the Institute for the Care of Mother and Child in Prague was subdivided into three groups: (1) no adverse immunity (CMI negative, n = 103); (2) CMI positive, without IP (n = 127); and (3) CMI positive, the couple underwent IP prior to the ET (n = 157). The female partners then experienced standard IVF (or ICSI, when necessary) ET treatment and the implantation was considered to be successful when the pregnancy was confirmed by ultrasound. Results: Implantation rate (IR) in untreated couples with positive CMI was 18.3%, while in immunomodulatorypretreated ones the IR reached 33.8%. In couples with negative CMI the IR was 37.2%. Conclusion: Our data suggest that activated CMI at the feto-maternal interface could be a significant brake on implantation and that immunomodulatory pretreatment increases the implantation rate in such a case. http://dx.doi.org/10.1016/j.jri.2013.12.109
33
Results: Compared with the value of the mid-luteal phase before pregnancy, the ratio of TNF-␣/IL-10, NK cytotoxicity, and the absolute count of CD3+ T cells and NK cells were significantly decreased at both week 12 and week 24 of the pregnancy. The TNF-␣ cytokine and absolute count of CD4+ T cells were significantly decreased at week 12 of the pregnancy; the IFN-␥ cytokine, the ratio of IFN-␥/IL-10, the percentage of NK cells and the absolute count of B cells were significantly decreased whereas the percentages of CD3 + T cells, CD4+ T cells, CD69+ T cells, and CD69+ NK cells were significantly increased at week 24 of the pregnancy. Conclusions: The decrease in TNF-␣/IL-10 ratio, NK cytotoxicity, NK cell and T cell counts probably contribute to the immune tolerance to the fetus and successful pregnancy in patients with a history of unexplained recurrent miscarriage. Furthermore, these parameters could be the potential markers for immunotherapy for unexplained recurrent miscarriage.
acteristic cytokines. Th 1 dominance points to elevated cellular immune response while Th 2 dominance represents humoral immune response and suppressive immunoregulation. Analysis of natural killer (NK) cells: If elevated NK cell numbers are detected in the blood and/or their reactivity is elevated indicates a generally upregulated immune reactivity of the patient. In the present work we summarize our experience with the 104 RSA patients investigated so far in our laboratory. We analyze the efficiency of the different measurements and emphasize the importance of the integrated evaluation of the results. Most informative results were concluded from the MLC measurement, where 32% of the patients showed hyper-reactivity against the partner and 77% of patients’ serum contained factors that enhanced the reactivity. In contrast, only 21 patients had components in their serum that blocked the reaction against the partner cells.
http://dx.doi.org/10.1016/j.jri.2013.12.105
http://dx.doi.org/10.1016/j.jri.2013.12.106
O31
O32
Immunological methods to investigate alloimmune habitual abortion
Macrophages are crucial for embryo implantation
˝ o˝ Petrányi 1 , Ádám David Fekete 1,∗ , Eszter Ujhelyi 1 , Gyoz 2 3 2 Galamb , András Széll , Attila Pajor , István Vályi-Nagy 1 1 United St. István and St. László Hospital and Outpatient Clinic, Budapest, Hungary 2 Semmelweis University, 2nd Department of Obstetrics and Gynaecology, Budapest, Hungary 3 G1 Laboratory, Budapest, Hungary
Recurrent spontaneous abortion (RSA) is repeated pregnancy loss at 6–12th week of gestation, despite normal implantation and normal embryonic development. In several cases where no anatomical, genetic, hematological, endocrine or infectious cause can be identified we speak of RSA of unknown cause. These cases can occur against a background of autoimmune/alloimmune conditions or disorders of immune regulation. Clinical observations suggest that immunotherapies used to treat RSA are effective only in those cases, where the immunological cause of RSA is accurately assessed. For this reason a national RSA committee was founded in 2013 at the 2nd Department of Obstetrics and Gynaecology. In cooperation with this committee we introduced a complex protocol for the investigation of immunological RSA. The protocol consists of the following: Flowcytometry cross-match: Detection of IgG molecules in the patient serum that react with the T or B lymphocytes of the partner showing HLA sensitization or immune regulation. Mixed leukocyte culture (MLC) and blocking antibody analysis: With this method we can determine the cellular reactivity of the patient against the partner. We can also measure whether the patients’ serum contains any factor that modifies this reactivity. Determination of Th 1–Th 2 ratio: The determination of Th 1 vs. Th 2 cell ratio is based on measuring their char-
Kenneth D. Beaman ∗ , Mukesh K. Jaiswal Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Among immune cells, the macrophages have the most potential as regulators of tissue homeostasis under various inflammation-associated conditions. During pregnancy proper, immune response is crucial to support trophoblast proliferation and differentiation for successful placental development. Our data show that semen initiates a receptive immune/inflammatory reaction in a maternal environment that will be vital for successful implantation. The sperm’s expression of vacuolar ATPase, particularly the a2 isoform, plays an important role in commencing progressive changes in a composition of immunocompetent cells at the endometrial mucosa, such as at the levels and in the phenotypes of endometrial macrophages. Once initiated, these changes result in establishment of the critical balance of inflammation at the maternal–fetal interface. This balance also allows the angiogenic portion of the inflammatory reaction to prevail, while limiting the destructive aspects of the immune response. An alteration of vacuolar ATPase expression in the male mouse via siRNA knock down or by monoclonal antibody administration results in changes related to the functional characteristics of testicular macrophages, namely modified cytokine production. These experiments mimic our earlier findings using the classic CBA/DBA model in a role of cytokine profile (systemic or testicular?) in male mouse determinative between success and failure in this recurrent miscarriage model and not an assumed adaptive immune difference. Perhaps the most interesting fact is that the alteration of cytokine production by testicular macrophages manifests in poor pregnancy outcome despite viable blastocysts being implanted.
34
Abstract / Journal of Reproductive Immunology 101–102 (2014) 18–39
This supports the idea that macrophages on both sides, maternal as well as paternal, are the chief immune cells that regulate the pregnancy outcome by controlling the inflammatory process. Moreover, paternal macrophages could be the key players in the pathogenesis of recurrent miscarriage that was previously thought to be caused only by female immune problems. http://dx.doi.org/10.1016/j.jri.2013.12.107 O33 Expression of granzyme B by non-cytotoxic cells in the decidual region of the placenta—a novel immunoregulatory mechanism during pregnancy? Christoph Scholz 2,∗ , Arthur Krause 1 , Thamara Beyer 1 , Anke Faul 2 , Emanuel Bauer 2 , Frank Reister 2 , Lisa Wiesmüller 2 , Bernd Jahrsdörfer 1 1
Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden-Württemberg-Hessen and Institute of Transfusion Medicine, Ulm University, Ulm, Germany 2 Department of Gynecology and Obstetrics, Ulm University, Ulm, Germany Certain lymphocyte subpopulations such as regulatory T cells can potently suppress T cell expansion in a granzyme B (GrB)-dependent manner. Recently, we found that plasmacytoid dendritic cells (pDC) and B cells (BC) can also secrete the cytotoxic serine protease GrB and potently suppress T cell proliferation by GrB-dependent degradation of the T cell receptor zeta chain. The fetomaternal interface constitutes a physiological immunoprivileged environment that allows the fetus to grow in spite of its semi-allogeneic character with respect to the maternal immune system. Here we hypothesized that non-cytotoxic cells in the decidual region of the placenta might be able to contribute to this immunoprivileged environment by expressing the immunoregulatory molecule GrB. Using flow cytometry, we were able to demonstrate that various non-cytotoxic cell subsets of the decidua express substantial intracellular levels of GrB. These cell subsets, which are primarily associated with antigen-presenting capacity or the capacity to differentiate into such cells included monocytes, pDC and BC. Importantly, GrB levels were significantly higher in the decidual region than in the villous stroma. Additionally, short term incubation (12 h) with various activating agents including specific cytokines (IL-3, IL-10, and IL-21), TLR agonists (CpG, LPS) or CD40 ligand resulted in consistent expression of GrB in decidual monocytes, pDC or B cells and in CD5+ cord blood B cells. Our results show that GrB-expressing non-cytotoxic cells, most of them with antigen-presenting capacity, are present at the fetomaternal interface, and may therefore play an important role in the induction of materno-fetal immunoprotection. http://dx.doi.org/10.1016/j.jri.2013.12.108
O34 Immunomodulatory pretreatment improves the success rate of the IVF-ET method in women suffering from immunologically induced infertility ˇ Jindrich Madar ∗ , Jitka Rezᡠcová, Andrea Kestlerová, Lucie Melicharová, Jaroslav Feyereisl Institute for the Care of Mother and Child, Prague, Czech Republic Problem: The in vitro fertilization-embryo transfer (IVF-ET) technique is a potent tool for the treatment of fertility disorders, but its success rate is limited by the failure of implantation. Nowadays, there is valid evidence that in many cases a discrepancy between the embryo and the mother’s immunity disturbs the implantation. In this presentation, we analyze the possibility of pre-treating the mother’s adverse immunity before the transfer of the embryo (ET). Methods: In this particular presentation we identified the “adverse immunity” as an increase in specific cell-mediated immunity (CMI) measured by the method of migration inhibition index (MII, Dimitrov et al., J. Immunol. Methods 154:147–153, 1992, slightly modified). Briefly, leukocytes separated from the woman’s blood were allowed to migrate either alone, or in the presence of antigens (sperm, trophoblast) under the agarose layer. After a 16-h incubation (37 ◦ C, 5% CO2 ), the zones of migration were measured by image analysis. When the migration in the presence of antigens was 65% or less of the control zones, the samples were considered to be CMI-positive. Immunomodulatory pretreatment (IP): At least 6 weeks prior to the ET, the couple used condom occlusion; the female partner was advised to allow any gynecological infections to heal and took a low dose of corticoids (5 mg prednisolone daily). Patients: A pool of 387 women treated by IVF-ET in the Department of Assisted Reproduction of the Institute for the Care of Mother and Child in Prague was subdivided into three groups: (1) no adverse immunity (CMI negative, n = 103); (2) CMI positive, without IP (n = 127); and (3) CMI positive, the couple underwent IP prior to the ET (n = 157). The female partners then experienced standard IVF (or ICSI, when necessary) ET treatment and the implantation was considered to be successful when the pregnancy was confirmed by ultrasound. Results: Implantation rate (IR) in untreated couples with positive CMI was 18.3%, while in immunomodulatorypretreated ones the IR reached 33.8%. In couples with negative CMI the IR was 37.2%. Conclusion: Our data suggest that activated CMI at the feto-maternal interface could be a significant brake on implantation and that immunomodulatory pretreatment increases the implantation rate in such a case. http://dx.doi.org/10.1016/j.jri.2013.12.109