Dynamics and reproductive effects of complement factors in the spontaneous abortion model of CBA/J × DBA/2 mice

The complement system is one component of innate immunity that could participate in fetal loss. We have already reported that adipsin, a complement activator in the alternative pathway, is stably expressed in the placenta and that an increase in this expression is related to spontaneous abortion. However, complement inhibitor Crry was concurrently expressed in the placenta, and the role of complement factors during pregnancy was not clear. In the present study, we examined the endogenous regulation of complement factors in placenta and serum by using another model mouse for spontaneous abortion and studied the effect of exogenous complement disruption on pregnancy. Compared to control mice, the CBA/J × DBA/2 model mice had higher expression levels of adipsin in the placenta and serum. Adipsin and complement C3 were localized in the metrial gland and labyrinth regions, and both positive reactive ranges were limited in the maternal blood current in normal implantation sites. These results suggest that extrauterine adipsin hematogenously reaches the placenta, activates complement C3, and promotes destruction of the feto-maternal barrier in aborted implantation sites. Crry was consistently expressed in the placenta and serum and reduced in the resorption sites of CBA/J × DBA/2 mice as compared to normal sites. Injection of recombinant adipsin increased the resorption rate and changed the expression of Th-type cytokines toward a Th1 bias. The present study indicates that adipsin could induce the fetal loss that accompanies the Th1 bias and may be a crucial cause of spontaneous abortion. In addition, the local expression of Crry prevents complement activation in placenta in response to a systemic increase of adipsin.     

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.     

Is Spontaneous Resorption in the DBA/2-Mated CBA/J Mouse due to a Defect in “Seed” or in “Soil”?

PROBLEM: Recurrent spontaneous resorption in DBA/2-mated CBA/J mice has been attributed to damage by NK-lineage cells and TNF-α beginning several days after implantation. However, some recent data have suggested CBA/J female mice have a high proportion of preimplantation embryo abnormalities resulting in defective in vitro implantation and impaired trophoblast outgrowth. Could spontaneous abortion be due to a defective embryo (“seed”) rather than a hostile post-implantation uterine lining (“soil”). METHOD: Mated CBA/J females were manipulated so as to have high spontaneous abortion rates and a high percent abnormal embryos, or low resorption rates and a low percent abnormal embryos. Embryos from low aborting females were transferred into high aborting female recipients that were pseudopregnant, and vice versa. RESULTS: Abnormal embryos from females with high abortion rates implanted in low aborting females and did not show any greater tendency to resorb than normally developed embryos in these recipients. By contrast, normal embryos to some extent and abnormal embryos to a much greater extent, gave a high abortion rate when the recipient female was a high aborter. CONCLUSION: Properties of the “soil” into which embryos implant determines the likelihood of success or failure (abortion). Abnormal pre-implantation embryos can be “rescued” by “good soil”; “sick soil” damages both normal and abnormal embryos. Defining the cellular and molecular mechanisms may be useful in understanding basic mechanisms leading to aborting and nonaborting pregnancy.     

A double replication of a “small-sample” study of the sexual behavior of DBA/2J male mice

The sexual behavior scores of three different groups of DBA/2J male mice, run at different times of the year, in two different places, and by different observers, were compared. Of 46 comparisons, two revealed statistically significant differences. It was hypothesized that one measure (% Move) was subject to much environmentally determined variation, while for the other measure (III), the significant difference may have been due to sampling error. Furthermore, substitution of the data resulting from either replication for the data of the DBA/2J males of the original study did not result in any changes as regards significant differences in sexual behavior among DBA/2J, C57BL, and BALB/c male mice. The results, in general, supported the hypothesis that intensive study of a small sample from an inbred strain would yield replicable results.     

ASRI2005‐89 During pregnancy,treg cells induce a privileged tolerant microenvironment at the fetal‐maternal interface by up‐regulating HO‐1, TGF‐β and LIF expression

The mechanisms underlying immune tolerance during pregnancy are poorly understood. We recently proposed a crucial role of T regulatory cells (Treg) in avoiding immunological rejection of the fetus, since we observed diminished number and function of Treg in abortion-prone mice. We further confirmed the protective role of Treg during pregnancy by transferring pregnancy-induced Treg into abortion-prone mice, which prevented rejection. Here, we analyzed the mechanisms involved in Treg-mediated protection. We transferred mice undergoing abortion with Treg from mice having normal pregnancies, including normal pregnant and abortion-prone mice as controls. We evaluated decidual lymphocytes for their cytokine profile. The mRNA levels for several tolerance-associated cytokines were also analyzed. As expected, Treg therapy prevented abortion, while expanding the peripheral and thymic Treg population. Locally, augmented foxp3 levels could be observed. Surprisingly, after therapy the decidual levels of the Th1 cytokines IFN-γ and TNF-α were not diminished. The transfer of Treg dramatically up-regulated the expression of LIF, TGF-β and HO-1 at the fetal-maternal interface. Our data suggest that Treg-treatment can not prevent T cell infiltration or high Th1 levels but is able to create a privileged tolerant microenvironment at the fetal-maternal interface, further shedding light onto the molecular mechanisms involved in pregnancy tolerance.     

Absence of in vitro responses to type II collagen by splenocytes from arthritic BALB/c mice is possibly caused by intrinsic CD25+ regulatory cells

Collagen-induced arthritis-resistant BALB/ c mice develop arthritis if a foreign protein is added to an emulsion of type II collagen (CII) and adjuvant. The IgG autoantibody activity to CII is increased, whereas no CII autoreactive T cells in vitro can be recorded. In this study, we have explored whether CD25⁺ cells inhibit T-cell autoreactivity to CII. We also followed the IgG anti-CII autoantibody activity and the IL-6 level in serum during the development of arthritis. BALB/ c mice were coimmunized with bovine CII (BCII) and keyhole limpet haemocyanin (KLH) in complete Freund's adjuvant and boostered 3 weeks later. Control animals were immunized with either BCII or KLH. Sera were collected prior to and during the development of arthritis and examined for IgG anti-CII antibody activity and IL-6 content. When all BCII–KLH immunized mice had developed arthritis, splenocytes were prepared, with and without CD25⁺ cells, and tested for BCII reactivity in vitro . The serum IgG, IgG1 and IgG2a anti-CII antibody activities and the IL-6 level were significantly higher in BCII–KLH immunized mice than in BCII-immunized animals that failed to develop arthritis. The BCII-specific IL-2 secretion in vitro was significantly increased in CD25-depleted splenocyte cultures prepared from arthritic BCII–KLH-immunized mice. Development of arthritis in BALB/ c mice induced by coimmunization with BCII/KLH results in increased levels of circulating IL-6 and IgG autoantibodies to CII. The arthritogenic BCII–KLH immunization potentiates BCII-specific IL-2 secretion by CD25-depleted splenocytes, but CD25⁺ cells hamper the outcome of their action, at least in vitro .     

Prevention of spontaneous abortion in the CBA x DBA/2 mouse model by intravaginal TGF-beta and local recruitment of CD4+8+ FOXP3+ cells.

PROBLEM: Activation of latent transforming growth factor (TGF)-beta in seminal plasma has been suggested by Robertson et al. to promote maternal tolerance to paternal antigens. A possible consequence reported by Tremellen et al. is increased pregnancy rates in women undergoing IVF. A decreased spontaneous abortion rate has also been postulated. Seminal plasma contains many factors besides TGF-beta, and a critical test of the hypothesis was required. The purpose of the present study was to directly test the effect of pure TGF-beta. METHOD OF STUDY: Pharmaceutical grade bioactive TGF-beta3 with a bovine serum albumin (BSA) carrier 0.1-1% in phosphate-buffered saline (PBS) was given into the vaginal tract of CBA/J female mice at the time of mating with DBA/2 males. One microgram Salmonella enteritidis lipopolysaccharide was given intraperitoneally to augment occult losses and spontaneous resorptions assessed on day 13.5 of pregnancy. The effect of TGF-beta3 on recruitment of lymphomyeloid cells to the vaginal wall and vaginal lumen of unmated mice in estrus was assessed using immunohistochemistry and flow cytometry. RESULTS: Two nanogram of intravaginal TGF-beta3 in 0.1% BSA-PBS or 20 ng in 1% BSA-PBS reduced abortion rates. Protection was comparable to that achieved by immunization with BALB/c spleen cells. Fraction V BSA, a binder of TGF-betas, had some activity, and could reduce availability of added TGF-beta3. CD11c dendritic cells, CD3+ T cells, and CD25+ cells were recruited to the vaginal wall by 48 hr after TGF-beta3 treatment, and cellularity of vaginal exudates increased. Foxp3+ cells were present in increased numbers, and appeared to be CD8+ and CD4+ 8+. Semen, but not TGF-beta3, stimulated a physiological polymorphonuclear leukocyte exudate. CONCLUSION: Intravaginal bioactive TGF-beta3 can enhance success of pregnancy in vivo in an established model of abortion. The result could be explained by the independent ability of TGF-beta to promote a regulatory T-cell response.     

Induction of specific cell‐mediated immune responses and protection in BALB/c mice by vaccination with outer membrane vesicles from a Brucella melitensis human isolate

Brucellosis is a worldwide bacterial zoonosis caused by Brucella spp. No approved vaccine is available for human use against the disease. In this study, outer membrane vesicles (OMVs) from a Brucella melitensis biovar 1 human isolate obtained in Iran were used to immunize BALB/c mice (n = 12) by 2 intramuscular injections with a 2‐week interval. Another group of 12 mice was used as non‐vaccinated controls. Two weeks after the last vaccination, six mice of each group were sacrificed, and proliferation and interferon gamma (IFNγ) production responses of their splenocytes were evaluated following in vitro stimulation with killed Brucella cells. The other mice were challenged with the virulent B. melitensis isolate. Two weeks later, mice were killed and spleens were cultured to determine the number of the challenge strain. The results showed proliferative response and IFNγ production of splenocytes from vaccinated mice (stimulation index: 2.18 ± 0.57, and 1519.35 ± 10.70 pg/mL, respectively) were significantly higher than those of control mice (stimulation index: 1.02 ± 0.02, and 210.01 ± 17.58 pg/mL, respectively). Numbers of the challenge strain in spleens of vaccinated mice were also significantly less than those in the controls with 1.6 units of protection. Our study revealed vaccination with OMVs of the B. melitensis isolate could induce specific immune responses and protection against infection in the mouse model suggesting their potential application for active immunization against brucellosis.     

Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19.

The study compared the immune and protective responses induced in BALB/c mice vaccinated with six salt-extractable periplasmic protein fractions (Brucella cell surface proteins [BCSP]) of Brucella abortus 19 and later challenge exposed with B. abortus 2308. BCSP70 was precipitated with ammonium sulfate at 70% saturation, and BCSP100 was precipitated with ammonium sulfate at 100% saturation by use of supernatant fluid of BCSP70 that had been precipitated with 70% ammonium sulfate. Four subfractions were separated from BCSP100 by anion-exchange high-performance liquid chromatography (HPLC). Monophosphoryl lipid A (MPL) from Salmonella typhimurium Re mutant strain was used as a potential immune response modifier in some vaccines. Reduced or increased numbers of CFU and increased spleen size in the principal groups of mice relative to that of the nonvaccinated control group were considered protectiveness or virulence (survival) criteria. Results indicated that vaccines prepared from BCSP70 and BCSP100 were moderately protective and immunogenic. The subfractions designated BCSP100-A through BCSP100-D purified by anion-exchange HPLC were not protective when MPL was not used as an immune response modifier. However, two subfractions were associated with significant (P < 0.05) increases in CFU per spleen and splenomegaly in vaccinated mice compared with those in nonvaccinated challenge-exposed mice. MPL enhanced protection or was neutral when used with BCSP70, BCSP100, BCSP100-C, and BCSP100-D. Serologic results of an enzyme-linked immunosorbent assay indicated that MPL modulated the immunoglobulin G responses induced by BCSP70, BCSP100, and subfraction BCSP100-B vaccines only. The overall results suggest that certain proteinaceous periplasmic fractions might serve as virulence or survival factors in B. abortus infections.     

Association of cellular and molecular alterations in Leydig cells with apoptotic changes in germ cells from testes of Graomys griseoflavus × Graomys centralis male hybrids

Spermatogenesis is disrupted in Graomys griseoflavus × Graomys centralis male hybrids. This study was aimed to determine whether morphological alterations in Leydig cells from hybrids accompany the arrest of spermatogenesis and cell death of germ cells and whether apoptotic pathways are also involved in the response of these interstitial cells. We used three groups of 1-, 2- and 3-month-old male animals: (1) G. centralis, (2) G. griseoflavus and (3) hybrids obtained by crossing G. griseoflavus females with G. centralis males. Testicular ultrastructure was analyzed by transmission electron microscopy. TUNEL was studied using an in situ cell death detection kit and the expression of apoptotic molecules by immunohistochemistry. The data confirmed arrest of spermatogenesis and intense apoptotic processes of germ cells in hybrids. These animals also showed ultrastructural alterations in the Leydig cells. Fas, FasL and calbindin D_28k overexpression without an increase in DNA fragmentation was detected in the Leydig cells from hybrids. In conclusion, the sterility of Graomys hybrids occurs with ultrastructural changes in germ and Leydig cells. The enhancement of Fas and FasL is not associated with cell death in the Leydig cells. Probably the apoptosis in these interstitial cells is inhibited by the high expression of the antiapoptotic molecule calbindin D_28k.     

In vitro production of interleukin-4 and interferon-gamma by lymph node cells from BALB/c mice infested with nymphal Ixodes ricinus ticks.

In this study we compared the ability of lymphocytes taken from axillary and brachial lymph nodes of BALB/c mice that had been infested once three times with 15 nymphal Ixodes ricinus ticks, to produce interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) after in vitro stimulation with concanavalin A (Con A). They released high levels of IL-4 and low levels of IFN-gamma. An increase of IFN-gamma between the first and the third tick infestation was observed. Salivary gland extracts from female I. ricinus ticks induced specific in vitro proliferation of lymphocytes from infested mice. IL-4 production was correlated with the salivary gland extracts' ability to stimulate tick-specific lymphocyte proliferation. Its levels remained high from the first to the third infestation. IFN-gamma production was not necessarily associated with tick salivary gland antigen stimulation. In BALB/c mice, anti-tick immune response induction is regional and the contribution of other similar secondary lymphoid organs is negligible. Only cells from the lymph nodes which drained the tick-fixation site proliferated in vitro in the presence of tick antigens, and when stimulated with Con A produced IL-4 and IFN-gamma.     

Specific and nonspecific antitumor immunity. III. Specific T lymphocyte-mediated cytolysis of P815 mastocytoma and SL2 lymphoma by draining lymph node cells from syngeneic tumor-bearing DBA/2J mice.

Tumor-specific cytolytic activity, as measured by the 51Cr release assay, has been demonstrated in the draining lymph node cells from DBA/2 mice bearing the syngeneic P815 mastocytoma or SL2 lymphoma. This lytic activity is mediated by cytolytic T lymphocytes (CTL), since cytotoxicity is eliminated by treatment of the effector cells with anti-Thy 1.2 (theta) serum plus complement but is enhanced or unaffected by anti-Thy 1.2 serum alone, antimouse immunoglobulin plus complement, normal or aggregated mouse immunoglobulin, or removal of adherent cells. The time course of the CTL response has been analyzed and is similar for both P815 and SL2, with a peak around Days 10 to 12 after tumor grafting. Detectable CTL activity then wanes despite continued antigenic stimulation from the growing tumor. The ability of the immunotherapeutic agent Corynebacterium parvum to augment such specific CTL responses is documented as one antitumor pathway by which this agent may act.     

In Vivo Immunosuppressive Effects of Recombinant Ovine Interferon-tau (Trophoblastin): r.oTP (r.oIFN-τ) Inhibits Local GVH Reaction in Mice (PLN Assay), Prevents Fetal Resorptions,and Favors Embryo Survival and Implantation in the CBA/J × DBA/2 Mice Combination

PROBLEM : Ovine trophoblastin protein, be it natural or recombinant (oTP, r.oTP), a member of the tau interferon family (r.oIFN-τ), has been shown to possess immunosuppressive properties in vitro. It acts as a cytostatic agent across species. Indeed, it was immunosuppressive when tested on human and murine lymphocytes in a variety of in vitro immune assays, as it is also on syngenic (ovine) lymphocytes. METHODS : In the present paper, we first verified that this property to act across species also occurred in vivo assays; r.oTP was able to down regulate a local GVH reaction assay (PLN assay) in mice. We then took advantage of these properties of r.oTP to investigate its in vivo effects during murine pregnancy as there is no ovine equivalent of the murine CBA/ J × DBA/2 resorption prone mating combination. RESULTS : When given in the postimplantation period, r.oTP drastically boosted resorptions in the CBA/J × DBA/2 matings, as did murine recombinant gamma interferon. However, the same r.oTP treatment in the peri-implantation period resulted in a reduction in resorptions in this spontaneous abortion system. CONCLUSION : The data suggested that r.oTP might have acted more by favouring implantation and embryo survival than by preventing the resorption process itself. The mechanisms possibly underlying these effects, as well as the putative uses of r.oTP evolving from these data, are discussed.     

1140918040Prevention of abortion in the CBAxDBA/2 model by intravaginal TGF-β is associated with local recruitment of predominantly CD8+ Foxp3+ T cells to the genital tract

Problem: Spontaneous abortions in DBA/2‐mated CBA/J mice can be prevented by an immune response to BALB/c, and CD4⁺25⁺ Treg cells as well as CD8⁺ T cells have been proposed to confer protection. Recently a 2 ng dose of intravaginal TGF‐β3 at the time of exposure to DBA/2 semen was shown to be effective. TGF‐β is known to facilitate development of Treg cells. Is there evidence for local Treg induction? Methods: The phenotype of cellular recruitment to the vaginal wall and uterus was established by immunostaining tissue sections from CBA/J females following intravaginal TGF‐β treatment. The phenotype of cells in vaginal washings 48 hr after TGF‐β was determined by flow cytometry. Results: Increased numbers of CD3⁺, CD25⁺, and CD11c⁺ cells were found in vaginal mucosa with increasing doses of TGF‐β. A 2 ng TGF‐β3 treatment at the time of estrus recruited Foxp3⁺ cells to the vaginal lumen, and the majority of these were CD8⁺; CD4⁺ cells were also present, but expressed only low levels of CD25 and CTLA4. A 20 ng dose recruited predominantly CD4⁺8⁺ Foxp3⁺ cells. Conclusion: Induction of Tregs to semen‐associated DBA/2 antigens may prevent pregnancy loss in the CBAxDBA/2 model without the need for BALB/c as an immunogen. The Treg phenotype in the genital tract is compatible with additional members of the Treg family that recognize Class I MHC and associated paternal peptides and prevent abortions.