Immunosuppressive effect of pregnant mouse serum on allostimulatory activity of dendritic cells

In normal pregnancy, the maternal immune system is directed towards tolerance or suppression in order to prevent rejection of the semi-allogenic fetus. Antigen-presenting cells, especially dendritic cells (DCs), are key cells in initiation and regulation of immune responses. The presence of potent immunostimulatory DCs in the decidual tissue of pregnancy has been demonstrated. The aim of this study was to determine how allostimulatory activity of DCs could be affected during pregnancy. DCs were isolated from spleen of pregnant or non-pregnant Balb/c mice and co-cultured with allogenic T lymphocytes prepared from brachial lymph nodes of C57BL/6 mice. Some cultures of non-pregnant female DCs were treated by 2.5% serum obtained from pregnant mice at early, middle or late gestational periods, and were used in the same mixed lymphocyte reaction (MLR) settings. Cell proliferation was measured by 3H-thymidine incorporation, and cytokine production measured in supernatants of MLR cultures using ELISA. The effect of pregnant mouse serum on expression of DC surface markers was evaluated by flow cytometry. No significant difference was found between stimulatory potential of splenic DCs from pregnant and non-pregnant mice in induction of allogenic T cell proliferative response. Moreover, serum of early or late pregnancy did not have any effect on DC function in comparison with non-pregnant mouse serum, while mid-pregnancy serum significantly inhibited allostimulatory activity of DCs. IFNgamma production in co-culture of DCs treated with pregnant mouse serum was significantly lower than that of the control group; however, no significant difference in IL-10 production was observed. Treatment of DCs with pregnant mouse serum did not influence the percentage of cells expressing MHC-II, CD86, CD8alpha or CD11b. However, a marked reduction of the mean fluorescence intensity of MHC-II was observed. Collectively, our results concerning the diminished capacity of DCs to induce production of Th1 cytokines and allogenic T cell proliferation after treatment with pregnant mouse serum reveal a new way of immunologic tolerance against the semi-allogenic fetus.     

Immunosuppressive effect of serum progesterone during pregnancy depends on the progesterone binding capacity of the lymphocytes

Cytotoxic activity and progesterone binding capacity of the lymphocytes, together with serum progesterone concentrations, were determined in women with normal pregnancy or with a clinical diagnosis of threatened abortion or threatened premature labour. The lymphocytes of women with threatened abortion or threatened premature labour showed significantly higher cytotoxic activity (P < 0.001) and significantly lower progesterone binding capacity (P < 0.001) than did lymphocytes obtained from the healthy pregnant women. Significant inverse correlation was found between progesterone binding capacity and cytotoxic activity of the lymphocytes (P < 0.001), but the progesterone concentration of the pregnancy serum appeared to have no influence on the other two parameters. The findings indicate that intact progesterone binding capacity of the lymphocytes is an essential factor for the manifestation of the blocking effect exerted by pregnancy serum on lymphocyte cytotoxicity in vitro.     

孕激素对卵巢癌细胞体外生长的影响

目的　探讨孕激素对人卵巢癌细胞体外生长的影响。方法　采用四甲基偶氮唑蓝MTT比色法观察孕酮对卵巢癌细胞株HO - 891 0体外生长的影响 ;免疫组化法观察孕酮作用后HO - 891 0雌激素受体、孕激素受体及雄激素受体的变化。结果　 1× 1 0 -6～ 1× 1 0 -5mol/L的孕酮作用 4 8h和 72h对HO - 891 0细胞生长有抑制 ;作用 2 1d后 1× 1 0 -5mol/L时出现明显的抑制作用 ;但是孕激素未影响HO - 891 0雌激素受体、孕激素受体及雄激素受体蛋白的表达 ,为孕激素临床应用提供间接依据。结论　孕激素可抑制卵巢癌细胞株的体外生长但未改变细胞的激素受体表达     

The effect of insulin on in vitro progesterone production of human granulosa cells.

Insulin appears to play an important role in regulating ovarian function in some mammals. The present study assessed the effect of insulin on progesterone production by human granulosa lutein (G-L) cells in vitro. G-L cells were isolated from individual follicles at the time of laparoscopy for oocyte retrieval in patients undergoing in vitro fertilization/embryo transfer (IVF/ET). G-L cells were purified and plated for culture. Control wells contained no additions, while insulin was added to test wells. Although G-L cells obtained from follicles which contain an oocyte that subsequently fertilizes in vitro produce more progesterone at three and six days of culture than G-L cells from follicles that contain an oocyte that does not fertilize in vitro, insulin is unable to increase further the progesterone production of G-L cells from follicles containing either fertilized or nonfertilized oocytes at days 3 and 6 in culture. Further analysis based on the stimulation protocol (follicle stimulating hormone, n = 13; clomiphene citrate/hMG, n = 19; hMG, n = 10) also failed to yield a significant difference in basal or insulin-stimulated progesterone secretion after three or six days in culture. The lack of an effect of insulin on progesterone production by G-L cells in vitro may indicate that these cells are maximally stimulated following hyperstimulation and cannot increase progesterone production further, or may signify that insulin has no effect on the in vitro luteinization of human G-L cells obtained from hyperstimulated cycles.     

靶向沉默Gata3对妊娠期哮喘小鼠树突状细胞表型及效应T细胞失衡表达的影响

目的:研究慢病毒介导siRNA靶向沉默Gata3基因表达对妊娠哮喘模型小鼠骨髓来源树突状细胞(DC)抗原递呈及其免疫生物学特性的影响。方法:以脂质体DNA沉淀法将重组慢病毒干扰质粒与包装质粒共转染AD293细胞,包装生成慢病毒感染体外诱导DC,RT-PCR、Western blot分析其对siGata3基因的整合转录效果;流式细胞术观察细胞表型变化。通过尾静脉注射将pSH1/siGata3转移至小鼠体内,ELISA法测定其肺泡灌洗液(BALF)中的炎性因子含量;MTT法验证混合淋巴细胞反应(MLR)后T细胞增殖能力。结果:经重组、包装携有Gata3特异siRNA慢病毒感染DC成功,其siRNA靶点Gata3 mRNA及蛋白的转录表达分别下调87.30%和81.33%(P0.05);同时降低DCs膜表面CD86荧光强度(P0.05)。减少pSH1/siGata3组小鼠BALF中IL-5、IL-17分泌而提升IL-12水平(P0.05);MLR证实,共培养感染DC激活T细胞的能力明显不足(P0.05)。结论:siRNA干扰可有效抑制模型小鼠免疫微环境特定Gata3通路,阻遏T细胞及其亚群分化由此来诱导DC免疫耐受,为妊娠期哮喘防治奠定基础并提供新的思路和手段。     

The effect of progesterone and RU486 on progesterone production in the ovulatory process of rats

To clarify the autoregulatory mechanism of progesterone (P) production in the ovulatory process, we examined the ovarian concentration of P 46 hrs after PMSG and the effects of P and RU486 (RU) injected 4-2 hrs before hCG administration on the serum concentrations of P and estradiol (E2), and ovarian 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activities in PMSG/hCG treated immature rats. The effect of RU on the number of ovulated ova was also studied. The ovarian concentration of P 46 hrs after PMSG was 0.96 +/- 0.03,ng/mg protein (mean +/- SEM). When P (100,mg/kg) was injected 2 hrs before hCG, ovarian 3 beta-HSD activities had significantly increased by 4 hrs after hCG. However, P at a dosage of 10 and 20,mg/kg had no effect on ovarian 3 beta-HSD activities. The administration of RU (20,mg/kg) 2 hrs before hCG significantly inhibited ovarian 3 beta-HSD activities measured 4 and 6 hrs after hCG (p less than 0.01 and p less than 0.05, respectively). In addition, the serum P concentration 4 hrs after hCG was significantly lower than that of the control (p less than 0.01). However, RU (20 mg/kg) in concomitant with hCG had no effect on ovarian 3 beta-HSD activities within 6 hrs after hCG. The suppression of ovarian 3 beta-HSD activities by RU was the concomitant reversed by the concomitant treatment with P (10 mg/kg). RU (10, 20, or 40 mg/kg) injected 2 hrs before hCG significantly reduced the number of ovulated ova (p less than 0.01, p less than 0.01 and p less than 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunosuppressive effect of serum in patients with ovarian carcinoma.

Cell-mediated immune response was measured in 23 patients with ovarian cystadenocarcinoma, 38 patients with benign ovarian tumor, and 44 healthy volunteers. The method used two indexes: the lymphocyte response per unit volume of peripheral blood to phytohemagglutinin (PHA) and the immunosuppressive effect of serum on the response of normal lymphocytes to PHA stimulation. The lymphocyte response per 50 microliter peripheral blood did not differ significantly between patients with ovarian cancer and healthy volunteers. The serum effect, in contrast, differed significantly between malignant and benign ovarian tumors, and was found to increase significantly even when the cancer masses were as small as about 5 x 5 x 5 cm in size, ie, in FIGO Stage I. It is our belief that the measurement of the serum effect in patients with any ovarian tumor enables the early detection of ovarian cancer.     

The effect of insulin on progesterone production and cellular growth in long-term cultures of human granulosa lutein cells

The direct action of insulin on human granulosa lutein cells (GLCs) in long-term cultures obtained from in vitro fertilization (IVF) cycles was investigated. Progesterone (P) secretion by GLC increased progressively in both basal and human chorionic gonadotropin (hCG; 100 mIU/ml) stimulated conditions up to 4 days in culture, and plateaued thereafter. Insulin (0.0025 mU/ml to 2500 mU/ml) had no effect on either basal or hCG stimulation during the culture period. GLC in culture formed a monolayer and multiplied at a rate of approximately once every 3 days. Neither morphology nor cell division was affected by insulin in supraphysiologic levels (25 mU/ml). These results suggest that GLC obtained from preovulatory follicles in an IVF program are already stimulated maximally by in vivo exposure to high doses of human menopausal gonadotropin (hMG)/hCG administered to the women. Contrary to its stimulatory effect on early preovulatory granulosa cells, insulin dose not affect P production, cellular morphology, or growth rate of luteinized granulosa cells.     

Influence of progesterone on myometrial contractility in pregnant mice treated with lipopolysaccharide

AIM: To evaluate the effect of progesterone on interleukin (IL)-6, prostaglandin (PG) E2 and nitric oxide (NO) metabolite (NOx) production and contractile activity by NO in pregnant mice treated with lipopolysaccharide (LPS). METHODS: Pregnant C57BL mice on day 14 of gestation were killed 6 h after i.p. injection of LPS (400 microg/kg) or vehicle. Progesterone (2 mg) was subcutaneously injected 2 h before LPS treatment. Uterine rings were equilibrated in Krebs-Henseleit solution (37 degrees C) bubbled with 20% O2 and 5% CO2 (pH 7.4) for sampling and isometric tension recording. IL-6, PGE2 and NOx productions were measured from the bathing solution. Changes in spontaneous contractile activity in response to cumulative concentrations of l-arginine, diethylamine/nitric oxide (DEA/NO, the NO donor), and 8-bromo-cGMP (8-br-cGMP) were compared. Integral contractile activity over 10 min after each concentration was calculated and expressed as percentage change from basal activity. Statistical analyses were performed using one-way anova followed by Dunnett's test (significance was defined as P < 0.05). RESULTS: Interleukin-6 (34.7 +/- 6.0 pg/g tissue), PGE2 (66.8 +/- 6.7 pg/g tissue) and NOx (51.0 +/- 5.4 pmol/2 mL/g wet tissue) production were significantly stimulated by LPS treatment (138.2 +/- 23.2, 147.0 +/- 29.0, 98.6 +/- 16.2, respectively; P < 0.05). L-arginine, DEA/NO and 8-br-cGMP concentration-dependently inhibited spontaneous contractions in uterine rings both in LPS-treated and -untreated animals. Treatment with LPS significantly attenuated the maximal inhibition induced by l-arginine, DEA/NO and 8-br-cGMP in uterine rings from pregnant mice. Progesterone significantly decreased the levels of IL-6 production (74.9 +/- 12.1, P < 0.05), but not PGE2 and NOx production, and contractile responses by l-arginine, DEA/NO and 8-br-cGMP. CONCLUSIONS: The administration of LPS is associated with increases in IL-6, PGE2 and NO, and these increases may or may not have a role to play in LPS-induced preterm labor. Progesterone reduced the LPS-induced increase in IL-6 production and this may be one of the ways that progesterone reduces the risk of preterm labor.     

The suppressive effect of progesterone on lymphocyte cytotoxicity: unique progesterone sensitivity of pregnancy lymphocytes

The effect of progesterone on the cytotoxic activity of lymphocytes obtained from healthy pregnant women, women with threatened pre-term delivery, healthy non-pregnant women and healthy male donors has been compared. The cytotoxic activity of lymphocytes from healthy pregnant women was significantly reduced by progesterone at concentrations present in the serum during pregnancy. In contrast, a 100-fold higher concentration of progesterone was required to diminish the cytotoxic activity of lymphocytes from women with threatened pre-term delivery and from healthy male donors. Individuals with lymphocytes of high and low progesterone sensitivity could be found amongst non-pregnant women. The results of investigations at the single cell level suggested that although progesterone did not inhibit the ability of the lymphocytes to bind to the target cells, it markedly reduced the target cell lysing capacity of the bound effector cells.     

Reversal by human chorionic gonadotropin of the inhibitory effect of clomiphene on progesterone production by granulosa-luteal cells in culture

The possibility of a direct ovarian effect of clomiphene citrate was assessed in vitro by measuring progesterone release from cultured human granulosa-luteal cells in both the presence and absence of hCG. Granulosa-luteal cells obtained by laparoscopic follicle aspiration following ovarian stimulation with hMG and hCG in women undergoing in vitro fertilization/embryo transfer were incubated for 72 hours in medium containing increasing concentrations of clomiphene (10(-9) - 10(-5) M) both without and with hCG (1 IU/mL). Clomiphene stimulated progesterone production in both the presence and absence of additional hCG at concentrations less than or equal to 10(7) M. In both the presence and absence of hCG a dose-dependent inhibition of progesterone production was observed with higher concentrations of clomiphene (greater than 10(6) M). hCG (1 IU/mL) significantly increased progesterone production in control cells and at all concentrations of clomiphene (10(-9)-10(-5) M) tested. Low clomiphene (10(-9)-10(-7) M) and hCG concentrations appeared to have a synergistic effect on progesterone production. Thus it appears that clomiphene has a direct ovarian effect on progesterone production, which is stimulatory at low concentrations and inhibitory at higher concentrations.     

The effect of intraperitoneal progesterone on postoperative adhesion formation in rabbits

The immunosuppressive and anti-inflammatory properties of progesterone (P) have been established. The authors investigated whether the intraperitoneal instillation of P would lessen postoperative adhesion formation in New Zealand white rabbits undergoing pelvic surgical procedures. In phase I, severe, peritoneal lesions were made in the right uterine horn (n = 48). Animals were randomized to receive equal volumes of either (1) Ringer's lactate (RL); (2) 32% dextran 70 (HY; Hyskon Division, Pharmacia, Piscataway, NJ); (3) 500 mg P in oil (PO); or (4) 500 mg aqueous P (PA) at initial laparotomy. In phase II, the distal right uterine horn, including the mesosalpinx, was excised and microsurgical anastomosis was accomplished (n = 45). Aqueous P was not used in phase II; otherwise, the same agents were tested. Six weeks later, the severity of the adhesions formed was graded. The mean adhesion scores for the RL and HY groups were low for the right side in both phases and did not differ (P greater than 0.05). In contrast, higher scores were observed in all the P groups, regardless of the P preparation used or the surgical procedure performed (P less than 0.05).     

Preliminary report on progesterone effect on peripheral estrone sulfate metabolism

The effect of progesterone (P) on peripheral estrone sulfate (E1S) was investigated in postmenopausal women using E1S constant infusions. The metabolic clearance rate of E1S (MCRE1S) was significantly lowered by P administration (p less than 0.01), while no significant changes were observed for E1S----E1 conversion ratio. The present findings suggest that P can affect the E1S peripheral fate inducing deep changes in estrogen equilibrium.     

The effect of in vivo progesterone administration on relaxin-inhibited rat uterine contractions

Progesterone pretreatment in vitro was previously shown to sensitize myometrium to the inhibiting effect of relaxin. The following experiments were performed to control for possible artifacts of the in vitro system and to place these studies on a more physiologic basis. Immature rats were treated with estrogen and either progesterone or vehicle only. Uterine horn segments were isolated and mounted in a muscle bath. After a baseline contraction pattern was established by means of electrical stimulation, porcine relaxin was added to the bath. At all dose levels of relaxin, greater inhibition of contraction amplitude occurred in uterine segments of progesterone-treated animals. Since both progesterone and relaxin are present in the circulation from the time of the missed menses in human pregnancy, this interaction suggests a physiologic synergism in the maintenance of early human pregnancy.     

Immunosuppressive effect of early pregnancy factor on early expression of cell surface membrane IgG

We have adopted a new assay to investigate the influence of early pregnancy factor (EPF) on the modulation of lymphocyte activity. Lymphocytes were attached to the plastic surfaces of microplates in serum-free medium in the presence of Sepharose-Con A. After 2-3 days incubation with EPF, and ELISA assay was used to detect the expression of surface membrane IgG (smIgG); this was done in the same microplates used for the culture, thus avoiding cell manipulation. Using only a few picograms of EPF a significant inhibition (in the range 26-40%) was obtained. The variation in the inhibition observed was mainly due to the different sources of lymphocytes used. Unrelated proteins and hormones, tested at the same concentration as EPF, did not show any inhibitory activity. Using the F(ab)2 fragment of anti-human IgG instead of the whole molecule the same levels of inhibition were obtained, suggesting that the observed inhibition by EPF was not due to a non-specific interaction between the anti-human IgG and the Fc receptors on the cell. Such inhibitory activity detected in vitro by this method provides additional support for a suppressive role for EPF during pregnancy.     

Modulatory influences of estrogen and progesterone on cervical dysplasia in mice model system.

The effectiveness of estrogen and progesterone on the different types of cervical dysplasia are poorly documented compared to their effects on the total process of cervical carcinogenesis. In the present study, an attempt has been made to evaluate the influences of estrogen and progesterone on the different types of cervical dysplasia which are believed to be the antecedents of cervical carcinoma induced by chemical carcinogen (20-Methy cholanthrene) in the mice model system. It was observed that moderate dysplasia is less aberrant than that of marked dysplasia which could not be modified by either of the hormones.