RESUMO
One route of human exposure to environmental chemicals is oral uptake. This is primarily true for chemicals that may leach from food packaging materials, such as bisphenols and phthalate esters. Upon ingestion, these compounds are transported along the intestinal tract, from where they can be taken up into the blood stream or distributed to mucosal sites. At mucosal sites, mucosal immune cells and in the blood stream peripheral immune cells may be exposed to these chemicals potentially modulating immune cell functions. In the present study, we investigated the impact of three common bisphenols and two phthalate esters on mucosal-associated invariant T (MAIT) cells in vitro, a frequent immune cell type in the intestinal mucosae and peripheral blood of humans. All compounds were non-cytotoxic at the chosen concentrations. MAIT cell activation was only slightly affected as seen by flow cytometric analysis. Phthalate esters did not affect MAIT cell gene expression, while bisphenol-exposure induced significant changes. Transcriptional changes occurred in â¼ 25 % of genes for BPA, â¼ 22 % for BPF and â¼ 8 % for BPS. All bisphenols down-modulated expression of CCND2, CCL20, GZMB and IRF4, indicating an effect on MAIT cell effector function. Further, BPA and BPF showed a high overlap in modulated genes involved in cellular stress response, activation signaling and effector function suggesting that BPF may not be safe substitute for BPA.
RESUMO
Various physiologically relevant processes are regulated by the interaction of the receptor tyrosine kinase (c-Kit) and its ligand stem cell factor (SCF), with SCF known to be the most important growth factor for mast cells (MCs). In spite of their traditional role in allergic disorders and innate immunity, MCs have lately emerged as versatile modulators of a variety of physiologic and pathologic processes. Here we show that MCs are critical for pregnancy success. Uterine MCs presented a unique phenotype, accumulated during receptivity and expanded upon pregnancy establishment. Kit(W-sh/W-sh) mice, whose MC deficiency is based on restricted c-Kit gene expression, exhibited severely impaired implantation, which could be completely rescued by systemic or local transfer of wild-type bone marrow-derived MCs. Transferred wild-type MCs favored normal implantation, induced optimal spiral artery remodeling and promoted the expression of MC proteases, transforming growth factor-ß and connective tissue growth factor. MCs contributed to trophoblast survival, placentation and fetal growth through secretion of the glycan-binding protein galectin-1. Our data unveil unrecognized roles for MCs at the fetomaternal interface with critical implications in reproductive medicine.
Assuntos
Mastócitos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Feminino , Galectina 1/deficiência , Galectina 1/genética , Galectina 1/metabolismo , Mastócitos/transplante , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Proteínas Proto-Oncogênicas c-kit/deficiência , Proteínas Proto-Oncogênicas c-kit/genética , Fator de Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Útero/anatomia & histologiaRESUMO
In a well-established murine abortion model, stress is thought to trigger fetal rejection by inducing a proinflammatory immune response via substance P (SP), being tumour necrosis factor (TNF)-alpha-producing CD8+ T cells involved. Interestingly, the SP metabolite SP5-11 also binds to SP receptors and mediates SP-like effects on immune cells at sites of inflammation. No data were available regarding the effects of SP5-11 on pregnancy outcome in the CBA/J x DBA/2J abortion-prone combination. We investigated the influence of SP5-11 in contrast to stress or SP on the abortion rate and the cytokine production by lymphocytes as well as on the levels of CD8+ T cells. Stress and SP boosted the abortion rate and increased the percentage of type 1 [TNF-alpha, interferon-gamma, interleukin (IL)-12] and type 2 (IL-4 and IL-10) cytokine-producing lymphocytes in blood and decidua, predominantly CD8+ T cells. Interestingly, SP5-11 did not significantly affect the abortion rate or cytokine production in the decidua, while increasing the Th1 and Th2 cytokine production systemically. Our data suggest that stress and SP induce abortion by augmenting the local levels of TNF-alpha, which seems therefore to be a potent trigger of miscarriage. On the contrary, the SP metabolite SP5-11 only affects the systemic cytokine production without boosting the abortion rate in this experimental model.
Assuntos
Aborto Espontâneo/imunologia , Linfócitos T CD8-Positivos/imunologia , Fragmentos de Peptídeos/farmacologia , Estresse Fisiológico/imunologia , Substância P/toxicidade , Fator de Necrose Tumoral alfa/metabolismo , Aborto Espontâneo/induzido quimicamente , Aborto Espontâneo/etiologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Citocinas/sangue , Citocinas/metabolismo , Decídua/citologia , Decídua/efeitos dos fármacos , Decídua/imunologia , Feminino , Contagem de Linfócitos , Troca Materno-Fetal/imunologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Fragmentos de Peptídeos/toxicidade , Gravidez , Substância P/metabolismo , Substância P/farmacologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Several burning questions remain unanswered in pregnancy-related research. Pro- and anti-inflammatory cytokines orchestrate an intriguing interaction leading either to the development of a normal individual or to its rejection. Augmented Th1 cytokines' production is involved in immunological rejection of the foetus. Excessive production of Th1 cytokines, particularly of tumour necrosis factor (TNF)-alpha, also triggers apoptosis. Thus, in the present work we investigated the incidence of apoptosis in a well-known experimental model of Th1-induced abortion, characterized by increased local TNF-alpha levels. Apoptosis of lymphocytes as well as their Th1 and Th2 cytokine production were analysed by flow cytometry. TNF-alpha mRNA levels were additionally analysed by real time reverse transcription-polymerase chain reaction (RT-PCR) in placental and decidual samples. Total placental apoptosis activity was investigated by measuring caspase-3 activity and by TdT-mediated dUTP nick end label staining. Immunohistochemistry, Western blot and real time RT-PCR were used to localize and quantify several anti- and pro-apoptotic molecules at the foetal-maternal interface. Despite elevated Th1 levels at the foetal-maternal interface, mice undergoing abortion presented comparable apoptotic rates. Interestingly, we found a significant upregulation of the anti-apoptotic Bcl-2 protein at the foetal-maternal interface from abortion-prone mice, while no changes could be observed for pro-apoptotic molecules. In the light of our results, we conclude that there is no evidence of increased apoptosis in mice undergoing immunological abortion in spite of elevated TNF-alpha levels. This is probably due to a selective upregulation of anti-apoptotic pathways (i.e. Bcl-2) at the foetal-maternal interface as a compensatory and/or protective mechanism.
Assuntos
Aborto Espontâneo/metabolismo , Placenta/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Aborto Espontâneo/imunologia , Animais , Decídua/imunologia , Decídua/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Placenta/imunologia , Reação em Cadeia da Polimerase , Gravidez , RNA Mensageiro/análise , Células Th1/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Regulação para CimaRESUMO
Dipeptidyl peptidase-IV (DPP-IV, CD26), a serine protease with broad distribution in mammalian tissues and known activity in serum, participates in T-cell activation and promotes a Th1-like cytokine response. Previous data on murine abortion indicate that DPP-IV may play a critical role in pregnancy failure by inducing a Th1 local response. Here, we investigated the possible participation of DPP-IV in the onset of human spontaneous abortion (SA). The systemic (peripheral blood) and local (decidua) percentages of CD4(+), CD8(+), CD26(+) and CD56(+) cells as well as the number of Th1 lymphocytes (CCR5(+) cells) were assessed in samples from women after SAs (n = 20) and from women with normally progressing pregnancies (NPs) (n = 27) using flow cytometry and immunohistochemistry. We further measured the DPP-IV activity and concentrations of Th1 (interferon-gamma and tumour necrosis factor-alpha), Th2 [interleukin-4 (IL-4), IL-10] and Th3 (transforming growth factor-beta2) cytokines in serum samples. We could not find any difference in the number of CD4(+), CD8(+), CD26(+), CD26(+)/CD4(+) or CD8(+)/CD26(+) blood cells between NP and SA patients. No differences in the Th1, Th2 or Th3 cytokine levels could be observed between both groups. However, the percentages of decidual CD26(+) lymphocytes as well as the number of decidual Th1 cells were significantly higher in SA samples compared to samples from patients with NP. Our data support the hypothesis that CD26(+) decidual lymphocytes with DPP-IV activity may play a critical role in SAs, as previously suggested in an abortion mice model. This abortive effect may be mediated by enhancing the levels of Th1 abortogenic cytokines only locally.
Assuntos
Aborto Espontâneo/imunologia , Decídua/imunologia , Dipeptidil Peptidase 4/imunologia , Linfócitos/imunologia , Adolescente , Adulto , Contagem de Células , Citocinas/imunologia , Dipeptidil Peptidase 4/sangue , Feminino , Humanos , Imuno-Histoquímica , Subpopulações de Linfócitos/imunologia , GravidezRESUMO
PROBLEM: We previously reported a diminished expression of the heme-degrading enzymes heme oxygenases (HO)-1 and HO-2 in decidua and placenta from mice undergoing Th1-mediated abortion, strongly indicating the protective effect of HO in murine pregnancy maintenance. Here we investigated whether the expression of HO-1 and HO-2 is also reduced at the feto-maternal interface of pathologic human pregnancies. METHOD OF STUDY: Immunohistochemistry was used to detect HOs expression in placental and decidual first-trimester tissue from patients with: spontaneous abortion (n = 14), choriocarcinoma (n = 14), hydatidiform mole (H-mole) (n = 12), compared with normally progressing pregnancies (n = 15). Further, we investigated early third-trimester decidual and placental tissue from patients with pre-eclampsia (n = 13) compared with fetal growth retardation (n = 14) as age-matched controls. RESULTS: In first trimester tissue, we observed a significant reduction of HO-2 expression in invasive trophoblast cells, endothelial cells, and syncytiotrophoblasts in samples from patients with spontaneous abortion compared with normal pregnancy. H-mole samples showed a diminished expression of HO-2 in invasive trophoblast cells and endothelial cells in comparison with NP, whereas choriocarcinoma samples showed no significant differences compared with the control. In third trimester tissue, HO-2 was also reduced in syncytiotrophoblasts and invasive trophoblast cells from pre-eclampsia compared with samples from fetal growth retardation. HO-1 expression was diminished in all pathologies investigated; however, the differences did not reach levels of significance. CONCLUSIONS: Our data indicate that HOs play a crucial role in pregnancy and low expression of HO-2, as observed in pathologic pregnancies, may lead to enhanced levels of free heme at the feto-maternal interface, with subsequent upregulation of adhesion molecules, allowing enhanced inflammatory cells migration to the feto-maternal interface.
Assuntos
Decídua/enzimologia , Heme Oxigenase (Desciclizante)/metabolismo , Placenta/enzimologia , Complicações na Gravidez/enzimologia , Aborto Espontâneo/enzimologia , Aborto Espontâneo/metabolismo , Aborto Espontâneo/patologia , Adulto , Coriocarcinoma/enzimologia , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Decídua/química , Decídua/patologia , Regulação para Baixo , Células Endoteliais/química , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Feminino , Retardo do Crescimento Fetal/enzimologia , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Heme Oxigenase-1 , Humanos , Mola Hidatiforme/enzimologia , Mola Hidatiforme/metabolismo , Mola Hidatiforme/patologia , Imuno-Histoquímica/métodos , Queratinas/análise , Proteínas de Membrana , Placenta/química , Placenta/patologia , Pré-Eclâmpsia/enzimologia , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Complicações na Gravidez/metabolismo , Trofoblastos/química , Trofoblastos/enzimologia , Trofoblastos/patologiaRESUMO
PROBLEM: The immune system contributes to the outcome of pregnancy by complex immunological interactions. Cytokines especially influence the immune milieu pro or contra pregnancy. T helper 1 (Th1) cytokines [tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] cause inflammation and together are thought to threaten the maintenance of pregnancy. It has been proposed that increased levels of these Th1 cytokines activate coagulation via up-regulating the novel prothrombinase, fgl2. This study further investigates the Th1 cytokine up-regulation of fgl2 expression in a pathophysiological, stress induced abortion model, and an inflammatory, interleukin-12 (IL-12) triggered abortion model. METHOD: The DBA/2J-mated CBA/J female mice were exposed to sonic sound stress or were injected with IL-12 during early gestation. On day 13.5 of pregnancy the uteri were removed and the resorption rate was calculated. We evaluated TNF-alpha, IFN-gamma, fgl2 as well as IL-12 messenger RNA (mRNA) expression in decidual samples of all mice by quantitative, real-time polymerase chain reaction (PCR). RESULTS: A similar resorption rate of 24% was detected in stressed mice, as well as in IL-12 injected mice compared with approximately 11% in non-stressed, non-injected control mice. In stressed mice compared with controls, we observed on day 13.5 up-regulated TNF-alpha, unchanged IFN-gamma down-regulated fgl2, and a slightly increased levels of IL-12. In the IL-12 triggered abortion model, we observed up-regulated levels of TNF-alpha, IFN-gamma and fgl2. CONCLUSION: These novel data suggest two distinct cytokine patterns leading to similar abortion rates. A physiological cascade associated with up-regulation of TNF-alpha, and an IL-12-triggered cascade characterized by persistent up-regulation of TNF-alpha and IFN-gamma as well as a persistent increase in fgl2.
Assuntos
Aborto Espontâneo/metabolismo , Interferon gama/metabolismo , Tromboplastina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Aborto Espontâneo/etiologia , Aborto Espontâneo/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Feminino , Inflamação/complicações , Interferon gama/efeitos dos fármacos , Interleucina-12/metabolismo , Interleucina-12/farmacologia , Camundongos , Modelos Animais , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Psicológico/complicações , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Regulação para CimaRESUMO
PROBLEM: In recent years, the central role of cytokines in the immune response has been widely studied. It is considered that a T helper (Th)1-type cytokine profile is associated with the rejection phenomenon, whereas a Th2-type cytokine profile is associated with immunological tolerance. In pregnancy, the enhanced Th2/Th1 ratio seems to be necessary to fetal protection. Taking into account that a Th2-type response means antibody production by B cells, and that these antibodies could induce degradation of the paternal antigens, we investigated the quality of the antibodies produced during pregnancy and their regulation. METHOD OF STUDY: Review of previous data. RESULTS: The regulation of protective antibodies by IL-6 in a dose-dependent fashion is proposed as a hypothesis. CONCLUSION: Cytokines play a central role in the success (or failure) of pregnancy. However, the quality of the synthesized antibodies is also a regulatory key. The preferential synthesis of asymmetric immunoglobulin G antibodies during pregnancy could be one of the several pathways that lead to a successful pregnancy
Assuntos
Formação de Anticorpos , Interleucina-6/sangue , Manutenção da Gravidez/imunologia , Células Th2/imunologia , Animais , Anticorpos/imunologia , Feminino , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , GravidezRESUMO
A multicascade of leukocyte-endothelial cell interactions is involved in the trafficking of inflammatory lymphocytes into tissue. The primary contact between leukocytes and endothelium is mediated by selectins. Ligands for P-Selectin are preferentially expressed on Th1 cells and thereby allow migration of these inflammatory cells through the vessel wall. Since a peripheral and local Th1-type cytokine profile is present in spontaneous human abortion (SA), opposed by a Th2 dominant situation in normal pregnancies (NP), we investigated (1) the phenotype of peripheral Th1 cells by flow cytometry, as well as the Th1-type cytokine levels by ELISA, (2) the decidual expression of P- and E-Selectin by immunohistochemistry (IHC), and (3) the phenotype of decidual immunocompetent cells by IHC in patients with NP or SA. We observed enhanced production of IFN-gamma and TNF-alpha in CD8(+), CD3(+), and CD56(+) blood cells, as well as an increase in the number of CCR5(+) cells in patients suffering from SA compared to those with NP. No difference was detectable with respect to the serum levels of the two cytokines. Using IHC methods, we observed increased staining intensity of P-Selectin(+) vessels in samples of SA patients. E-Selectin was only weakly expressed in decidual endothelial cells, with no difference between NP and SA. In SA samples, E-Selectin(+) stromal cells were exclusively present. We further detected increased numbers of decidual CD8(+), CD3(+), CCR5(+), and CD56(+) cells in SA patients. We propose that Th1 lymphocyte migration into decidua is enhanced in SA due to upregulated P-Selectin expression in decidual vessels. This increase of Th1-producing lymphocytes might be involved in the rejection of trophoblasts.
Assuntos
Aborto Espontâneo/imunologia , Decídua/imunologia , Selectina-P/biossíntese , Células Th1/citologia , Regulação para Cima , Aborto Espontâneo/sangue , Adolescente , Adulto , Biomarcadores , Complexo CD3/imunologia , Antígeno CD56/imunologia , Antígenos CD8/imunologia , Decídua/citologia , Selectina E/biossíntese , Feminino , Humanos , Imunocompetência , Interferon gama/sangue , Contagem de Linfócitos , Gravidez , Receptores CCR5/imunologia , Células Th1/classificação , Células Th1/imunologia , Fator de Necrose Tumoral alfa/análiseRESUMO
PROBLEM: Recurrent spontaneous abortion (RSA) could be interpreted as the cause for the incapacity of the mother to recognize paternal antigens to produce the desired protective response. The practise of alloimmunization was introduced in an attempt to induce in the mother the production of an alloimmune response; some authors proposed an association between cytokines and RSA. The production of IL6 and its soluble receptor (sIL6R) before and after lymphocyte immunotherapy was evaluated in sera of 33 patients suffering from two or more RSA, and in sera of 47 women with normal pregnancy. METHOD OF STUDY: The immunization of RSA patients was achieved by injection of four doses of 10(5) mononuclear cells (MNC) from the husband, at weekly intervals, before pregnancy. The IL6 and sIL6R levels were measured using sandwich ELISAs and the results evaluated by Tukey-Kramer multiple comparison-tests. RESULTS: Our data show no significant differences between IL6 and sIL6R serum levels of normal pregnant women and RSA pregnant women with white-cell immunization before pregnancy. In contrast, the sera of pregnant RSA patients without allogeneic therapy show higher values. We also found significant differences between IL6 levels in non-pregnant RSA women with and without immunotherapy. CONCLUSION: These results show that the alloimmunization with paternal white cells leads the serum IL6 and sIL6R-levels to the values observed in the course of normal pregnancy, suggesting a role for IL6 and sIL6R in the modulation of the immune response's quality.