Immune regulation at the interface during early steps of murine implantation: involvement of two new cytokines of the IL-12 family (IL-23 and IL-27) and of TWEAK

PROBLEM: An important subset of implantation defects/early abortion seems to be linked with a deregulation of the interleukin (IL)-12/IL-15/IL-18 system as well as tumor necrosis factor (TNF)- and natural killer (NK)-controlled/mediated networks at the decidual placental interface, both in case of deficient or excess expression. The presence of TNF in high amounts in the pre/peri-implantation uterus and its pivotal role during pregnancy are difficult to reconcile with its abortive effects in ongoing pregnancy. We therefore searched for regulators of the IL-12/IL-18 family of cytokines as well as for antagonist(s) of TNF with potentially selective effects on implantation. METHOD OF STUDY: We first used Swiss mice to verify the presence in the murine reproductive tract of 'new members' of the IL-12 family of cytokines, IL-23 and IL-27, as well as of tumor necrosis factor-like WEAK inducer of apoptosis (TWEAK), described as acting with TNF as Yin and Yang of innate immunity in murine placenta/ decidua at days 0-12.5. We then compared expression by RT-PCR in the CBA x DBA/2, and CBA x BALB/c murine mating combinations. Finally, we performed in vivo neutralization experiments of TWEAK and IL-27. RESULTS: Immunohistochemistry (IHC) studies showed that IL-23, IL-27, and TWEAK were expressed at the interface. For RT-PCR, IL-23 expression peaked at day 9.5 in the non-aborting mating combination, a peak absent in the aborting one, and thus difficult to explain except by invoking a feed back on EB13 (Epstein-Barr virus-induced gene 3 cytokine). Most important, an immediate post-mating IL-27 hyper expression was seen in the CBA x DBA/2 mating compared to CBA x BALB/c one. The difference in expression resurged and was statistically very significant by days 6.5-9.5, compatible with an early activation of inflammation on day 0.5 which would then peak again in the 'resorption window' where takes place the early NK/mph activation described by Baines et al. A significant TWEAK expression was present in both strains from days 0.5 to 4.5 peaking in both cases in the first days when it is known that intra uterine TNF also reaches high levels as a component of post-mating inflammation. However, it was lower from day 1.5 in the abortion-prone CBA x DBA/2 mating combination, and almost absent by days 6.5-9.5 when compared to the non-aborting CBA x BALB/c mating combination. In both mating combinations, neutralization of TWEAK-enhanced resorption rates, but surprisingly so did IL-27 neutralization. CONCLUSION: TWEAK is likely offering protection against the deleterious effects of TNF in implantation explaining embryo survival in a TNF-rich environment, and equal number of implants in both strains. However, there is a clear difference of protection in abortion-prone mating peaking in the abortion/resorption window but starting early, and therefore possible links with the prevention of abortion in CBA x DBA/2 matings by interfering with complement activation as recently described by Girardi et al. are discussed, as well as consequences for our current view of feto-maternal 'seed and soil' interplay. The apparently paradoxical effects of IL-27 neutralization are also discussed.     

Regulation of interleukin-6 fetoplacental levels could be involved in the protective effect of low-molecular weight heparin treatment on murine spontaneous abortion

PROBLEM: CBA/J x DBA/2 abortion rate could be the consequence of a deficient local production of T helper (Th2) cytokines, which cause fetal wastage via fgl2 prothrombinase. Heparin reduces significantly the abortion rate in mice and recurrent spontaneous abortion (RSA) patients. We proposed to determine the effect of enoxaparin on the levels of local interleukin (IL)-6 during murine pregnancy. METHOD OF STUDY: Recombinant human IL-6 (rhIL-6) or enoxaparin were inoculated in CBA/J x DBA/2 pregnant mice on days 6.5-12.5. IL-6 levels in sera as well as in culture supernatants of day 9.5 fetoplacental units of CBA/J x BALB/c control mice or CBA/J x DBA/2 abortion combination were determined by enzyme-linked immunosorbent assay (ELISA) test. RESULTS: CBA/J x DBA/2 fetoplacental units secreted significantly lower levels of IL-6 with regard to CBA/J x BALB/c normal units. rhIL-6h and enoxaparin treatments decreased the resorption rate and regulated IL-6 fetoplacental levels. CONCLUSION: This study suggests that regulation of IL-6 fetoplacental levels could be involved in heparin-mediated anticoagulation protection against abortion.     

过继转输胚胎抗原耐受T细胞诱导自然流产孕鼠母胎免疫耐受

探讨过继转输胚胎抗原耐受T细胞对小鼠自然流产模型妊娠预后及宿主孕鼠免疫细胞对父系抗原免疫耐受状态的影响。以CBA/J×BALB/c为正常妊娠模型 ,CBA/J×DBA/ 2为自然流产模型 ,将自然流产模型CBA/J孕鼠于孕 4d (着床期 )分别腹腔注射大鼠抗小鼠CD80和CD86mAb或大鼠同型IgG。于孕 9d ,应用免疫磁珠阴性分选三组孕鼠的脾脏T细胞 ,并将三组T细胞分别转输至孕 4d的CBA/J×DBA/ 2孕鼠。于宿主孕鼠孕第 9天 ,采用单向混合淋巴细胞反应分析宿主孕鼠脾脏免疫细胞对父系抗原的增殖能力 ,并用流式细胞术分析经父系抗原刺激的宿主孕鼠脾脏T细胞内IL 2表达水平。于孕 1 4d分别观察宿主孕鼠的胚胎吸收率。结果显示 ,过继转输胚胎抗原耐受T细胞和转输正常妊娠模型孕鼠的T细胞均可诱导宿主孕鼠脾脏免疫细胞对父系抗原的增殖能力及IL 2的表达显著下降 (P <0 0 5 ) ,孕 1 4d胚胎吸收率也显著下降 (P <0 0 5 )。这些结果表明 ,于孕早期过继转输胚胎抗原耐受T细胞和转输正常妊娠模型孕鼠的T细胞能诱导宿主孕鼠母 胎免疫耐受 ,防止母体对胚胎的免疫排斥 ,从而使自然流产模型的妊娠预后达到正常妊娠水平。     

Ly-49D transfected NK cells show reduced interleukin-10 production in response to H-2d and increased lytic activity: implication by interaction using class I MHC alloantigen+ target cells in pregnancy

PROBLEM: Mating of CBA/J (H-2k) with DBA/2 (H-2d) males leads to a high rate of spontaneous resorption (about 40%), which is not seen in other mating combinations, such as CBA/J X BALB/c. The activation of natural killer cells (NK cells) seems to be a key mechanism for the maternal-fetal intolerance in allogeneic pregnancy, and recurrent spontaneous abortion. The effect of expression of the NK cell activating receptor Ly49D recognizing BALB/c or DBA/2 class I MHC was investigated. METHOD OF STUDY: Intracellular interleukin (IL)-100 production was detected and target cell survival rates were calculated after 22 hr coincubation of rat NK cells transfected, or not. with a murine Ly49D receptor, with either male BALB/c or male DBA/2 splenocytes, by using flow cytometry. RESULTS: Ly49D negative rat NK cells produced 13.7% more IL-10 than Ly49D positive rat NK cells, and more splenocytes were killed by Ly49D transfected rat NK cells (survival rate 2.45%) than by Ly49D negative rat NK cells (survival rate 4.36%). CONCLUSION: After physiological stimulation with BALB/c or DBA/2 splenocytes, rat NK cells are able to synthesize IL-10. Recognition of mouse splenocyte major histocompatibility complex (MHC) by Ly49D mice receptor decreased IL-10 production. The observed increase in killing activity might be a result of this phenomenon. NK cell activation via the Ly49D receptor might play an important role in pregnancy failure, but cannot explain why CBA/J X DBA/2 matings are abortion prone, and CBA/J X BALB/c matings are abortion resistant.     

Reduced stathmin-1 expression in natural killer cells associated with spontaneous abortion

Female CBA/J mice impregnated by male DBA/2J mice (CBA/J×DBA/2J matings) are prone to spontaneous abortion, although the reason for this is unclear. In this study, the stathmin-1 expression pattern was evaluated in uterine natural killer (uNK) cells purified from CBA/J×DBA/2J matings. Results were compared with those in a CBA/J×BALB/c control group that yields successful pregnancies. The mean ± SD percentage of stathmin-1(+) cells in the CD49b(+) uNK cell population was lower in CBA/J×DBA/2J mice (0.7% ± 0.4%) than in control CBA/J×BALB/c mice (4.9% ± 1.5%, P < 0.01) using flow cytometry, and the intracellular stathmin-1 level in uNK cells was lower in CBA/J×DBA/2J mice than in control mice using Western blot analysis. Co-localization of lectin from Dolichos biflorus agglutinin (DBA-lectin) and stathmin-1 was confirmed using multivision immunohistochemical analysis. The frequency of stathmin-1(+)DBA-lectin(+) cells was lower in CBA/J×DBA/2J mice than in CBA/J×BALB/c mice. A similar trend in the frequency of stathmin-1(+)CD56(+) cells was seen in patients with unexplained spontaneous abortion compared with normal early pregnancy. A neutralizing antibody against stathmin-1 further increased the percentage of embryo loss in CBA/J×DBA/2J matings. These results provide evidence that stathmin-1 expression in uNK cells at the maternal-fetal interface may help modulate uNK cell function and may be beneficial for a successful pregnancy.     

免疫治疗对母胎交界面NK细胞表面CD69表达水平的影响及其与鼠胚和鼠仔预后的关系

目的:研究反复自然流产(recurrent spontaneous abortion,RSA)模型CBA/J×DBA/2小鼠母胎交界面NK细胞表面CD69分子的表达水平,并评价淋巴细胞免疫治疗(lymphocyteimmunotherapy,LIT)对CD69表达水平的影响及其与鼠胚和鼠仔预后的关系。方法:评价CBA/J×DBA/2、C57BL/6×DBA/2和BALB/c×DBA/2小鼠胚胎和鼠仔的预后,绘制出生后1-21 d鼠仔离乳前生长曲线和Kaplan-Meier生存分析曲线。并且采用PE-CD69和FITC-DX5双色流式细胞术检测在有或无LIT的情况下,母胎交界面NK细胞表面CD69分子的表达水平。此外,对CD16/CD32⁺NK细胞亚群等也进行检测。结果:在孕期母鼠体重平均增长幅度、平均每窝产仔数、新生鼠仔出生第1 d平均体重和胚胎吸收率等方面,CBA/J×DBA/2小鼠与生殖力正常的对照组相比均有显著差异。在CBA/J×DBA/2小鼠中,代表活化型NK细胞的CD69⁺DX5⁺细胞比率也显著高于对照组。而有效的LIT能够显著降低CD69在母胎交界面NK细胞表面的表达水平,并相应地显著降低胚胎吸收率。结论:CD69⁺DX5⁺细胞在胚胎被排斥的机制中可能具有重要作用。有效的LIT通过降低CD69分子在母胎交界面NK细胞表面的表达水平而显著降低流产率。     

原因不明复发性流产中CD4+ Notch1+T细胞与IL-10的相关性分析

探讨原因不明复发性流产（URSA）中CD4＋Notch1＋T细胞（Notch1＋占CD4＋T细胞的比例）与白介素-10（IL-10）表达水平的相关性。以雌性CBA/J×雄性Balb/c为正常妊娠模型,以雌性CBA/J×雄性DBA/2J为自然流产模型,采用流式细胞术检测6例未孕CBA/J雌性小鼠、6例正常妊娠模型孕14天CBA/J雌性小鼠和6例自然流产模型孕14天CBA/J雌性小鼠脾细胞中CD4＋Notch1＋T细胞,同时运用ELISA法检测其血清中IL-10的表达水平。相比于未孕组,正常妊娠模型中CD4＋Notch1＋T细胞比例减少,而自然流产模型中CD4＋Notch1＋T细胞比例增加,正常妊娠模型与自然流产模型中CD4＋Notch1＋T细胞比例差异有统计学意义（P〈0.05）。CD4＋Notch1＋T细胞比例与IL-10负相关（r=-0.568,P〈0.05）。结论：CD4＋Notch1＋T细胞可能参与原因不明复发性流产发病机制,拮抗Notch1＋表达有可能成为治疗URSA新途径。     

ORIGINAL ARTICLE: How Should Data on Murine Spontaneous Abortion Rates be Expressed and Analyzed?

Problem Spontaneous abortions in the CBA x DBA/2 model are normally reported as number of resorptions/total number of implantations (R/T), pooling data from individual mice. The significance of differences between groups has been determined using non-parametric statistics (e.g. chi-square or Fisher's Exact test) based on a priori predictions. Recently, it has been argued that medians with box plots should replace the accepted standard, but this deprives readers of data needed to verify P-values, and leads to inferences incompatible with biological and statistical reality. Method of study Raw data on 173 individual CBA x DBA/2 matings were analyzed by median and mean, along with R/T data from 18 independent experiments containing 5-10 mice per group. Raw data from 19 CBA x BALB/c matings were similarly analyzed. Results Individual CBA x DBA/2 mouse resorption rates showed a non-Gaussian distribution, but the mean and median differed by <0.5%. Resorption data from 6 and 12 independent pools of mice were normally distributed. Only the mean enabled a between-group P-value calculation. CBA x BALB/c matings gave a median of 0 and mean of 5.1%; the data were not normally distributed, but that was because of a bimodal distribution. One group of mice had 0 abortions, and the second a mean of 13.9% abortions, and the data from the latter group were normally distributed. Conclusion Although it is possible to compare individual mice, and even individual implantation sites, in resorption (abortion) studies, as the relevant question is the significance of differences between treatment groups of mice, and reproducibility, the established classical method of reporting R/T should continue to be provided. In CBA x BALB/c matings, where abortion rates are low, using the median is misleading and may obscure the existence of two distinct populations.     

IL-6 as a regulatory factor of the humoral response during pregnancy

Problem Regulatory factors seem to be essential to achieve transition from implantation window to placental vascularization. A novel function of interleukin (IL)-6 in the promotion of Th2 differentiation and inhibition of Th1 polarization has been demonstrated. Considering that Th2 response promotes antibody synthesis, we postulate that IL-6 could be modulating the quality of this response during pregnancy by increasing the proportion of blocking asymmetric antibodies. Method of study We investigated expression of blocking-asymmetric-IgG during pregnancy of CBA/J x DBA/2 abortion model treated with IL-6, with regards to CBA/J x BALB/c. We also determined asymmetric-IgG production in IL-6-deficient pregnant mice. Results We found that IL-6 treatment increased asymmetric-IgG in multiparous placentas from abortion combination whereas diminished abortion rate. Moreover, asymmetric-IgG proportion was diminished in sera from IL-6-deficient pregnant mice. Conclusion Modulation of asymmetric antibody synthesis could be another mechanism implicated in the beneficial effect of IL-6 in prevention of murine recurrent abortion.     

Host genetic background determines whether IL-18 deficiency results in increased susceptibility or resistance to murine Leishmania major infection

Interleukin-18 (IL-18) plays an important role in innate and acquired immunity. IL-18 gene deficient (IL-18-/-) mice of the 129 x CD1 strain were reported to be more susceptible to Leishmania major infection than the wild-type mice. In contrast IL-18-/- mice of the C57BL/6 background were found to be as resistant as the wild-type (WT) mice. To resolve this discrepancy, IL-18 gene deficiency was introduced by backcrossing on to the highly susceptible BALB/c, or the moderately resistant DBA/1 backgrounds. Here we have demonstrated that BALB/c IL-18-/- mice were more resistant to L. major infection than WT BALB/c mice, whereas DBA/1 IL-18-/- mice were markedly more susceptible than their WT littermates. BALB/c IL-18-/- mice produced less IFNgamma and IL-4, whereas DBA/1 IL-18ko mice produced more IFNgamma and IL-4 than their respective WT controls. These result clearly demonstrate that the role of IL-18 in resistance or susceptibility to L. major is determined by host genetic background.     

环孢霉素A对小鼠妊娠失败模型妊娠预后的影响

目的 :探讨环孢霉素A(CyclosporinA ,CsA)对小鼠妊娠失败模型及正常妊娠模型妊娠预后的影响。方法 :于孕 4d分别给小鼠妊娠失败模型及正常妊娠模型腹腔注射不同剂量 (0 ,5 ,10 ,15mg kg)CsA ,于孕 14d处死各组小鼠 ,观察各实验组胚胎吸收率、胎鼠及胎盘重量变化。结果 :①CsA可使小鼠妊娠失败模型胚胎吸收率显著降低 ,接近于小鼠正常模型胚胎吸收率 ;同时胎鼠及胎盘重量无显著性差异 ,但有增加趋势。②CsA对小鼠正常妊娠模型胚胎吸收率无明显影响 ;胎鼠及胎盘重量亦无显著性差异。结论 :于孕早期 (胚胎着床期 )腹腔注射CsA可使小鼠妊娠失败模型胚胎吸收率恢复至正常水平 ,从而可能成为保胎的潜在药物。     

Therapy with dendritic cells influences the spontaneous resorption rate in the CBA/J x DBA/2J mouse model

PROBLEM: DBA/2J-mated CBA/J female mice are prone to a high incidence of fetal abortions. This fetal wastage can be dramatically reduced by immunizing the female mice with BALB/c, but not with DBA/2J spleen cells during early gestation. Nevertheless, the underlying mechanisms remain to be elucidated. Recently, dendritic cells (DC) have been described at the feto-maternal interface in the human uterus. In this work, we studied the effect of adoptive transfer of DC on the maintenance of pregnancy in the CBA/J x DBA/2J model. METHODS: Bone marrow-derived DC were generated from virgin female CBA/J mice (6-8 weeks old). CBA/J females were inoculated with DC twice before mating. Four different experimental groups were included: (i) no treatment control, (ii) mice injected with culture medium [granulocyte-macrophage colony-stimulating factor (GM-CSF)], (iii) immunized with DC and (iv) immunized with paternal DBA/2J antigens lisate-pulsed DC, n = 5. RESULTS: The control abortion rate was 23.8%, and with GM-CSF alone was 17.6%. Following inoculation of syngeneic DC abortion rates were reduced to 2.2%, but protection was short-lived. Abortion rates with DC pulsed with DBA/2J antigens was 5%. Serum of interleukin (IL)-6 levels were lower in the latter two groups up to the time of abortion. The kinetics of immunoglobulin G asymmetric antibodies synthesis was modified, but there was no correlation between asymmetric antibodies production and the lowering of abortions rates. CONCLUSION: Syngeneic DC prevented abortions and this was linked to a decrease in IL-6 levels, but not with levels of asymmetric antibodies.     

Altered Modulation of the In Vitro Antibody Synthesis by Placental Factors from the CBA/J × DBA/2 Abortion-Prone Mating Combination

PROBLEM: The in vitro immunomodulating effect of placental culture supernatants (PSs) obtained from two H-2k × H-2d allogeneic crossbreedings, the CBA/J × DBA/2 abortion-prone mating combination, and the reproductively normal pregnancy CBA/J × BALB/c crossbreeding were compared, and the influence of previous deliveries was evaluated. The behavior of placentae obtained from CBA/J females with two previous pregnancies by BALB/c males was also investigated. METHOD OF STUDY: Supernatants of cultures of murine placentae were added to a mouse immunoglobulin (Ig) G1 hybridoma culture which produced anti-dinitrophenol (anti-DNP) antibodies. The quantity of monoclonal antibody produced, the nature of these antibodies, and the proliferation of the hybridoma cells were studied. RESULTS: CBA/J × DBA/2 placental factors obtained from multiparous females induced a diminished asymmetric IgG antibody production without varying the quantity of antibody produced. In contrast, PSs obtained from the nonresorption-prone CBA/J × BALB/c mating combination with the same number of previous deliveries enhanced the production of both symmetric and asymmetric anti-DNP molecules and also increased the proportion of asymmetric blocking monoclonal antibodies (mAbs) synthesized by the hybridoma. Both of the PSs analyzed had induced similar inhibition of ³H-thymidine uptake. PSs obtained from the abortion-prone mating combination whose CBA/J females had two previous pregnancies by BALB/c males showed similar immunomodulating effects to those observed using multiparous CBA/J × BALB/c placentae. CONCLUSIONS: We propose that the placenta produces soluble factors that participate in the regulation of antibody synthesis by the mother during gestation. Such a placental immunomodulating effect appears to be altered in the CBA/J × DBA/2 abortion-prone mating combination and could be corrected by previous pregnancies by BALB/c males. These observations suggest that placental factors would be relevant to the protection of the fetus and might play an important role in the immune equilibrium between mother and fetus. Asymmetric antibody production as a Th2 responsiveness was also discussed.     

Differential production of IL-12 in BALB/c and DBA/2 mice controls IL-4 versus IFN-{gamma} synthesis in primed CD4 lymphocytes

The profile of cytokines produced by CD4 T cells is profoundlyinfluenced by the presence of IL-12. Here we demonstrate thatduring re-stimulation of antigen-specific immune responses invitro, antigen-primed lymph node cells from DBA/2 mice produced3- to 30-fold more IL-12 than did cells from BALB/c mice, whichare identical at the major histocompatibility locus. The straindifferences in IL-12 production were observed only in antigen-drivenresponses (and not in responses induced by bacterial products),and were dependent upon an interaction between CD4 T cells andlymph node adherent cells. In addition, differences in the quantityof IL-12 produced by DBA/2 and BALB/c antigen-presenting cells(APC) was not dependent on differential production of IFN- byT cells, since APC from DBA/2 mice still produced much greaterquantities of IL-12 than did BALB/c APC when each was culturedwith the same H-2^d-restricted T_h2 clones, in the complete absenceof IFN, or when each was cultured with primed (BALB/c x DBA/2)F₁T cells. The level of IL-12 produced in the cultures criticallyaffected cytokine production in CD4 T cells, since neutralizationof endogenous IL-12 in DBA/2 cultures, which are predisposedtowards developing T_h1 responses, reduced IFN- production andenhanced IL-4 synthesis to levels normally seen in BALB/c cultures,which are predisposed toward developing Th2 responses. We proposetherefore that differential production of antigen-driven IL-12is a mechanism by which the genetic background in DBA/2 andBALB/c mice can affect the pattern of cytokine synthesis byT cells during the development of adaptive immune responses.     

Immunogenetic studies of spontaneous abortion in mice. III. Non-H-2 antigens and gestation

CBA/J (H-2k) females, mated with DBA/2 J (H-2d) or DBA/1 J (H-2q) males, exhibit a high rate of fetal resorption. In contrast, when H-2 identical CBA substrains (i.e. CBA/Ca and CBA/N) are used, this phenomenon is not observed. On the other hand, before mating with DBA/2 J males, pre-immunization of CBA/J females with spleen cells coming from BALB/c J or (DBA/2 x BALB/c J) F1 males (and not from other H-2d identical males whatever their Mls alleles) has significantly decreased the fetal resorption rate. Thus, immunization against determinants other than classical H-2d (K, I, D, L) antigens (transmitted as a dominant character and different from Mls determinants) can elicit anti-abortive effects. Furthermore, it was observed that the spleen cell endowed with the anti-abortive effects was neither a T nor a B lymphocyte. In contrast, peritoneal cells were able to reproduce the phenomenon, indicating that it may be mediated by a cell of the macrophage-monocyte lineage. Finally, a first gestation was substituted for allo-immunization of CBA/J females. The anti-abortive effects of a first pregnancy by BALB/c J males (and not by other H-2k syngeneic or H-2d allogeneic males) was observed in the course of a second pregnancy sired by DBA/2 J males. These data can be interpreted in terms of maternal recognition of an antigen present on both macrophages and trophoblast cells and necessary for a successful gestation, which is coded for by genes outside the K, I, D, L regions.     

IL-12 directs severe renal injury,crescent formation and Th1 responses in murine glomerulonephritis

Glomerular crescent formation characterizes severe glomerulonephritis (GN). Evidence suggests that crescent formation results from a delayed-type hypersensitivity-like Th1 response. As IL-12 directs Th1 responses, we tested the hypothesis that IL-12 is important in crescentic GN. Neutralization of IL-12 attenuated crescent formation and cell-mediated injury in C57BL/6 mice sensitized to and challenged with sheep anti-mouse glomerular basement membrane (GBM) globulin. Recombinant IL-12 induced severe crescentic GN with enhanced Th1 responses in C57BL/6 mice in which non-crescentic GN was induced by injecting anti-GBM globulin into naive mice. BALB/c mice do not develop significant crescent formation in these models, due either to regulatory effects of IL-4, or to deficits in IL-12 production/responsiveness. Administering IL-12 to BALB/c mice with GN induced Th1 responses and crescent formation, whereas IL-4-deficient BALB/c mice did not develop cell-mediated crescentic injury when GN was induced in sensitized mice. These results establish a central role for IL-12 in severe crescentic GN.     

维生素D3对自然流产模型小鼠胚胎丢失率及外周血免疫细胞的影响

目的 探讨维生素D3（VitD₃）对自然流产模型小鼠胚胎丢失率及外周血免疫细胞的影响。 方法 采用CBA/J×BALB/c建立正常妊娠小鼠模型,采用CBA/J×DBA/2建立自然流产小鼠模型,实验动物分5组：正常组、自然流产组、VitD3低剂量组、中剂量组和高剂量组,每组15只。VitD₃各组自妊娠第1天开始腹腔注射VitD₃溶液,单次VitD₃给药剂量分别为1μg、4μg、16μg。记录和观察各组小鼠胚胎丢失情况,检测各组血清调节性T细胞（Treg）细胞因子：白细胞介素-10（IL-10）,辅助性T细胞17（Th17）细胞因子：白细胞介素-17A（IL-17A）以及各组外周血Treg、Th17。 结果 与正常组相比,自然流产组胚胎丢失率显著升高（χ2=16.045,P＜0.05）;与自然流产组比较,VitD₃低剂量组、中剂量组和高剂量组胚胎丢失率显著降低（χ2=5.881、13.704、15.663,P＜0.05）。与正常组相比,自然流产组Treg、IL-10、Treg/Th17、IL-10/IL-17A显著降低,Th17、IL-17A显著升高,差异均有统计学意义（P＜0.05）;与自然流产组比较,VitD₃低剂量组、中剂量组和高剂量组Treg、IL-10、Treg/Th17、IL-10/IL-17A显著升高,Th17、IL-17A显著降低,差异均有统计学意义（P＜0.05）。 结论 VitD₃能够降低自然流产模型小鼠胚胎丢失率,可能与调节Treg/Th17平衡有关。     

环孢素A诱导妊娠失败模型孕鼠外周母-胎免疫耐受

目的探讨环孢素A(cyclosporin A,CsA)能否诱导妊娠失败模型CBA/J×DBA/2孕鼠外周母-胎免疫耐受。方法分别使用CBA/J×DBA/2及CBA/J×BALB/c作为妊娠失败模型和正常妊娠模型,CBA/J小鼠于妊娠第4天(着床期)腹腔注射不同剂量(0、5、10、15mg/kg)CsA,妊娠第9天和第14天时分别采用单向混合淋巴细胞反应分析孕鼠外周脾脏免疫细胞对父系抗原的增殖能力,并用RT-PCR分析细胞IL-2mRNA含量以研究脾脏细胞母-胎免疫耐受状态;妊娠第14天观察两组的胚胎吸收率。结果(1)于妊娠第4天腹腔注射5、10、15mg/kg CsA后,妊娠失败模型胚胎吸收率分别下降至2.86%、5.75%及11.24%,明显低于对照组(P<0.001,P<0.01,P<0.01);而对妊娠成功模型胚胎吸收率无显著性影响(P>0.05)。(2)混合淋巴细胞反应研究显示,于妊娠第4天腹腔注射CsA,可使妊娠第9、14天的妊娠失败模型孕鼠外周脾脏免疫细胞对父系抗原的增殖能力显著下降(P均<0.01);RT-PCR分析显示,孕鼠脾细胞受父系抗原刺激后IL-2转录显著下降(P均<0.01)。结论于孕早期腹腔注射CsA可诱导妊娠失败模型孕鼠外周免疫细胞对父系抗原的特异性免疫耐受,从而使自然流产模型孕鼠的妊娠结局达到正常妊娠水平,提示CsA可能成为治疗妊娠失败的有效药物。     

IL-12 prevents neonatal induction of transplantation tolerance in mice

We investigated the effect of IL-12 on the induction of transplantation tolerance by neonatal injection of allogenic cells. We first observed that injection of newborn BALB/c mice with IL-12 and (A/J × BALB/c) F1 spleen cells prevented the Th2 alloimmune response induced by neonatal inoculation of F1 cells alone and allowed the differentiation of T cells secreting high amounts of IL-2 and IFN-γ in mixed lymphocyte cultures with donor-type stimulators. Furthermore, IL-12 administration resulted in the emergence of anti-donor cytotoxic T lymphocyte responses although at lower levels than in control uninjected mice. In parallel, we found that mice injected at birth with IL-12 and F1 cells did not develop chimerism and were able to reject a donor-type skin graft as efficiently as control mice. We conclude that IL-12 inhibits the Th2 polarization of the newborn response to alloantigens and prevents thereby the establishment of transplantation tolerance.     

Age,Environment, and Lymphocyte Immunization Influence the Spontaneous Resorption Rate in the CBA/J × DBA/2J Mouse Model

PROBLEM: This study was designed to examine the influence of age, environment, and lymphocyte immunotherapy on fetal resorption rates in the CBA/J × DJA/2J mouse model. METHODS: After weaning, CBA/J female mice were randomly allocated to two different environments: room A was a conventional housing facility, and room B was a specific pathogen-free room. They were further divided into two groups in each room according to age (> three months and ≤ three months), and mated with CBA/J, DBA/2J, or BALB/c males. The fetal resorption rates were observed. The immunization study was conducted using CBA/J females greater than three months old in room B. Preimmunization with various preparations and dosages of lymphoctyes was performed 1 wk before mating with DBA/2J males, and fetal resorption rates were measured to investigate the effect of immunotherapy. RESULTS: In room A, the fetal resorption rates (9.4 to 11.8%) were not significantly different among the various mating combinations using CBA/J females greater or less than three months of age. In room B, CBA/J females older than three months and mated with DBA/2J males had a higher rate of fetal resorption than those younger than three months (28.9 versus 14.2%) and had higher rate of fetal resorption than those housed and mated in room A (28.9 versus 10.9%). This fetal resorption rate was also higher than those seen with other mating combinations. Preimmunization with male BALB/c splenic lymphocytes (optimal dose 1 × 10⁷ cells) was effective in decreasing fetal resorption rate (7.9%), whereas administration of normal saline, or immunization with male CBA/J or DBA/2J lymphocytes was not (20.7 to 27.0%). CONCLUSION: Age and environment influence the spontaneous resorption rate in the CBA/J × DBA/2J mouse model. The high fetal resorption rate of older CBA/J females mated with DBA/2J males in a pathogen-free environment can be reduced by immunization with BALB/c splenic lymphocytes. These results suggested that a variety of mechanisms might initiate early pregnancy failure and that immunological modulation during implantation might be a nonspecific factor.