RESUMO
The involvement of NF-κB in the regulation of teratogen-induced apoptosis has not been established yet. Therefore, we tried to assess the involvement of the p65 subunit of NF-κB in the embryonic response to the anti-cancer drug Doxorubicin (DOX). Thus, exposure of p65 knockout (p65(-/-)) or wild type (WT) mouse embryonic fibroblasts (MEFs) to DOX resulted in a decrease in cell survival, culture density and cell proliferation, which was found to be more prominent in p65(-/-) MEFs. Those phenomena were accompanied by a DOX-induced increase in the proportion of apoptotic cells, which was demonstrated only in p65(-/-) cells and a G2/M arrest, which was found to be more prominent in WT cells. Furthermore, DOX-treated WT and p65(-/-) MEFs differed in their expression of various apoptosis-associated molecules, when the former demonstrated a decrease in the percentage of p65-positive and a more prominent decrease in the percentage of p53-positive cells, while a decreased percentage of IκBα-positive and a more prominent decrease in the percentage of bcl-2-positive cells was detected among the latter. The fact that the response of the cells to the teratogen was clearly p65-dependent implicates this molecule to be involved in the response of the embryonic cells to DOX.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Fator de Transcrição RelA/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fator de Transcrição RelA/genéticaRESUMO
Bax was shown previously to regulate apoptotic cell death in various experimental systems, however, its involvement in teratogen-induced apoptosis is not clear yet. Therefore, we explored the involvement of Bax in the response of mouse embryonic fibroblasts (MEFs) to the anti cancer drug methotrexate (MTX), using Bax wild type (WT) and knockout (Bax(-/-)) MEFs. Our results demonstrated a significant teratogen-induced dose- and time-dependant decrease in the survival and culture density of both cell lines, which were found to be somewhat more prominent in WT cells. Exposure to MTX resulted also in decreased cell proliferation of WT but not Bax(-/-) cells and accordingly, we observed an accumulation of cells in the S phase and an increased percentage of cells in the Sub-G(1) phase of the cell cycle and the appearance of condensed nuclei, which were found to be somewhat more prominent in WT MEFs. In parallel, WT MEFs demonstrated a MTX-induced increase in the percentage of Bax-positive cells and a significant decrease in the percentage of bcl-2-, p65- or IkappaBalpha-positive cells, which were not detected in Bax(-/-) MEFs. Altogether, the differential sensitivity of WT or Bax(-/-) MEFs to MTX suggests a possible involvement of this molecule in the response of embryonic cells to teratogens.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Metotrexato/toxicidade , Teratogênicos/toxicidade , Proteína X Associada a bcl-2/metabolismo , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Metotrexato/administração & dosagem , Camundongos , Camundongos Knockout , Inibidor de NF-kappaB alfa , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Fator de Transcrição RelA/metabolismoRESUMO
NF-kappaB was shown previously to regulate apoptotic cell death processes in various experimental systems. However, its role in controlling teratogen-induced cell death has not been established yet. Therefore, the objective of the present study was to explore the involvement of the p65 subunit of NF-kappaB in the response of mouse embryonic fibroblasts (MEFs) to heat shock, using p65 knockout (p65-/-) cells. Indeed, we found p65-/- MEFs to be more susceptible to the exposure to heat shock, as compared with wild-type (WT) MEFs, as they demonstrated a more prominent decrease in cell survival and proliferation as well as the appearance of cells undergoing apoptotic cell death. These heat-shock-induced effects were preceded by a decrease in p65 expression in WT cells, which was accompanied by a decrease in IkappaBalpha expression in WT MEFs, while disappearing completely in p65-/- MEFs and accordingly, by an increase in p-IkappaBalpha expression in both cell lines, which was found to be more prominent in p65-/- MEFs. Interestingly, the heat shock-induced decrease in p65 expression was accompanied by an increase in HSP70 expression in both cell lines. However, it was again found to be more prominent in p65-/- MEFs. Taken together, our results suggest a protective role for the p65 subunit of NF-kappaB in mechanisms underlying the response of embryonic cells to heat shock.
Assuntos
Febre/fisiopatologia , Fibroblastos/fisiologia , Resposta ao Choque Térmico/fisiologia , NF-kappa B/fisiologia , Fator de Transcrição RelA/fisiologia , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/fisiologia , Proteínas I-kappa B/genética , Proteínas I-kappa B/fisiologia , Camundongos , Camundongos Knockout , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Fator de Transcrição RelA/genéticaRESUMO
Insulin-dependent diabetes mellitus is a well-known teratogen, which might cause growth retardation, malformations and fetal death. We have previously shown, that potentiation of the maternal immune system (immunopotentiation) might protect the embryo from diabetes teratogenicity. Therefore, in the present study we further inquired whether diabetes teratogenicity might be associated with alterations in the level of immune effector cells in systemic and local lymphoid organs as well as in the uterus throughout pregnancy and whether the protection exerted by maternal immunopotentiation might be realized through its effect on those cells. Streptozotocin-induced diabetes in ICR mice was found to reduce pregnancy rate and fetal weight while increasing the resorption rate and the percentage of litters with malformed fetuses. These teratogenic effects were accompanied by a decreased percentage of cells expressing Mac-1, Thy-1.2, CD4 or CD8 in the spleen and inguinal as well as paraaortic lymph nodes, except for Mac-1 expression by splenocytes, which increased significantly in the beginning of pregnancy and decreased later. A different pattern was observed in the uterus, when the percentage of cells expressing these markers tended to increase in the beginning of pregnancy and decrease later. Intrauterine immunopotentiation with rat splenocytes was found to improve the reproductive performance of diabetic animals. This protective effect was accompanied by a general normalization of the level of the various cell surface markers, when in most cases their expression returned to that found in nonimmunopotentiated mice. Our results suggest that the protection exerted by maternal immunopotentiation on the embryo against diabetes teratogenicity might be mediated via its effect on the level of immune effector cells localized to uterus and lymphoid organs, which was found to be altered in diabetic mice.
Assuntos
Anormalidades Congênitas/patologia , Diabetes Mellitus Experimental/complicações , Tecido Linfoide/patologia , Macrófagos/patologia , Gravidez em Diabéticas/complicações , Subpopulações de Linfócitos T/patologia , Útero/patologia , Animais , Anormalidades Congênitas/etiologia , Anormalidades Congênitas/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Feminino , Peso Fetal , Feto/imunologia , Feto/patologia , Citometria de Fluxo , Imunização , Linfonodos/imunologia , Linfonodos/patologia , Tecido Linfoide/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Gravidez , Gravidez em Diabéticas/imunologia , Gravidez em Diabéticas/patologia , Ratos , Ratos Long-Evans , Baço/imunologia , Baço/patologia , Subpopulações de Linfócitos T/imunologia , Útero/imunologiaRESUMO
AIMS/HYPOTHESIS: Activation of apoptosis in embryos is thought to be a key event in the pathogenesis of diabetes-induced embryopathies such as early embryonic death and inborn structural anomalies. TNF-alpha can activate apoptotic and anti-apoptotic signalling cascades, indicating its ability to contribute to and counteract diabetes-induced maldevelopment. To investigate how TNF-alpha regulates the response of embryos to diabetes-induced embryopathic stress, we used streptozotocin-induced diabetic TNF-alpha knockout mice. MATERIALS: To evaluate the reproductive performance, mated diabetic female mice were examined on days 4 and 8 of pregnancy for the presence of blastocysts or embryos in uterine horns. To evaluate the teratogenic effect, the female mice were killed on day 18 of pregnancy and fetuses were examined for gross external anomalies. In addition, apoptotic nuclei were localised by the TUNEL assay and DNA-binding activity of the transcription factor NF-kappaB was evaluated by electrophoretic mobility shift assay in 10- and 11-day-old embryos respectively. RESULTS: Severely diabetic TNF-alpha(+/+) female mice had a much greater decrease in pregnancy rate but a lower incidence of malformed fetuses in litters than severely diabetic TNF-alpha(-/-) female mice. Also, the intensity of excessive apoptosis was higher, but the amount of active NF-kappaB complexes was lower in malformed TNF-alpha(-/-) embryos than in TNF-alpha(+/+) embryos. CONCLUSIONS/INTERPRETATION: TNF-alpha contributes to death of peri-implantation embryos and possibly protects postimplantation embryos exposed to diabetes-induced teratogenic stimuli via activation of NF-kappaB-mediated anti-apoptotic signalling. It seems that TNF-alpha prevents the birth of malformed offspring in severely diabetic mice.
Assuntos
Anormalidades Congênitas/prevenção & controle , Diabetes Mellitus Experimental/patologia , Gravidez em Diabéticas/patologia , Teratogênicos , Fator de Necrose Tumoral alfa/fisiologia , Animais , Apoptose , Anormalidades Congênitas/patologia , Desenvolvimento Embrionário e Fetal , Feminino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Gravidez , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genéticaRESUMO
Diabetes-induced early embryonic death is accompanied by an increased expression of tumour necrosis factor alpha (TNF-alpha) in the embryonic microenvironment. The aim of the present study was to evaluate whether diabetes-induced embryopathic stress may also alter the expression of TNF-alpha produced by the embryo itself. As a model, whole postimplantation embryos were cultured for 24 h in a medium with high concentrations of glucose, one of the main diabetes-associated teratogenic metabolites. An anomaly such as an open neural tube was used as an end-point characterizing the glucose-induced teratogenic effect and the number of somites was counted to evaluate growth retardation induced by glucose. The expression of TNF-alpha (by immunohistochemistry), apoptosis (by TdT-mediated dUTP nick-end labelling; TUNEL) and the activity of caspases 3 and 8 (by a fluorometric assay) were evaluated in normal and malformed embryos. Ninety-seven per cent of the embryos exposed to 1300 mg glucose dl(-1) exhibited an open neural tube. The percentage of malformed embryos was smaller in media containing 800 and 500 mg glucose dl(-1) (68 and 37%, respectively) but it still exceeded significantly the value registered in embryos developing in a normoglycaemic medium (12%). In addition, a significant decrease in the number of somites was observed in embryos developing in media containing 1300 and 800 mg glucose dl(-1). Malformed embryos exhibited a greater number of nuclei that were positive in the TUNEL assay as well as a higher amount of active caspase 8 compared with normal embryos (with closed neural folds). TNF-alpha expression was detected in the neuroepithelial layer of the neural tube of the malformed embryos, whereas the expression of this cytokine was weak, if detectable, in normal embryos. Together, these findings indicate that TNF-alpha produced by the embryo may be involved in regulating the response of embryos to diabetes-generated embryopathic stress.
Assuntos
Anormalidades Induzidas por Medicamentos/imunologia , Embrião de Mamíferos/imunologia , Glucose/toxicidade , Teratogênicos/toxicidade , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Caspases/metabolismo , Células Cultivadas , Meios de Cultura , Embrião de Mamíferos/química , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos ICR , Fator de Necrose Tumoral alfa/análiseRESUMO
PROBLEM: The mechanisms mediating pregnancy loss induced by various agents are far from being understood. Thus, we investigated the possible involvement of one such mechanism, the apoptotic process, in pregnancy loss induced by lipopolysaccharide (LPS) or cyclophosphamide (CP) as well as the associated changes in the apoptosis-regulating gene products p53 and bcl-2. METHOD OF STUDY: Pregnancy loss was induced by LPS or CP on days 9 or 12 of pregnancy, respectively. LPS- or CP-associated apoptosis was assessed by the TdT mediated dUTP-biotin nick end labeling (TUNEL) method as well as by DNA fragmentation analysis, while p53 or bcl-2 expression was evaluated by immunohistochemistry. RESULTS: Lipopolysaccharide treatment initiated a resorption process that was accompanied by the appearance of apoptotic cells in the uterus, which increased in number by 24 hr after treatment. Induction of pregnancy loss with CP resulted in the appearance of some apoptotic cells in the uterus, reaching a peak at 72 hr after treatment. DNA fragmentation analysis revealed a DNA ladder at 24 hr after LPS as well as 72 hr after CP treatment. Immunohistochemical analysis demonstrated a continuous p53 expression in the uterus of LPS- or CP-treated mice, which was somewhat elevated at the peak of the apoptotic process. On the other hand, bcl-2 expression in LPS-treated mice could be reciprocally correlated with the apoptotic process, appearing only at its initiation or completion, while in CP-treated mice it was continuously expressed except for some elevation at the completion of the apoptotic process. CONCLUSIONS: Our results suggest a possible role for the apoptotic process in mechanisms mediating pregnancy loss and indicate an involvement of p53 and bcl-2 in its regulation.
Assuntos
Aborto Espontâneo/patologia , Apoptose/fisiologia , Útero/fisiologia , Animais , Apoptose/efeitos dos fármacos , Ciclofosfamida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Feminino , Imunossupressores/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos ICR , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Útero/patologiaRESUMO
It is believed that failure of the maternal immune system to actively support embryonic development, through production of the appropriate cytokine network, might be responsible for embryonic death. Thus, the aim of this study was to evaluate the possible involvement of cytokines such as tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta2 (TGF-beta2), which are crucial for normal embryonic development, in the early stages of mechanisms that mediate induced pregnancy loss. The early stages of the resorption process induced by lipopolysaccharide (LPS) were characterized by blood accumulation in the vicinity of the embryo, preceding any visible embryonic damage. At that time, immunohistochemical analysis revealed an increased expression of TNF-alpha in the primary and secondary decidua, which was reduced as the resorption process was completed. In contrast, TGF-beta2 expression was decreased in the primary and secondary decidua, as well as in the glandular epithelium, at all the times assessed. Maternal immunopotentiation with granulocyte macrophage-colony stimulating factor (GM-CSF), which controls maternal immune activities supporting normal embryonic development, decreased the resorption rate in LPS-treated mice while normalizing the expression of TNF-alpha and TGF-beta2 in the uterus of these animals throughout the ongoing resorption process. These results indicate a possible role for maternal immunopotentiation with GM-CSF in the mechanisms mediating the early stages of pregnancy loss, possibly via modulation of TNF-alpha and TGF-beta2 activity.
Assuntos
Aborto Espontâneo/imunologia , Adjuvantes Imunológicos/administração & dosagem , Citocinas/análise , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Útero/imunologia , Animais , Feminino , Idade Gestacional , Imuno-Histoquímica/métodos , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C3H , Modelos Animais , Gravidez , Fator de Crescimento Transformador beta/análise , Fator de Necrose Tumoral alfa/análiseRESUMO
Early embryonic deaths as well as malformed newborns are among complications of the diabetic pregnancy. Cytokines and growth factors operating in the embryonic vicinity are found to be among factors that determine the sensitivity of embryos to external and internal detrimental stimuli, including diabetes. Transforming Growth Factor-beta2 (TGF-beta2) has been shown to be essential for embryonic development and survival. In the present work, we evaluated the pattern of TGF-beta2 expression in the uterus of streptozotocin-induced diabetic mice, demonstrating a decreased reproductive performance and elevated percentage of litters with severely malformed fetuses. Since stimulation of the maternal immune system was found to increase the resistance of mouse embryos to the teratogenic effect of diabetes, the effect of immunopotentiation on the expression of the cytokine was also investigated. TGF-beta2 expression was studied at the mRNA level by using the in situ hybridization technique and at the protein level by using the immunohistochemical analysis. A clear decrease in TGF-beta2 mRNA expression in the uterus of diabetic mice was observed at examined time points: days 1, 5, and 9 of pregnancy. Also, an evident reduction in TGF-beta2, the protein expression in the uterus of diabetic mice, was demonstrated at these time points. Maternal immunopotentiation that improved the reproductive performance of diabetic mice and reduced the number of the litters with malformed fetuses was also accompanied by a clear increase in the level of TGF-beta2 mRNA expression in the pregnant uteri. The above results clearly demonstrate that the embryotoxic effect of diabetes is accompanied by an alteration of TGF-beta2 expression. Immunopotentiation that was shown to improve the reproductive performance of the diabetic mice was accompanied by a partial normalization of TGF-beta2 expression in embryonic vicinity.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Perda do Embrião/imunologia , Fator de Crescimento Transformador beta/metabolismo , Útero/metabolismo , Animais , Perda do Embrião/genética , Feminino , Regulação da Expressão Gênica , Técnicas Imunoenzimáticas , Hibridização In Situ , Camundongos , Camundongos Endogâmicos ICR , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Long-Evans , Fatores de Tempo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta2RESUMO
PROBLEM: Tumor necrosis factor (TNF)-alpha mRNA and protein are expressed in the pregnant uterus of streptozotocin-induced diabetic mice at various stages of pregnancy. We intend to characterize their pattern and to evaluate whether the potentiation of the maternal immune system alters the pattern of the expression of the cytokine. METHOD OF STUDY: Diabetes was induced in ICR mice by streptozotocin injection. To modulate maternal immune responses, ICR mice were injected intrauterine with rat splenocytes 3 weeks before mating. The expression of TNF-alpha mRNA and protein was evaluated by in situ hybridization and immunohistochemistry techniques. RESULTS: The population of diabetic mice used in this study demonstrated a reduction in pregnancy rate and an increased number of litters with severely malformed fetuses. It has been observed that these disturbances are associated with a clear increase in TNF-alpha mRNA and protein expression in the uterus of these mice. Maternal immunopotentiation, while improving reproductive performance of these diabetic mice, was found to be accompanied by a reduced expression of TNF-alpha, both at the mRNA and protein level. CONCLUSIONS: The results of the present study suggest a possible involvement of TNF-alpha in mechanisms underlying diabetes-associated dismorphogenesis. Normalization of TNF-alpha expression by maternal immunopotentiation might represent a mechanism mediating its protective effect against diabetes-induced embryotoxic insult.
Assuntos
Diabetes Mellitus Experimental/imunologia , Expressão Gênica , Gravidez em Diabéticas/imunologia , Fator de Necrose Tumoral alfa/genética , Útero/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Gravidez , RNA Mensageiro , Ratos , Ratos Long-Evans , Estreptozocina/efeitos adversos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PROBLEM: The mechanisms mediating pregnancy loss are far from being understood, but it is believed that modulation of the maternal immune system, that is known to support pregnancy, might serve as a means for the treatment of habitual abortions. Thus, we examined the effect of the anti bacterial agent ciprofloxacin, which was shown to affect maternal immunoreactivity, on pregnancy loss in the resorption-prone CBA/JxDBA/2J mouse combination as well as associated changes in Interleukin (IL)-3 and Granulocyte macrophage-colony stimulating factor (GM-CSF) production. METHOD OF STUDY: CBA/J females mated to DBA/2J males were treated with ciprofloxacin for 5 consecutive days. On day 15 of pregnancy, the number of resorbed embryos was recorded and IL-3 mRNA expression as well as IL-3 and GM-CSF protein production by splenocytes were assayed by northern blotting and ELISA, respectively. RESULTS: Ciprofloxacin treatment resulted in a significant reduction in the resorption rate as compared to the effect of the control antibiotic ceftazidime or PBS only, while not affecting the number of implantation sites/mouse. Also, splenocytes from ciprofloxacin-treated CBA/J mice exhibited an increased level of IL-3 mRNA transcripts as well as an elevation in IL-3 and GM-CSF protein production. CONCLUSIONS: Our data demonstrate the ability of ciprofloxacin to reduce pregnancy loss in the CBA/JxDBA/2J mouse model, possibly via elevation of IL-3 and GM-CSF production.
Assuntos
Aborto Animal/imunologia , Anti-Infecciosos/farmacologia , Ciprofloxacina/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Interleucina-13/biossíntese , Animais , Anti-Infecciosos/administração & dosagem , Ciprofloxacina/administração & dosagem , Feminino , Interleucina-13/genética , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBARESUMO
This study was aimed at characterizing the temporal patterns of cell responses and p53 protein expression in the limbs, head, and liver of embryos responding to cyclophosphamide (CP)-induced teratogenic insult. ICR murine embryos were examined 24, 48, or 72 h after injection of 40 mg/kg CP on day 12 of pregnancy. The cellular events and temporal pattern of p53 protein expression were determined by FACS analysis and by TUNEL (apoptosis) in the head, limbs, and liver of the embryos. All tested organs showed apoptosis and a significantly decreased proportion of live cells after 24 h. Subsequent events were organ-dependent. In the liver, there were no dysmorphic events at any time and excessive cell death had been almost compensated for by 48 h. Compensation was preceded by G(1) arrest and accompanied by an increased level of p53 protein in surviving cells. Excessive cell death in the head and the limbs resulted in structural anomalies. In the head, there was an increased level of p53 protein and G(1) arrest after 24 h and the number of live cells at 48 h was equal to that seen in earlier samples, despite apoptosis. In the limbs, however, only isolated viable cells were seen by 48 h, but there was no increased level of p53 protein or G(1) arrest. Results of this study suggest that the differential sensitivity of tested organ systems to CP may be associated with differences in cellular events following CP-initiated cell death. They also suggest that the input of p53 in determining the response of these organ systems to CP-induced teratogenic insult may be different. Teratogenesis Carcinog. Mutagen. 19:353-367, 1999.
Assuntos
Morte Celular/efeitos dos fármacos , Ciclofosfamida/toxicidade , Teratogênicos/toxicidade , Proteína Supressora de Tumor p53/biossíntese , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Extremidades , Feminino , Cabeça , Marcação In Situ das Extremidades Cortadas , Fígado/citologia , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Especificidade de Órgãos , GravidezRESUMO
CSF-1 plays an important role in female reproduction and normal embryo development. To understand further CSF-1 function in normal and, especially, in compromised pregnancy, we studied the pattern of its mRNA expression as well as expression of its receptor (c-fms) in the uteroplacental units of mice with induced (cyclophosphamide (CY)-treated) and spontaneous (CBA/J x DBA/2J mating combination) pregnancy loss. RNase protection analysis demonstrated the presence of two forms of CSF-1 mRNA in the uteroplacental unit corresponding to 1400- and 263-bp protective fragments. Densitometric analysis demonstrated that the level of 1400-bp mRNA form was decreased by 40% in the uteroplacental units of mice with CY-induced pregnancy loss compared with the control mice. About 20% decrease in 263-bp protective fragment was registered in resorbing versus non-resorbed placenta of CBA/J females mated to DBA/2J males. As judged by in situ hybridization assay, CSF-1 mRNA transcripts were localized in the uterine epithelium and stroma, while c-fms mRNA was found mainly in the trophoblast. The number of metrial gland cells as well as the number of uterine leucocytes expressing CSF-1 and c-fms mRNAs was substantially lower in the uteroplacental unit of mice with pregnancy loss than in control animals. Maternal immunostimulation, while significantly decreasing the resorption rate in mice with CY-induced pregnancy loss, also strengthened CSF-1 mRNA expression at the fetomaternal interface and resulted in reconstitution in the number of CSF-1+ uterine leucocytes and metrial gland cells. These data suggest a role for uterine CSF-1 in the physiology of normal and compromised pregnancy and demonstrate a possible involvement of CSF-1-associated signalling in mechanisms of placenta and endometrium repair following immunopotentiation.
Assuntos
Aborto Induzido , Aborto Espontâneo/imunologia , Fator Estimulador de Colônias de Macrófagos/biossíntese , Placenta/imunologia , Útero/imunologia , Animais , Ciclofosfamida/farmacologia , Feminino , Expressão Gênica , Fator Estimulador de Colônias de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos , Placenta/patologia , Gravidez , Ratos , Ratos Long-Evans , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Útero/patologiaRESUMO
It was already shown that stimulation of the maternal immune system by allogeneic or xenogeneic leukocytes is capable of affecting embryonic responses to teratogenic insults and various cytokines, including granulocyte macrophage-colony stimulating factor (GM-CSF), were implicated as mediators of this effect. Therefore, in the present study we tried to assess the ability of GM-CSF to modulate teratogenic activity, along with possible changes in systemic as well as local maternal immune responses, that might be involved in the process. Thus, the percentage of cyclophosphamide (CP)-treated embryos exhibiting limb malformations was shown to decrease significantly following GM-CSF administration. This effect was found to be comparable to that demonstrated by intrauterine leukocytes administration. GM-CSF treatment resulted in a significant enhancement in maternal splenocytes Con-A-induced proliferation, as well as Interleukin-2 (IL-2) and IL-3 production. Examination of leukocyte cell surface antigens expressed by splenocytes revealed no statistically-significant changes in the level of the T lymphoid antigens Thy-1, CD5, CD4, CD8 and CD3, the macrophage antigen Mac-1, and the adhesion molecules LFA-1alpha, LFA-1beta and L-Selectin, following GM-CSF immunostimulation. In parallel, immunohistochemical analysis of the uteroplacental unit revealed Mac-1 and to a lesser extent LFA-1beta-positive cells localized to the myometrium and the placenta in both the control and the GM-CSF-treated groups, while no cells expressing Thy-1, CD3, CD4, CD8, or LFA-1alpha could be demonstrated. Our results suggest a possible role for GM-CSF in modulating teratogen-induced effects, a process in which maternal immune responses such as splenocytes proliferation and cytokine production might be involved.
Assuntos
Anormalidades Induzidas por Medicamentos/prevenção & controle , Ciclofosfamida/toxicidade , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Teratogênicos/toxicidade , Animais , Linhagem Celular , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos ICR , Placenta/imunologia , Gravidez , Ratos , Ratos Long-Evans , Baço/imunologia , Útero/imunologiaRESUMO
Acute immunosuppression induced by total body irradiation (TBI) or cyclophosphamide (Cy) treatment, followed by syngeneic bone marrow transplantation (SBMT), was reported to be effective in inducing long-term tolerance in some autoimmune disease models. We examined the efficacy of this approach in the mouse model of experimental autoimmune uveoretinitis (EAU). Development of EAU induced by the interphotoreceptor retinoid binding protein (IRBP) was abolished almost completely by either TBI or Cy treatment, followed by SBMT, instituted one week after priming. In parallel, IRBP-specific delayed-type hypersensitivity (DTH) responses and lymph node cell proliferation were strongly suppressed or abolished. However, when these IRBP-immunized, lymphoablated and BM reconstituted mice were rechallenged with the immunizing antigen seven weeks after the primary immunization, they were not protected from developing disease, despite the fact that DTH and lymph node cell proliferation to the antigen were suppressed relative to controls. TBI treatment appeared somewhat more effective than Cy treatment as judged by its more profound effect on immunological responses. These results demonstrate the ability of acute immunosuppression followed by reconstitution of the immune system to inhibit the development of EAU, although long-term protection from disease was not achieved.
Assuntos
Doenças Autoimunes/terapia , Autoimunidade/imunologia , Transplante de Medula Óssea/imunologia , Terapia de Imunossupressão , Retinite/terapia , Uveíte/terapia , Animais , Autoantígenos/farmacologia , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/patologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Medula Óssea/efeitos da radiação , Ciclofosfamida/farmacologia , Proteínas do Olho/farmacologia , Feminino , Hipersensibilidade Tardia/induzido quimicamente , Hipersensibilidade Tardia/patologia , Hipersensibilidade Tardia/prevenção & controle , Tolerância Imunológica/imunologia , Imunossupressores/farmacologia , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Retinite/induzido quimicamente , Retinite/patologia , Proteínas de Ligação ao Retinol/farmacologia , Transplante Isogênico , Uveíte/induzido quimicamente , Uveíte/patologia , Irradiação Corporal TotalRESUMO
An elevated expression of TNF-alpha in embryonic microenvironment was found to be associated with postimplantation loss. In this work, we examined the pattern of TNF-alpha expression at both the mRNA and the protein level as well as the distribution of TNF-alpha receptor mRNA in the uteroplacental unit of mice with induced (cyclophosphamide-treated) or spontaneous (CBA/J x DBA/2J mouse combination) pregnancy loss. RNase protection analysis demonstrated an increase in TNF-alpha mRNA expression in the placentae of mice with pregnancy loss compared with that in control mice. TNF-alpha messages were localized to the uterine epithelium and stroma as well as the giant and spongiotrophoblast cells of the placenta. The intensity of the hybridization signal in placentae of mice with pregnancy loss was substantially higher than that in control mice. The up-regulation of TNF-alpha mRNA was accompanied by an increase in the expression of TNF-alpha receptor I mRNA in the same cell populations. The elevation of TNF-alpha production was also demonstrated at the protein level. Western blot analysis showed an increased level of the 18- and 26-kDa TNF-alpha protein species in the uteroplacental unit of mice with pregnancy loss. Immunostaining revealed TNF-alpha-positive leukocytes located in the uterus and placenta. Finally, we found that immunization of mice with cyclophosphamide-induced pregnancy loss while decreasing the resorption rate in these females resulted in a decline in TNF-alpha expression at the fetomaternal interface. These data clearly suggest an involvement of TNF-alpha in pathways leading to both spontaneous and induced placental death.
Assuntos
Aborto Espontâneo/imunologia , Placenta/imunologia , RNA Mensageiro/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Útero/imunologia , Aborto Espontâneo/induzido quimicamente , Animais , Ciclofosfamida , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Placenta/fisiopatologia , Gravidez , Fator de Necrose Tumoral alfa/imunologia , Útero/fisiopatologiaRESUMO
OBJECTIVE: To evaluate the immunomodulatory potential of ciprofloxacin in mice with experimental antiphospholipid syndrome (APS). METHODS: Ciprofloxacin or ceftazidime (control antibiotic) was given to mice with experimentally induced APS. The titers of autoantibodies, levels of cytokines, and number of cytokine-producing cells were determined by enzyme-linked immunosorbent assay. Myeloid progenitor cells were determined by granulocyte-macrophage colony-forming unit, and interleukin-3 (IL-3) messenger RNA (mRNA) was tested by Northern analysis. RESULTS: A decrease in the incidence of pregnancy loss and an improvement in the clinical manifestations of APS were noted in the mice treated with ciprofloxacin, compared with the mice given ceftazidime. The effect of ciprofloxacin was found to be associated with increased serum levels of IL-3 and with increased IL-3 mRNA transcription in the splenocytes. Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was documented by elevated titers in the sera and elevated numbers of colony-forming cells in the bone marrow. CONCLUSION: Ciprofloxacin prevents the manifestations of experimental APS. This effect may be associated with increased IL-3 levels and GM-CSF expression.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anti-Infecciosos/farmacologia , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Ciprofloxacina/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-3/metabolismo , Animais , Formação de Anticorpos/efeitos dos fármacos , Síndrome Antifosfolipídica/patologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Ceftazidima/farmacologia , Contagem de Células , Ensaio de Unidades Formadoras de Colônias , Feminino , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Baço/efeitos dos fármacos , Baço/patologiaRESUMO
PROBLEM: The role of tumor necrosis factor (TNF)-alpha produced by embryonic cells in normal and abnormal development is poorly understood. To assess to what extent TNF-alpha may be involved in the process of induced dysmorphogenesis, the expression of TNF-alpha and TNF-alpha receptor (TNFRI) mRNA as well as TNF-alpha protein was evaluated in embryos responding to a cyclophosphamide (CP)-induced teratogenic insult. The effect of maternal immunostimulation increasing the embryo's tolerance to CP on TNF-alpha expression was also investigated. METHOD OF STUDY: ICR female mice were treated intraperitoneally with 40 mg/kg CP on day 12 of pregnancy. The immunostimulator, xenogeneic rat splenocytes, was injected intrauterine 21 days before mating. Embryos were collected on days 13, 14, or 15 of pregnancy. TNF-alpha mRNA, TNFRI mRNA, and TNF-alpha protein expression were evaluated by in situ hybridization and immunostaining techniques in control, teratogen-treated, and immuno-stimulated teratogen-treated embryos. RESULTS: CP-treated embryos showed severe external brain and craniofacial anomalies already visible on day 14 of pregnancy. TNF-alpha mRNA transcripts were detected in cells of the brain and the head of 13-day embryos, which preceded the occurrence of CP-induced external craniofacial anomalies. On day 15 of pregnancy, when severe craniofacial anomalies increased, a significant increase in the intensity of TNF-alpha, TNFR1 mRNA transcripts, and TNF-alpha protein expression were observed in cells of the malformed regions of the head and the brain. In other nonmalformed organs of CP-treated embryos such as the liver (not macroscopically different from controls), neither TNF-alpha nor TNFR1 transcripts were detected. Immunostimulation substantially diminished the severity of CP-induced brain and craniofacial anomalies, decreased the resorption rate, and was associated with decreased intensity of TNF-alpha mRNA transcripts detected on day 15 of pregnancy in the head and the brain of CP-treated embryos. CONCLUSIONS: TNF-alpha expressed in the embryo may be one of the molecules promoting the formation of CP-induced brain and craniofacial anomalies. The decrease of TNF-alpha expression in embryos of immunostimulated females may be one of the mechanisms responsible for the increased tolerance to the teratogenic insult.
Assuntos
Ciclofosfamida/farmacologia , Embrião de Mamíferos/efeitos dos fármacos , Teratogênicos/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Encéfalo/anormalidades , Encéfalo/embriologia , Anormalidades Craniofaciais/embriologia , Ciclofosfamida/administração & dosagem , Embrião de Mamíferos/imunologia , Feminino , Imunização , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos ICR , Gravidez , RNA Mensageiro/metabolismo , Ratos , Receptores do Fator de Necrose Tumoral/biossíntese , Baço/citologia , Baço/imunologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
This study focused on the role of adhesion molecules in early pregnancy in mice. Injection of anti-Mac-1 antibodies during early pregnancy resulted in early pregnancy loss (only 30.7% of mice in the group injected with anti-Mac-1 antibody were pregnant compared with 87.5% of controls), while mice treated with anti-Mac-1 antibodies during late pregnancy did not show a significant abortive effect (68.8% mice in the treated group were pregnant compared with 92.9% of control mice). Anti-LFA-1 alpha, LFA-1 beta or mouse Ag-Eb antibodies, when injected during early pregnancy, caused a nonsignificant decrease in pregnancy rate ranging between 15% and 25% (P > 0.05), while anti-Thy-1.2 antibodies demonstrated a marginal effect only. Staining of uterine tissue sections, collected on days 4-6 of pregnancy, with anti-Mac-1 antibodies, demonstrated antibody bound to cells in the deep endometrium and in the myometrium but not in the uterine area close to the lumen or on the surface of the blastocyst. These results indicate a possible role for the Mac-1 antigen in early pregnancy.
Assuntos
Aborto Espontâneo/imunologia , Anticorpos/administração & dosagem , Implantação do Embrião/imunologia , Antígeno de Macrófago 1/imunologia , Animais , Moléculas de Adesão Celular/imunologia , Endométrio/química , Feminino , Idade Gestacional , Imuno-Histoquímica , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno de Macrófago 1/análise , Camundongos , Camundongos Endogâmicos ICR , Miométrio/química , Gravidez , Antígenos Thy-1/imunologiaRESUMO
It is known that programmed cell death (apoptosis) is an important physiological determinant of embryonic development. In parallel, it may be one of the major events involved in induced teratogenesis. The present study was designated to evaluate to what extent is apoptosis involved in the formation of some final abnormalities induced by cyclophosphamide (CP) in ICR mice. The level of apoptosis in limbs, tail, liver, and whole embryo was assessed 24 h after administration of various doses of CP (day 12 of pregnancy) by flow cytometric analysis and by DNA fragmentation assay. In parallel, the rate of limb and tail malformations, resorptions, and growth retardation induced by various doses of CP was evaluated in animals sacrificed on day 19 of pregnancy using routine teratological methods. A striking correlation between the rate of CP-induced apoptosis in limb and tail cells and the severity of limb and tail anomalies was found after administration of CP ranging from 10 to 40 mg/kg. Thus, the percent of apoptotic cells collected from limbs and tails increased from 18 to 78%. In parallel, the severity of limb and tail anomalies increased from digit anomalies to amely and from crooked to short or absent tail. CP-induced embryolethality and fetal growth retardation also correlated with the level of apoptosis in cells collected from whole embryos but to a lesser extent. These results claim that CP-induced apoptosis is one of the inevitable events in the pathway leading to the formation of CP-induced abnormalities and also suggest that the extent of the involvement of apoptosis in the formation of different types of final abnormalities, may be different.