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Cytotoxic response of uterine natural killer cells in abnormal pregnancy
A4
Abstracts / Journal of Reproductive Immunology 75 (2007) A3–A17
L3 Cytotoxic response of uterine natural killer cells in abnormal pregnancy A.T. Yamada Department of Histology & Embryology, Institute of Biology, University of Campinas, Campinas, SP, Brazil The new paradigm of innate immune responsive natural killer (NK) cells comprises the high cytokine producing, low cytotoxic CD56bright /CD16dim subset and high cytotoxic CD56dim /CD16bright subset as two functionally distinct populations of human blood circulating NK (cNK) cells. The uterine natural killer (uNK) CD56bright /CD16dim cells found in the decidualized uterus produce cytokine that sustain pregnancy, but they also accumulate all cytolytic mediators in their secretory lysosome granules and seem to comprise a third subset of NK lymphocytes. In pregnant mice, these uNK cells are recognized as Dolichos biflorus (DBA)-lectin positive cells to distinguish from cNK cells. UNK cells are the most abundant lymphocyte population in the decidualized endometrium of pregnant women and rodent uteri and all experimental evidence suggests these cells mediate the Th2 type-positive regulation of homeostasis at the maternal–fetal interface during pregnancy. On the other hand, several indirect evidences suggest the aggressive involvement of uNK in the miscarriages and interruption of gestations in women of unknown etiology. However, if the uNK cells are responsive to any stimuli to develop cytolytic activity in the pregnant uterus or the mechanism of down-regulation controlling their cytotoxicity in the uterine environment still remains to be proved experimentally. To evaluate the uNK cell behavior in the abnormal pregnancy, we established an experimental model of pregnancy interruption in mice provoked by surgical damage of embryo. The uNK cell in the embryo-damaged uterus changes drastically with disruptions of their secretory lysosome granules and releasing of perforin and proteoglycans from secretory compartment as soon as 30 min after embryo lesion. The expression of perforin, granzyme A, HSP70, IFN-␥ and TNF-␣ decreased during first 2 h after lesion and gradually recovered after 6 h. Interestingly, the uNK cells express iNOS, eNOS and nNOS in the normal pregnancy that probably contributes to NO production at the maternal–fetal interface to increase vascular permeability and angiogenesis. After embryo lesion, the iNOS and nNOS contents decreased drastically during first 2 h and recovered after 6 h as evaluated both by immunocytochemistry and Western blot. It means a quick and strong consumption of these enzymes committed on production of NO that affected drastically the permeability of local blood vessels, inducing hyperemia followed by hemorrhage. Similar effects were detected in animals inoculated with LPS, but these were inhibited by pre-treatment of animals with L-NAME both in LPS treated and embryo-damaged animals. All these evidences rescue the uNK cells as cytolytic effective cell and any unbalance of homeostasis at maternal–fetal interface during pregnancy can change the uNK cell amiable behavior in the pregnancy. Grants: CNPq and FAPESP. doi:10.1016/j.jri.2007.06.005 L4 Laboratory evaluation of NK cell parameters in women with reproductive failures E. Ntrivalas, A. Gilman-Sachs, J. Kwak-Kim, K. Beaman Clinical Immunology Laboratory, Rosalind Franklin University of Medicine and Science/The Chicago Medical School, Chicago, IL, USA Natural killer (NK) cells belong to the innate immunity and can function either by direct cytotoxicity against specific targets or by cytokine production. NK cells with the characteristic phenotype of CD56bright/CD16− are present in the human decidua in early pregnancy and play a role in implantation by controlling trophoblast invasion and vascular remodeling. In peripheral blood, CD56dim/CD16+ NK cells predominate in numbers, while the CD56bright/CD16− representing less than 2% of the total NK population. Peripheral blood NK cells, being easily accessible, have been used for diagnostic purposes of reproductive failures. It has been shown that peripheral blood NK cell numbers and cytotoxicity are often elevated in women with recurrent spontaneous abortions, compared with normal controls. Additionally, women with reproduc-
tive failures showed an increase in the activation markers CD69 and CD161 in their peripheral blood NK cells, indicating elevated NK cell cytotoxicity. NK cells regulate their actions through a balance of activating and inhibitory receptors that are expressed in their membrane and can recognize specific ligands on target cells. Trophoblast cells lack expression of the classical MHC class I. They only express the less polymorphic HLA-C and the non-classical HLA-G and -E. It is generally accepted that upon engagement of NK cell inhibitory receptors with MHC class I ligands, expressed on specific targets, an inhibitory signal is delivered to the NK cell. Killer immunoglobulin-like receptors (KIRs, also referred to as CD158) are expressed on the NK cell membrane and can be either inhibitory or activating (depending on their cytoplasmic tail). Previously, we found that peripheral blood NK cells of women with implantation failures had decreased expression of CD94, CD158a and CD158b inhibitory receptors, indicating impaired inhibition. Loss of interaction of CD94 and KIRs with their ligands (HLA-E and HLA-C, respectively) that are expressed in trophoblast cells may lead to increased NK cell cytotoxicity. Finally, NK cells produce cytokines, such as TNF-␣, IFN-␥, IL-10 and GM-CSF that can have an effect in implantation. Previously, we found that peripheral blood NK cells down-regulated intracellular expression of TNF-␣ when in contact with JEG-3 choriocarcinoma cells. Certain cytokine gene polymorphisms may influence the expression levels of cytokines. Recently, we found that the expression of the TNF-␣ promoter −308G/A and the IL-6 promoter −174G/C were different between women with reproductive failures and normal controls. In conclusion, NK cells are important for the success or failure of reproduction. The laboratory evaluation of peripheral blood NK cell parameters can assist in the diagnosis of reproductive failures. The various methods will be discussed. doi:10.1016/j.jri.2007.06.006 L5 IL-6 in reproduction G. Gutierrez, G. Junovich, V. Dubinsky, T. Gentile Dep. Inmunolog´ıa, Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires, IDEHU, CONICET, Buenos Aires, Argentina IL-6 in the prevention of abortion. IL-6 has been described as a factor involved in the prevention of recurrent abortion (Jasper, 2007). Up to now, classification of this cytokine as Th1 or Th2 is very controversial due to IL-6 having different roles depending on the dose as well as on the stage of gestation (Jauniaux, 1996). It has been demonstrated that it is necessary for implantation and its deficiency during placental vascularization could be associated with recurrent abortion. However, Interleukin 6 is involved in the pro-inflammatory process at the end of gestation (Agarwal, 2000; Schwab, 1991). This controversy has been resolved by considering that IL-6 is the factor which shifts the Th1/Th2 balance towards Th2 (Diehl, 2002). Considering this, we investigated placental IL-6 production on the high resorption rate in the CBA/j × DBA/2 murine abortion model. During pregnancy, CBA/j females mated with DBA/2 males, showed an increase in inflammatory cytokines which is associated with high abortion rate (Clark, 1980). We found that, during placental vascularization, CBA/j × DBA/2 feto-placental units are deficient in IL-6 levels and supplementation with recombinant IL-6 during this stage normalizes IL-6 levels and diminishes resorption rate (Guti´errez, 2004). IL-6 in the immunomodulatory effect of anticoagulant and antioxidant treatment of recurrent abortion. Taking into account that vitamin E is able to diminish the production in vitro of inflammatory cytokines (Tan, 2005), and that enoxaparin protective effect of CBA/j × DBA/2 recurrent abortion is associated with prevention of placental IL-6 deficiency (Guti´errez, 2004), we decided to study the effect of vitamin E on abortion rate and IL-6 production. Results showed that vitamin E supplementation during pregnancy was able not only to prevent abortion but to increased in vivo synthesis of placental IL-6. Moreover, considering that the vitamin E is able to induce the synthesis of the transcription factor inducible by hypoxia HIF 1-␣ which activates transcription of vascular endothelial growth factor VEGF (Zhang, 2004), we studied VEGF production during vitamin E and enoxaparin treatment. Results showed that both treatments increase VEGF levels, facilitating an angiogenic response and showing similar immunomodulatory protective effect. In conclusion, we postulate that IL-6 could be involved in the regulation of the inflammatory predominance of the implantation window to favor placental angiogenesis. Placental IL-6 deficiency proper of this model could be associated with exacerbated inflammatory response
Abstracts / Journal of Reproductive Immunology 75 (2007) A3–A17
L3 Cytotoxic response of uterine natural killer cells in abnormal pregnancy A.T. Yamada Department of Histology & Embryology, Institute of Biology, University of Campinas, Campinas, SP, Brazil The new paradigm of innate immune responsive natural killer (NK) cells comprises the high cytokine producing, low cytotoxic CD56bright /CD16dim subset and high cytotoxic CD56dim /CD16bright subset as two functionally distinct populations of human blood circulating NK (cNK) cells. The uterine natural killer (uNK) CD56bright /CD16dim cells found in the decidualized uterus produce cytokine that sustain pregnancy, but they also accumulate all cytolytic mediators in their secretory lysosome granules and seem to comprise a third subset of NK lymphocytes. In pregnant mice, these uNK cells are recognized as Dolichos biflorus (DBA)-lectin positive cells to distinguish from cNK cells. UNK cells are the most abundant lymphocyte population in the decidualized endometrium of pregnant women and rodent uteri and all experimental evidence suggests these cells mediate the Th2 type-positive regulation of homeostasis at the maternal–fetal interface during pregnancy. On the other hand, several indirect evidences suggest the aggressive involvement of uNK in the miscarriages and interruption of gestations in women of unknown etiology. However, if the uNK cells are responsive to any stimuli to develop cytolytic activity in the pregnant uterus or the mechanism of down-regulation controlling their cytotoxicity in the uterine environment still remains to be proved experimentally. To evaluate the uNK cell behavior in the abnormal pregnancy, we established an experimental model of pregnancy interruption in mice provoked by surgical damage of embryo. The uNK cell in the embryo-damaged uterus changes drastically with disruptions of their secretory lysosome granules and releasing of perforin and proteoglycans from secretory compartment as soon as 30 min after embryo lesion. The expression of perforin, granzyme A, HSP70, IFN-␥ and TNF-␣ decreased during first 2 h after lesion and gradually recovered after 6 h. Interestingly, the uNK cells express iNOS, eNOS and nNOS in the normal pregnancy that probably contributes to NO production at the maternal–fetal interface to increase vascular permeability and angiogenesis. After embryo lesion, the iNOS and nNOS contents decreased drastically during first 2 h and recovered after 6 h as evaluated both by immunocytochemistry and Western blot. It means a quick and strong consumption of these enzymes committed on production of NO that affected drastically the permeability of local blood vessels, inducing hyperemia followed by hemorrhage. Similar effects were detected in animals inoculated with LPS, but these were inhibited by pre-treatment of animals with L-NAME both in LPS treated and embryo-damaged animals. All these evidences rescue the uNK cells as cytolytic effective cell and any unbalance of homeostasis at maternal–fetal interface during pregnancy can change the uNK cell amiable behavior in the pregnancy. Grants: CNPq and FAPESP. doi:10.1016/j.jri.2007.06.005 L4 Laboratory evaluation of NK cell parameters in women with reproductive failures E. Ntrivalas, A. Gilman-Sachs, J. Kwak-Kim, K. Beaman Clinical Immunology Laboratory, Rosalind Franklin University of Medicine and Science/The Chicago Medical School, Chicago, IL, USA Natural killer (NK) cells belong to the innate immunity and can function either by direct cytotoxicity against specific targets or by cytokine production. NK cells with the characteristic phenotype of CD56bright/CD16− are present in the human decidua in early pregnancy and play a role in implantation by controlling trophoblast invasion and vascular remodeling. In peripheral blood, CD56dim/CD16+ NK cells predominate in numbers, while the CD56bright/CD16− representing less than 2% of the total NK population. Peripheral blood NK cells, being easily accessible, have been used for diagnostic purposes of reproductive failures. It has been shown that peripheral blood NK cell numbers and cytotoxicity are often elevated in women with recurrent spontaneous abortions, compared with normal controls. Additionally, women with reproduc-
tive failures showed an increase in the activation markers CD69 and CD161 in their peripheral blood NK cells, indicating elevated NK cell cytotoxicity. NK cells regulate their actions through a balance of activating and inhibitory receptors that are expressed in their membrane and can recognize specific ligands on target cells. Trophoblast cells lack expression of the classical MHC class I. They only express the less polymorphic HLA-C and the non-classical HLA-G and -E. It is generally accepted that upon engagement of NK cell inhibitory receptors with MHC class I ligands, expressed on specific targets, an inhibitory signal is delivered to the NK cell. Killer immunoglobulin-like receptors (KIRs, also referred to as CD158) are expressed on the NK cell membrane and can be either inhibitory or activating (depending on their cytoplasmic tail). Previously, we found that peripheral blood NK cells of women with implantation failures had decreased expression of CD94, CD158a and CD158b inhibitory receptors, indicating impaired inhibition. Loss of interaction of CD94 and KIRs with their ligands (HLA-E and HLA-C, respectively) that are expressed in trophoblast cells may lead to increased NK cell cytotoxicity. Finally, NK cells produce cytokines, such as TNF-␣, IFN-␥, IL-10 and GM-CSF that can have an effect in implantation. Previously, we found that peripheral blood NK cells down-regulated intracellular expression of TNF-␣ when in contact with JEG-3 choriocarcinoma cells. Certain cytokine gene polymorphisms may influence the expression levels of cytokines. Recently, we found that the expression of the TNF-␣ promoter −308G/A and the IL-6 promoter −174G/C were different between women with reproductive failures and normal controls. In conclusion, NK cells are important for the success or failure of reproduction. The laboratory evaluation of peripheral blood NK cell parameters can assist in the diagnosis of reproductive failures. The various methods will be discussed. doi:10.1016/j.jri.2007.06.006 L5 IL-6 in reproduction G. Gutierrez, G. Junovich, V. Dubinsky, T. Gentile Dep. Inmunolog´ıa, Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires, IDEHU, CONICET, Buenos Aires, Argentina IL-6 in the prevention of abortion. IL-6 has been described as a factor involved in the prevention of recurrent abortion (Jasper, 2007). Up to now, classification of this cytokine as Th1 or Th2 is very controversial due to IL-6 having different roles depending on the dose as well as on the stage of gestation (Jauniaux, 1996). It has been demonstrated that it is necessary for implantation and its deficiency during placental vascularization could be associated with recurrent abortion. However, Interleukin 6 is involved in the pro-inflammatory process at the end of gestation (Agarwal, 2000; Schwab, 1991). This controversy has been resolved by considering that IL-6 is the factor which shifts the Th1/Th2 balance towards Th2 (Diehl, 2002). Considering this, we investigated placental IL-6 production on the high resorption rate in the CBA/j × DBA/2 murine abortion model. During pregnancy, CBA/j females mated with DBA/2 males, showed an increase in inflammatory cytokines which is associated with high abortion rate (Clark, 1980). We found that, during placental vascularization, CBA/j × DBA/2 feto-placental units are deficient in IL-6 levels and supplementation with recombinant IL-6 during this stage normalizes IL-6 levels and diminishes resorption rate (Guti´errez, 2004). IL-6 in the immunomodulatory effect of anticoagulant and antioxidant treatment of recurrent abortion. Taking into account that vitamin E is able to diminish the production in vitro of inflammatory cytokines (Tan, 2005), and that enoxaparin protective effect of CBA/j × DBA/2 recurrent abortion is associated with prevention of placental IL-6 deficiency (Guti´errez, 2004), we decided to study the effect of vitamin E on abortion rate and IL-6 production. Results showed that vitamin E supplementation during pregnancy was able not only to prevent abortion but to increased in vivo synthesis of placental IL-6. Moreover, considering that the vitamin E is able to induce the synthesis of the transcription factor inducible by hypoxia HIF 1-␣ which activates transcription of vascular endothelial growth factor VEGF (Zhang, 2004), we studied VEGF production during vitamin E and enoxaparin treatment. Results showed that both treatments increase VEGF levels, facilitating an angiogenic response and showing similar immunomodulatory protective effect. In conclusion, we postulate that IL-6 could be involved in the regulation of the inflammatory predominance of the implantation window to favor placental angiogenesis. Placental IL-6 deficiency proper of this model could be associated with exacerbated inflammatory response