Journal of Reproductive Immunology

Editorial Board

2010-01-01

Notch activation enhances IFNγ secretion by human peripheral blood and decidual NK cells

2009-11-26

Abstract: NK cells specialize in killing tumor cells and virally infected cells and also possess non-cytotoxic functions, which include secretion of a variety of cytokines and growth factors. Their activity is mediated by a vast repertoire of inhibitory and activating NK receptors. Recently, it was demonstrated that ligation of the Notch receptor plays a significant role not only in T cell development but also in human T cell and mouse NK cell activation. However, the involvement of Notch triggering in human NK cell activity has not yet been determined. Here we show that Notch1 and Notch2, but not Notch3 and Notch 4, are expressed in human peripheral blood NK cells and in decidual NK cells. We demonstrate that in peripheral blood NK cells only the Notch ligand Delta4 could interact with Notch, whereas in decidual NK cells both Delta1 and Delta4 can interact with Notch. Finally, we show that Notch activation in these cells leads to increased secretion of IFNγ. We therefore present here a new function of the Notch receptors as enhancers of peripheral blood NK cell and decidual NK cell functions.

Herpes simplex virus (HSV)-specific T cells activated in the absence of IFN-gamma express alternative effector functions but are not protective against genital HSV-2 infection

2009-11-18

Abstract: Interferon gamma (IFNγ) is important for immune resistance to herpes simplex virus (HSV) infection. To examine the influence of IFNγ on the development of HSV-specific immune responses and test for IFNγ-independent adaptive immune mechanisms of protection, IFNγ-deficient mice (IFNγ−/−) were immunized with thymidine kinase-deficient HSV-2 (HSV-2 333tk−). HSV-specific cellular and humoral responses were elicited in immunized IFNγ−/− mice resulting in increased resistance relative to non-immune C57BL/6J (B6) mice following challenge with fully virulent HSV-2. CD8+ T cells from IFNγ−/− mice displayed cytotoxic activity and secreted TNFα. HSV-specific CD4+ T cells from immunized IFNγ−/− mice secreted IL-4, TNFα, and IL-17, but unlike T cells from HSV-immune B6 mice, could not clear virus from genital tissue following adoptive transfer. HSV-immune IFNγ−/− mice produced predominantly IgG1 HSV-specific antibodies while immune B6 mice produced predominantly IgG2c antibodies. Transfer of equivalent amounts of HSV-specific antibodies from either strain to naïve mice imparted equivalent early resistance against infection of the genital epithelia. However, protection against neurological symptoms mediated by immune B6 antibodies was superior late in infection. Taken together, these results demonstrate that the limited resistance of HSV-immune IFNγ−/− mice to HSV-2 infection resulted from the action of HSV-specific Ab rather than IFNγ-independent effector functions of T cells. Further, protection against neurological manifestations of HSV-2 infection was superior in mice receiving Ab from immune B6 mice suggesting that Ab-mediated protective mechanisms involving IFNγ-induced IgG subclasses were more effective once virus had spread to neural tissues.

Fibroblast growth factor-inducible 14 (Fn14) is expressed in the lower genital tract and may play a role in amplifying inflammation during infection

Eugene S. Han, Samrawit Mekasha, Robin R. Ingalls — 2009-11-23

Abstract: TNF-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) cytokine superfamily which regulates a number of cellular responses, including inflammation and proliferation. TWEAK is primarily secreted by phagocytic cells and its receptor, fibroblast growth factor-inducible 14 (Fn14), is expressed on non-lymphoid cells, including epithelial, endothelial and mesenchymal cells. The TWEAK/Fn14 pathway is highly conserved from an evolutionary standpoint, and has been shown to play a role in tissue regeneration and inflammation in the liver, kidney, lung and skeletal muscle. We hypothesized that TWEAK/Fn14 might have a physiological role in regulating infection-induced inflammation in the lower female genital tract. To test this hypothesis, we examined expression of the receptor Fn14 in relevant cells and tissue. Receptor function was tested by treating cells with recombinant TWEAK, with and without other known proinflammatory stimuli. Flow cytometric analysis of vaginal and cervical epithelial cells revealed that Fn14 was highly expressed at the cell surface. We also detected both Fn14 and TWEAK in whole cervical tissue by RT-PCR. Treatment of vaginal and cervical epithelial cells with recombinant TWEAK led to a weak induction of the chemokine IL-8. However, TWEAK potentiated the effects of IL-1ß, the TLR2 ligand Pam3CysSK4, and live Neisseria gonorrhoeae in a synergistic manner. These data reveal a novel pathway for regulation of microbial-induced inflammation in the female reproductive tract and suggest that interference with the TWEAK/Fn14 pathway might be an approach to abrogate excessive infection-induced inflammation caused by sexually transmitted pathogens.

Identification of human sperm proteins that interact with human zona pellucida3 (ZP3) using yeast two-hybrid system

Rajesh K. Naz, Latha Dhandapani — 2009-11-23

Abstract: Sperm proteins that interact with zona pellucida 3 (ZP3) have not been clearly identified in humans. In the present study, the yeast two-hybrid (Y2H) system was used to identify human sperm proteins that interact with human ZP3. Human ZP3 cDNA was cloned into pAS2-1 yeast vector and used as bait to find reactive proteins in the human testis cDNA library. Six specific clones were obtained that were further confirmed for interaction using the mammalian two-hybrid system. These six clones showed homologies with several proteins in the GenBank database. Of these, the strongest ZP3-interacting protein, that shows 97% homology with ubiquitin associated protein-2 like (UBAP2L), was tested in the hemizona assay. UBAP2L antibodies significantly (p<0.001) inhibited human sperm–zona binding in this assay. We conclude that the Y2H system is a useful strategy for identifying novel genes encoding proteins that interact with ZP proteins. To our knowledge, this is the first study using the Y2H system to identify sperm proteins that interact with human oocyte ZP3. Novel proteins identified using this system may find applications in elucidating the fertilization cascade, development of a new generation of non-steroidal contraceptives, and specific diagnosis and treatment of human infertility.

Heat shock proteins on the human sperm surface

Soren Naaby-Hansen, John C. Herr — 2009-11-18

Abstract: The sperm plasma membrane is known to be critical to fertilization and to be highly regionalized into domains of head, mid- and principal pieces. However, the molecular composition of the sperm plasma membrane and its alterations during genital tract passage, capacitation and the acrosome reaction remains to be fully dissected. A two-dimensional gel-based proteomic study previously identified 98 human sperm proteins which were accessible for surface labelling with both biotin and radioiodine. In this report twelve dually labelled protein spots were excised from stained gels or PDVF membranes and analysed by mass spectrometry (MS) and Edman degradation. Seven members from four different heat shock protein (HSP) families were identified including HYOU1 (ORP150), HSPC1 (HSP86), HSPA5 (Bip), HSPD1 (HSP60), and several isoforms of the two testis-specific HSP70 chaperones HSPA2 and HSPA1L. An antiserum raised against the testis-specific HSPA2 chaperone reacted with three 65kDa HSPA2 isoforms and three high molecular weight surface proteins (78–79kDa, 84kDa and 90–93kDa). These proteins, together with seven 65kDa HSP70 forms, reacted with human anti-sperm IgG antibodies that blocked in vitro fertilization in humans. Three of these surface biotinylated human sperm antigens were immunoprecipitated with a rabbit antiserum raised against a linear peptide epitope in Chlamydia trachomatis HSP70. The results indicate diverse HSP chaperones are accessible for surface labelling on human sperm. Some of these share epitopes with C. trachomatis HSP70, suggesting an association between genital tract infection, immunity to HSP70 and reproductive failure.

Expression and function of Toll-like receptors in human endometrial epithelial cell lines

2009-11-18

Abstract: In mammals, Toll-like receptors (TLRs) are the principal family of innate immune pattern recognition receptors (PRRs). The main function for TLRs is the detection of molecular patterns associated with invading pathogens. We investigated TLR expression and function in three established human endometrial epithelial cell lines, including hTERT-EEC, HEC-1B and Ishikawa cells, and clarified the application of these endometrial cell lines as in vitro models for studying TLR expression and function in the female reproductive tract. TLR gene expression was examined by RT-PCR and protein localization by immunohistochemistry. Our results showed that TLR expression in these cell lines is comparable to published literature on TLR expression in primary human endometrial tissue. TLR function was investigated by the detection of IL-6 and IL-8 production by ELISA in response to TLR2, TLR3, TLR5, TLR7 and TLR9 ligands. We found that hTERT-EEC cells were responsive to TLR5 ligand and HEC-1B cells respond to TLR3 and TLR5 ligands. In contrast, Ishikawa cells respond only to PMA/I which was used as a positive control for IL-8 production. Finally, we investigated the influence of flagellin as a TLR5 stimulant on TLR5 expression in these cell lines by QPCR. Our results showed that the endometrial cell lines showed a tendency for increased TLR5 expression in response to flagellin stimulation and in hTERT-EEC cells this tendency was statistically significant. These results suggest that hTERT-EEC, HEC-1B and Ishikawa cell lines can be used as in vitro models to investigate innate immune responses of endometrial cells in the female reproductive tract.

γδTCR+ cells of the pregnant ovine uterus express variable T cell receptors and contain granulysin

Annette Fox, Jill F. Maddox, Mike J. de Veer, Els N. Meeusen — 2009-11-18

Abstract: γδ T cells are a prominent granulated cell population in the ruminant pregnant uterus and both their number and granule size increase dramatically during pregnancy. Anchor-RT-PCR was used to assess TCRδ gene usage by γδ T cells from the uterine epithelium of pregnant sheep. The TCRδ genes obtained exhibited distinct combinatorial and junctional diversity and only two out of nine V–D–J rearrangements sequenced were identical. Furthermore, two of the Vδ elements used are also expressed in peripheral blood, indicating that γδTCR use in sheep epithelia is neither restricted nor site-specific, similar to humans but in contrast to findings in mice. Protein analysis of purified, granulated uterine γδ T cells revealed the presence of large amounts of the antimicrobial peptide, granulysin. The results of the present study indicate that ovine uterine γδTCR+ intraepithelial lymphocytes have the potential to recognise diverse antigens and may have a role in protecting the uterus from infection during pregnancy and parturition. A similar protective role for γδ T cells may exist in the human decidua parietalis.

Spermadhesin PSP-I/PSP-II heterodimer induces migration of polymorphonuclear neutrophils into the uterine cavity of the sow

2009-11-23

Abstract: Seminal plasma (SP) is a complex fluid which exerts biological actions in the female reproductive tract. In pigs, SP elicits endometrial inflammation and consequent immune changes after mating. This study tested whether heparin-binding spermadhesins (HBPs) and the heterodimer of porcine sperm adhesions I and II (PSP-I/PSP-II) in SP recruit different lymphocyte subsets (CD2+, CD4+ and CD8+ T cells) or polymorphonuclear leukocytes (PMNs) to the superficial endometrium or luminal epithelium and lumen, respectively, of oestrous sows. In Experiment 1, endometrial biopsies were taken between 2 and 120min after infusion of uterine horns with HBPs, PSP-I/PSP-II or saline and evaluated by immunohistochemistry or histology. In Experiment 2, the uterus of oestrous sows was infused with PSP-I/PSP-II or saline to assess PMN numbers in the uterine lumen 3h later. PSP-I/PSP-II elicited CD2+ T cell recruitment from 10min, and CD8+ T cells from 60min after infusion, while HBPs increased CD4+ T cell recruitment by 120min. PSP-I/PSP-II but not HBPs induced PMN migration to the surface epithelium by 10min. PMN numbers were elevated 5-fold by 30min and 7-fold from 60min, with PMNs detectable in the lumen from 30min after infusion. Six-fold more PMNs were collected from the uterine lumen of PSP-I/PSP-II-infused sows compared to controls at 3h after infusion. These data show that PSP-I/PSP-II heterodimer in seminal plasma has a predominant role in triggering the recruitment of uterine PMNs and T cells after mating, initiating a cascade of transient and long-lasting immunological events.

Gene transcription of TLR2, TLR4, LPS ligands and prostaglandin synthesis enzymes are up-regulated in canine uteri with cystic endometrial hyperplasia–pyometra complex

2009-11-18

Abstract: Escherichia coli (E. coli) is the most frequent bacterium isolated in cases of cystic endometrial hyperplasia–pyometra complex, the most frequent endometrial disorder in the bitch. Toll-like receptors (TLRs) play an essential role in the innate immune system. The aim of this study was to compare transcription of genes encoding TLR2, TLR4 and LPS ligands (CD14, MD-2, LBP), prostaglandin synthesis enzymes (COX1, COX2, PGES1 and PGFS), and to compare COX1 and COX2 protein expression and PGE2 and PGF2α endometrial content in the endometrium of canine diestrous uteri with (n=7) or without (n=7) pyometra. All cases of pyometra were hyperplastic and E. coli was the only isolated bacteria, while diestrous normal uteri did not present signs of cystic endometrial hyperplasia and were negative for bacteriology. Except for COX1, transcription of all genes was significantly higher in pyometra than in normal endometria. COX1 protein was observed in both normal and pyometra uteri, but COX2 protein was only present in pyometra cases. Endometrial PGE2 and PGF2α content were significantly higher in pyometra than in normal diestrous endometria. In conclusion, data obtained in this study provides evidence that pyometra-isolated E. coli induces the up-regulation of TLR2 and TLR4 genes in the canine diestrous endometrium. This up-regulation, which is probably the result of the stimulation by LPS and lipoprotein E. coli constituents, leads to the endometrial up-regulation of PG synthesis genes. This, in turn, results in a higher endometrial concentration of PGE2 and PGF2α, which may further regulate the local inflammatory response.

A role for IL-17 in induction of an inflammation at the fetomaternal interface in preterm labour

2009-11-19

Abstract: Chorioamnionitis (CAM) is a major cause of preterm delivery. Inflammatory cytokines and chemokines play important roles in the pathogenesis of preterm delivery. Interleukin (IL)-17 is a key cytokine which induces inflammation and is critical to host defense. In this study, we examined the role of IL-17 in the pathogenesis of preterm delivery. The levels of cytokines including IL-17, IL-8 and tumor necrosis factor (TNF) α were measured by ELISA in amniotic fluid from 154 cases of preterm labor. Flow cytometry and immunohistochemical staining were performed to determine the distribution of IL-17-producing cells. IL-8 secretion was evaluated in primary cultured human amniotic mesenchymal (HAM) cells and human amniotic epithelial (HAE) cells stimulated with IL-17, TNFα or IL-1β. We also studied the signaling pathway of IL-17 and TNFα in HAM cells. Levels of inflammatory cytokines in amniotic fluid were higher in preterm delivery cases than in term delivery cases. Furthermore, IL-8, IL-17 and TNFα levels were significantly higher in the preterm cases with CAM stage II or III than those without CAM. Flow cytometry and immunohistochemical staining revealed that CD3+CD4+ T cells were the main source of IL-17 in the chorioamniotic membrane. Interestingly, TNFα-induced IL-8 secretion was enhanced by IL-17 in a dose-dependent manner in HAM cells. The IKK inhibitor BMS-345541 and mitogen-activated protein kinase (MAPK) inhibitors p38, JNK and p42/44 (ERK1/2 pathway) reduced IL-8 secretion by IL-17-stimulated and TNFα-stimulated HAM cells. These results indicate that IL-17, produced by T cells, promotes inflammation at the fetomaternal interface in preterm delivery.

High-mobility group box protein 1 and its signalling receptors in human preterm and term cervix

2009-11-23

Abstract: The objective of this study was to identify possible changes in mRNA and protein expression of high-mobility group box protein 1 (HMGB1) and its suggested receptors – receptor for advanced glycation end-products (RAGE) and Toll-like receptor 2 (TLR2) and TLR4 – in human cervix during pregnancy, term and preterm labor. Cervical biopsies were taken from 58 women: 20 at preterm labor, 24 at term labor, 10 at term not in labor and 4 from non-pregnant women. Real-time RT-PCR was used to quantify mRNA expression, and immunohistochemistry and ELISA for protein analysis. HMGB1, RAGE, TLR2 and TLR4 proteins were localized and their mRNA expression was detected in the cervix. There was more extranuclear HMGB1 in the cervical epithelium and stroma in preterm and term labor compared to the term not in labor. TLR2 mRNA expression was upregulated 5-fold in term labor and 3-fold in preterm labor compared to term not in labor and non-pregnant controls. There was lower expression of TLR2 and TLR4 mRNAs in preterm labor compared to term. Lower mRNA expression of HMGB1 was found in the subgroup with preterm premature rupture of membranes than in the rest of the preterm group, where levels were significantly higher than in term labor. In conclusion, extranuclear expression of HMGB1 during labor suggests a possible role of HMGB1 during the process of cervical ripening. Changes in expression of mRNAs encoding HMGB1, TLR2 and TLR4 in preterm labor suggest differences in the mechanism of cervical ripening at preterm and term delivery.

Anti-β2 glycoprotein-I antibody increases the risk of pregnancy-induced hypertension: a case-controlled study

2009-11-23

Abstract: The aim of this study was to evaluate whether anti-β2 glycoprotein-I antibody (anti-β2GPI) of the IgG or IgM classes is associated with the development of pregnancy-induced hypertension (PIH) or preeclampsia in the Japanese population. In a case-controlled cohort study, peripheral blood was obtained at 8–14 weeks of gestation from a consecutive series of 1155 women. The case group comprised 36 patients who later developed PIH during the pregnancy. Of the 36 PIH patients, 13 had severe PIH, 18 had preeclampsia and 11 had severe preeclampsia. One hundred and eleven age- and parity-matched women whose pregnancies ended in normal delivery without obstetric complications were selected as controls. We found that a titer of anti-β2GPI IgG≥1.0U/ml was a risk factor for severe PIH (P=0.023, OR 5.7 95%CI 1.4–22.8). In addition, titers of anti-β2GPI IgM≥1.2U/ml was found to be a risk factor for PIH (P=0.001, OR 8.8 95%CI 1.6–47.5). In women positive for anti-β2GPI but negative for lupus anticoagulant, anti-cardiolipin, phosphatidylserine-dependent anti-prothrombin, or kininogen-dependent anti-phosphatidylethanolamine antibodies, the presence of anti-β2GPI was not a significant risk factor for development of PIH or preeclampsia. In conclusion, the presence of anti-β2GPI antibody represents a risk factor for developing PIH and severe PIH. This finding supports the utility of anti-β2GPI determination as one of the laboratory criteria for anti-phospholipid syndrome classification. The usefulness of anti-β2GPI measurement among women without other anti-phospholipid antibodies requires further study.

Placental blood leukocytes are functional and phenotypically different than peripheral leukocytes during human labor

2009-09-04

Abstract: Rupture of the fetal membranes during human labor is associated with an inflammatory process localized to the maternal–fetal interface. There is evidence that specific leukocytes subsets are attracted to the choriodecidua, and that after homing they condition a local inflammatory microenvironment, possibly being directly involved in rupture of the membranes. In this study our aim was to compare the phenotypes and function of leukocytes located in the placental intervillous blood with peripheral leukocytes obtained before or after labor, including expression of modulators of inflammation in these cells. Flow cytometry revealed that the proportion of CD14+ cells is increased in intervillous blood, suggesting the participation of monocytes/macrophages during labor. Real time qRT-PCR showed that at term gestation and particularly during labor, placental blood leukocytes adopt a different expression pattern of pro-inflammatory cytokines than leukocytes in peripheral blood, including IL-1β and IL-1RA. During labor, both placental and peripheral leukocytes increase their secretion of matrix metalloproteinase-9. Moreover, we showed that placental leukocytes respond differently than peripheral leukocytes to bacterial lipopolysaccharide, secreting differential amounts of TNF-α, IL-1β and IL-6. Finally, a preliminary proteomic characterization of placental leukocytes revealed a significantly higher number of individual proteins than in peripheral leukocytes. Our results support the existence of selective subsets of leukocytes recruited to the maternal–fetal interface that may participate in the triggering of parturition.

Cytokine production by peripheral blood mononuclear cells of women with a history of preterm birth

2009-11-18

Abstract: Preterm birth is associated with elevated production of pro-inflammatory cytokines such as TNFα at the maternal–fetal interface. Previous studies have suggested that women with a history of preterm birth produce aberrantly strong inflammatory responses to bacterial lipopolysaccharide (LPS). However many intrauterine infections in women are associated with pathogens including Ureaplasma urealyticum, Mycoplasma hominis and Streptococcus agalactiae (group B streptococcus) that contain pro-inflammatory factors other than LPS. We evaluated whether peripheral blood leukocytes from women with a history of preterm birth produce elevated amounts of TNFα upon stimulation with pathogens associated with preterm birth and if pre-treatment with aspirin, an anti-inflammatory medication, decreases the ex vivo production of this cytokine. Heat-killed bacteria elicited increased TNFα production from leukocytes in a dose-dependent manner, but no differences in TNFα production between leukocytes from women with preterm birth and control women with term birth were detected. In women who consumed aspirin each day for one week, TNFα production was increased in leukocytes from control women stimulated with Escherichia coli and U. urealyticum, but was reduced or unchanged in leukocytes from women with preterm birth. Similar trends were observed for a subset of samples stimulated with U. urealyticum and assayed for IL-6, IL-10, IL-1β and TNFα by bead array. We conclude that leukocytes from women with a history of preterm birth do not have elevated pro-inflammatory responses to pathogens, and that reproductive history is associated with different effects of aspirin on pro-inflammatory cytokine production.

International Congress in Reproductive Immunology Cairns

2010-01-01