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Endocrinology Vol. 142, No. 2 823-829
Copyright © 2001 by The Endocrine Society

ARTICLES

Pregnancy and Murine Thyroiditis: Thyroglobulin Immunization Leads to Fetal Loss in Specific Allogeneic Pregnancies1

M. Imaizumi, A. Pritsker, M. Kita, L. Ahmad, P. Unger and T. F. Davies

Division of Endocrinology and Metabolism, Departments of Medicine (M.I., A.P., M.K., L.A., T.F.D.) and Pathology (P.U.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Dr. T. F. Davies, Mount Sinai Medical Center, Box 1055, 1 Gustave L. Levy Place, New York, New York 10029. E-mail: terry.davies{at}mssm.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid autoantibodies are risk factors in human pregnancy. To investigate the influence of autoimmune thyroiditis on pregnancy, we have studied the impact of murine experimental autoimmune thyroiditis (EAT) on pregnancy outcome by using thyroglobulin (Tg) immunized CBA/J (H2k) female mice. When Tg immunized mice were mated with BALB/c (H2d) males, only 57% (47/83) of pregnant mice maintained their conceptions compared with >85% of other strain combinations (P < 0.05). We also found that MHC class II antigens were expressed on placental cells from Tg immunized pregnant mice but not in control normal pregnancies. Furthermore, the frequency and severity of thyroiditis, assessed by histological analyses, was also increased in Tg immunized mice mated with the BALB/c strain compared with syngeneic pregnancies (P < 0.05). In these pregnant mice mated with BALB/c, interleukin-4 secretion by mitogen-stimulated spleen cells was significantly suppressed and interferon-{gamma} secretion by mixed lymphocyte reactions with BALB/c cells was significantly increased. These data demonstrated enhanced Th1 cell proliferation and fetal loss in CBA/J X BALB/c pregnancies. We concluded, therefore, that pregnancy loss was increased in experimental autoimmune thyroiditis in a manner that was dependent on paternal antigens. These observations have broad implications for understanding the immunology of pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MANY AUTOIMMUNE diseases have been shown to be affected by pregnancy. In normal pregnancy, the maternal immune system undergoes major adjustments to maintain what may be an immunologically foreign fetus with 50% paternal genes (1). In normal pregnancy, along with a dampening of the immune system, the maternal immune responses have been shown to shift dramatically. For example, increased production of the immunosuppressive cytokines, interleukin (IL)-4 and IL-10 has been noted, moving the immune response away from Th1 cell-mediated immunity (2). Such immune changes have tended to suppress autoimmune tissue destruction. Several Th1 type human autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, and the autoimmune thyroid diseases, may be suppressed during human pregnancy (3, 4). However, the titers of thyroid autoantibodies also greatly decrease during pregnancy and a Th2-type of disorder, hyperthyroid Graves’ disease, is also suppressed (5). Hence, in practical terms it would appear that both arms of the immune response are reduced and not just Th1.

Autoimmune diseases themselves may also influence the pregnancy. For this discussion, it is particularly relevant that we, and others, have reported the presence of thyroid autoantibodies as a predictor for an increased risk of human pregnancy loss, using these measurements as immunological markers and not as markers of thyroid dysfunction (6, 7). Similarly, in experimental autoimmune encephalomyelitis (EAE), the onset of disease was found to induce pregnancy loss in rabbits and rats (8). However, the mechanisms by which such autoimmune responses induced pregnancy loss remains unclear. One area of investigation has been the relevance of the disparity in major histocompatibility (MHC) class II antigens between mother and fetus. In humans, it has been reported by some investigators that the more different the fetal HLA class II genes, the greater the need for immune restraint and the greater the amelioration of rheumatoid arthritis during pregnancy (4). How such a difference in HLA class II antigens between mother and fetus was able to influence pregnancy has not been determined. On the other hand, in murine pregnancy, the influence of fetal alloantigens (including MHC class II) on autoimmune disease has not been extensively studied, although a specific combination, CBA/J (H-2k) female x DBA2 (H-2d) male, has been reported as a murine model of spontaneous abortion (9).

Because all types of autoimmune thyroid disease are markedly suppressed by the development of pregnancy, we have tried to generate a suitable animal model in which to further explore the interaction of pregnancy and autoimmune thyroiditis. Murine experimental autoimmune thyroiditis (EAT) in susceptible strains (such as CBA/J) has been extensively investigated as a model of human autoimmune thyroiditis (Hashimoto’s disease) (10). This model, initiated by immunization with thyroglobulin (Tg), has a number of features of considerable advantage to the investigation of the immune response to pregnancy such as a reliable, predictable and easily accessible end organ abnormality. We have used several strains of males for mating with Tg immunized CBA/J females and investigated the influence of fetal alloantigens (including the different MHC class II) on EAT and vice versa. This has allowed us to determine the role of paternal antigen on the development of Tg induced EAT as well as the role of EAT on pregnancy outcome.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of murine thyroglobulin (Tg)
Murine Tg (mTg) was prepared from frozen mouse thyroids (Pel-Freez Biologicals, Rogers, AR) as previously described (11). Thyroids were homogenized in PBS and centrifuged at 1,000 x g at 4 C for 10 min and the supernatant ultracentrifuged at 425,000 x g for 1 h. The supernatant was then fractionated on a Sephadex G-200 column. The absorbance at 280 nm of the fractions was measured, and the first major peak was pooled and concentrated.

Immunization and mating protocol
CBA/J (H2k), BALB/c(H2d), C3H/He (H2k) and C57BL/6 (H2b) (8 weeks old unless stated otherwise) were purchased from The Jackson Laboratory (Bar Harbor, ME). All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. CBA/J female mice were immunized with mTg (50 µg) and followed by lipopolysaccharide (LPS) from Salmonella enteritidis (Sigma, St. Louis, MO) (20 µg) iv 3 h later. As controls, CBA/J female mice were immunized with LPS (20 µg) iv only. The same immunization schedule was repeated 1 week later. A week after the last immunization, immunized CBA/J female mice were mated with four different strain males (CBA/J or BALB/c or C3H/He or C57BL/6) for a week. Mice with vaginal plugs were counted as originally pregnant. Pregnancies were then allowed to proceed and mice were killed in the third week of their pregnancy (4 weeks after the last immunization), and fetuses were counted by intrauterine examination and their thyroid and spleen were examined. In some experiments, pregnant mice were proceed to deliver and pups were counted after delivery.

Thyroid histology
Thyroids were removed and fixed in 10% formalin in PBS and stained by hematoxylin and eosin. The severity of thyroiditis was graded as follows; 0.5: small focal areas of inflammatory cells; 1.0: focal collections of mononuclear cells with some follicular destruction; 2.0: diffuse infiltration of thyroid follicles involving approximately 40% or less of thyroid tissue examined; 3.0: destruction of more than 40% of thyroid tissue. The thyroid grading was performed by the pathologist in a manner that was blinded as to the experimental groups from which the tissue came. Percentages of thyroiditis were calculated as % of mice with thyroiditis and the mean thyroiditis grades were calculated by total thyroiditis grades divided by total number of mice. A thyroiditis index (TI) was calculated as the mean thyroiditis grades x % of thyroiditis showing both frequency and severity of thyroiditis.

Thyroid function tests
Total thyroxine (T4) was measured by blood spot assay (Diagnostic Products Corp., Los Angeles, CA) and murine TSH was measured in randomly selected animals by heterologous RIA using mouse TSH serum standard as described (12). For TSH measurement, serum from more than 5 mice in each group were mixed. TSH of control mice and total T4 were measured individually.

Detection of mTg antibodies
Murine sera were analyzed using 96-well plates (Immulon 2, Dynatech Corp. Laboratories, Chantilly, VA) which had been coated overnight with 10 µg/well of purified mTg in carbonate-bicarbonate buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6). After washing and blocking with 5% BSA/PBS, wells were incubated with individual mouse sera (1:1000 dilution). After further washing, bound antibodies were detected using alkaline phosphatase-labeled sheep antimouse IgG (Sigma; 1:500 dilution). After a final wash, p-nitrophenyl phosphate substrate was added, and absorbance at 405 nm was measured after development (13).

Cytokine assays
Spleens were removed and single cell suspensions were prepared for cytokine assays. Spleen cells were cultured in RPMI 1640 (BioWhittaker, Inc., Walkersville, MD) supplemented with 50 µM 2-mercaptoethanol, 10 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD) and 10% FBS (HyClone Laboratories, Inc., Logan, UT) at 37 C in 5% CO2. For each cytokine assay, 2 x 106 cells/well of immunized spleen cells and 2 x 106 cells/well of irradiated (1000rad) syngenic feeder cells were cultured together with 5 µg/ml of Concanavalin A (Con A) (Sigma). Culture supernatants were collected after 3 days and assayed for interferon-{gamma} (IFN-{gamma}) by ELISA and IL-4 by RIA. For the measurements of IFN-{gamma}, 96-well plates was coated overnight with 5 µg/well of purified anti-IFN-{gamma} (PharMingen, San Diego, CA). After washing and blocking with 1% BSA/PBS, wells were incubated with standard control IFN-{gamma} (Roche Molecular Biochemicals, Indianapolis, IN) and individual media. After further washing, wells were incubated with biotinylated anti-IFN-{gamma} (PharMingen). Bound antibodies were detected using streptavidin-peroxidase (POD, Roche Molecular Biochemicals) and 2,2'-Azino-di-[3-ethylbenzthiazoline sulfonate (6)] diammonium salt (ABTS) (Roche Molecular Biochemicals), and absorbance at 405 nm was measured after development. Sensitivity of this assay was more than 3 ng/ml. For measurements of IL-4, 96-well plates was coated overnight with 5 µg/well of purified anti-IL-4 (PharMingen). After washing and blocking with 1% BSA/PBS, wells were incubated with standard control IL-4 (Roche Molecular Biochemicals) and individual media. After further washing, wells were incubated with 125I labeled anti-IL4 and counted. The sensitivity of this assay was more than 12 pg/ml.

Mixed lymphocyte reactions (MLRs)
Spleens were removed from CBA/J or BALB/c mice and single cell suspensions prepared for MLRs. 2 x 105 cells/well of CBA/J responder spleen cells and 4 x 105/well irradiated (1000rad) CBA/J or BALB/c cells were cultured together in 96-well plates for 4 days and assayed for proliferative responses by adding 3H-thymidine (1 µCi/well) 18 h before harvest. Supernatants were also collected after 4 days’ incubation and assayed IFN-{gamma} and IL-4 as described above.

Immunohistochemistry of placental MHC class II expression
Frozen placental sections were fixed in acetone and kept at -70 C until used. Sections were stained with 1:200 diluted mouse antimouse IAk and IAd or mouse IgG2b{kappa} (PharMingen) as a negative control using the M.O.M. immunodetection Kit (Vector Laboratories, Inc., Burlingame, CA).

Data analysis
Data were analyzed by Student’s t test or {chi}-square test. Probability values less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tg immunization induced allogeneic pregnancy loss
The percentage of pregnant mice determined by detecting vaginal plugs in the control and Tg immunized groups varied from 60–87% (Table 1Go). However, in the group of Tg immunized CBA/J females that were mated with BALB/c males, only 57% of the pregnant mice maintained a fetus, whereas > 85% (range 87–100%) of other groups had one or more fetuses (P < 0.05) (Table 1Go). This was a consistent observation in three separate experiments involving 120 female mice. There were very few dead fetuses on intrauterine examination at the third week of their pregnancy, suggesting that any pregnancy loss had been early.


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  		Table 1. Summary of pregnancy outcomes in Tg-immunized and nonimmunized CBA/J females mated with males of four different strains

 
Enhanced EAT in allogeneic pregnancies
No lymphocytic infiltration was found in either pregnant or nonpregnant control mice (immunized with LPS alone). According to the combined data of Tg immunized mice from two separate experiments that had consistent results, there was no statistically significant difference in the percentage of mice with thyroiditis, mean thyroiditis grades or thyroiditis indices between nonpregnant mice and pregnant mice mated with CBA/J males (Fig. 1Go, A–C). However, the percentage of thyroiditis was increased in pregnant mice mated with BALB/c males (16/24, 67%) compared with the pregnant mice mated with CBA/J males or BALB/c mice (8/24, 33%) (P < 0.05) (Fig. 1AGo). The mean thyroiditis grades were also significantly increased in pregnant mice mated with BALB/c (0.50 ± 0.10) compared with pregnant mice mated with CBA/J (0.23 ± 0.09) (P < 0.05) (Fig. 1Go, B and D) and as further reflected by the large difference in the derived thyroiditis index (Fig. 1CGo). Hence, both pregnancy loss and worsening of thyroiditis was only seen in the BALB/c mated groups.



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  		Figure 1. Thyroid histology of Tg-immunized group mated with different strain males during pregnancy. The data were shown as combined data from two separate experiments that had consistent results. A, Percentage of mice with thyroiditis. *, P < 0.05. B, Data expressed as thyroiditis grades (expressed as mean ± SD). *, P < 0.05. C, Data expressed as a thyroiditis index calculated as % thyroiditis x the mean thyroiditis grade. D, Typical examples of thyroid histology in CBA/J-mated pregnant mice (a) and BALB/c-mated pregnant mice (b). Note that the intrathyroidal lymphocytic infiltration was more severe in BALB/c-mated pregnancies (b) than CBA/J-mated pregnancies (a) (H&E, magnification x100).

 
Thyroid function in pregnant mice with thyroiditis
To know the influence of Tg immunization and pregnancy on thyroid function, serum total T4 and TSH were measured. As shown in Table 2Go, serum T4 was significantly decreased in pregnant mice compared with controls. However there was no significant difference between nonimmunized and Tg immunized pregnant mice. Furthermore, there was no difference in serum T4 between controls and the Tg immunized pregnant mice without fetus who had had pregnancy loss. Serum TSH levels were within the control range (<5–97 µU/ml) in all groups. These data indicated that pregnancy loss was not related to their thyroid function.


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  		Table 2. The influence of Tg immunization and pregnancy on the thyroid function

 
Immunological profiles of pregnant mice with thyroiditis
Induction of antibodies to mTg. All mTg-immunized mice developed IgG antibodies to mTg, which were absent in nonimmunized mice (Fig. 2Go). Unlike the distinct differences in the histological appearance of the thyroid glands, we were unable to detect any difference in the titer of antibodies between pregnant mice mated with CBA/J and BALB/c.



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  		Figure 2. Anti-mTg levels during pregnancy in Tg immunized and nonimmunized groups mated with CBA/J or BALB/c. Results were expressed as mean OD ± SD (405 nm) of a 1:1000 dilution of serum. Inset shows appropriateness of 1:1000 dilution.

 
Cytokine secretion by spleen cells. To judge the changes in the maternal immune response, we examined mitogen (ConA) stimulated cytokine secretion by spleen cell preparations from Tg immunized pregnant mice in their third week. There were no significant differences in IFN-{gamma} secretion between pregnant mice that were mated with CBA/J males and those mated with BALB/c males (Fig. 3AGo). In contrast, IL-4 secretion was significantly decreased in pregnant mice that had been mated with BALB/c males (P < 0.05) (Fig. 3BGo), suggesting a fall in Th2 cells. This was evidence of a shift in the balance of the immune response to Th1 and was consistent with the increased degree of thyroiditis seen in the Tg immunized BALB/c group as well as the induced pregnancy loss.



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  		Figure 3. The secretion of IFN-{gamma} (A) and IL-4 (B) by Con A stimulated spleen cells of Tg-immunized pregnant mice mated with CBA/J or BALB/c males. Pregnant mice spleen cells (2 x 106 cells/well) and irradiated (1000rad) syngenic feeder cells (2 x 106 cells/well) were cultured together with 5 µg/ml of Con A. Culture supernatants were collected after 3 days and assayed for IFN-{gamma} by ELISA and IL-4 by RIA as described in Materials and Methods. *, P < 0.05.

 
Proliferative activity and cytokine secretion in mixed lymphocyte reactions (MLRs)
As expected, the proliferative activity of control and Tg immunized nonpregnant CBA/J spleen cells was increased when they were stimulated by allogeneic BALB/c spleen cells (P < 0.01) (Fig. 4AGo). There were no differences in proliferative responses between control and Tg-immunized mice. To evaluate the influence of pregnancy on this mixed lymphocyte reactivity to syngeneic and allogeneic antigens, spleen cells from pregnant mice were also stimulated with CBA/J and BALB/c spleen cells (Fig. 4BGo). The proliferative activity of all CBA/J responder spleen cells was again greater with BALB/c (P < 0.05) (Fig. 4BGo). In addition, it appeared that the allogeneic pregnancies had an enhanced lymphocyte reactivity to the alloantigens (P < 0.05).



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  		Figure 4. Mixed lymphocyte reactions (MLRs) of CBA/J pregnant female spleen cells stimulated by irradiated normal CBA/J or BALB/c spleen cells. CBA/J responder spleen cells (2 x 105 cells/well) and irradiated (1000rad) CBA/J (empty column) or BALB/c (filled column) cells (4 x 105/well) were cultured together for 4 days and assayed for proliferative responses by adding 3H-thymidine. Proliferation activities were expressed as mean stimulation index (SI) ± SD. SI = cpm/cpm (no stimulation). *, P < 0.05; **, P < 0.02; ***, P < 0.01. A, MLRs of nonpregnant control or Tg-immunized spleen cells. B, MLRs of pregnant nonimmunized spleen cells mated with CBA/J or BALB/c males.

 
In addition to T cell proliferation, we examined cytokine secretion in the MLR cultures. IFN-{gamma} was not detectable when CBA/J spleen cells were stimulated by syngeneic spleen cells (Fig. 5Go). However, we found that IFN-{gamma} secretion was increased when CBA/J spleen cells were stimulated by irradiated BALB/c spleen cells, suggesting that the T cell proliferation seen in these MLRs was Th1 in origin. There were again no differences between control nonimmunized mice and Tg-immunized. IL-4 was not detectable in any of these cell cultures (data not shown).



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  		Figure 5. Assay of IFN-{gamma} in MLR supernatants. Nonpregnant nonimmunized (control) or Tg-immunized spleen cells were cultured with irradiated CBA/J (empty column) or BALB/c cells (filled column) for 4 days (the same culture condition as Fig. 4AGo). Supernatants were collected from MLRs of and assayed for IFN-{gamma} by ELISA. ***, P < 0.01.

 
MHC class II expression on placental cells
Because the presence of MHC class II expression on placental cells has been reported to relate to fetal rejection (14, 15), we examined whether Tg immunized placentae expressed these antigens. Six of eight placenta from eight different Tg immunized pregnant mice were found to show mild to strong staining for MHC class II antigen, whereas 1 of 10 placenta from nonimmunized pregnant mice was mildly positive (Fig. 6Go). Negative controls (isotype matched IgG) gave no significant staining (data not shown). Unlike the different rates of pregnancy loss between syngeneic and allogeneic pregnancies, placenta from both groups showed this positive staining.



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  		Figure 6. MHC class II antigen expression on placental cells. These photomicrographs show staining for MHC class II expression on placenta from a nonimmunized mouse (A) and a Tg-immunized mouse (B). Note the positive brown staining on the labyrinthine trophoblast zone (La) close to the spongiotrophoblast zone (Sp) of the placenta from a Tg-immunized mouse (B) (magnification x100). Inset (C) shows an example of positive cells at higher magnification (x200).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In contrast to our hypothesis that an alloantigenic fetus would cause a greater suppression of the murine immune response to thyroglobulin, we found a distinctly different pattern involving activation of Th1 T cells. We found that the frequency and severity of thyroiditis was increased in pregnant CBA/J (H2k) mice mated with BALB/c (H2d) males. Furthermore, Tg immunization reduced fertility only when mice were mated with this specific strain (BALB/c), whereas H2k (CBA/J and C3H/He) and H2b (C57BL/6) males did not induce the exacerbation of thyroiditis or pregnancy loss. These data indicated that only specific strain combinations induced an exacerbation of thyroiditis as well as increased pregnancy loss. Furthermore, we showed that fetal alloantigens in the mouse exacerbated specific parts of the immune response rather than inducing a generalized immune modulation.

In humans, abnormal autoimmune responsiveness has been suggested as an important factor in reduced fertility and pregnancy loss. Antiphospholipid, antinuclear, and anti- single-stranded DNA antibodies have been associated with recurrent pregnancy loss (16). The presence of thyroid autoantibodies (antithyroid peroxidase and antithyroglobulin) during pregnancy has been reported to be a marker of increased risk of pregnancy loss (6, 7). However, the mechanism of such increased risk has remained unclear. In the current studies, Tg immunization was associated with a reduction in successful pregnancies when mice were mated with a specific strain (BALB/c) of males, whereas it had no influence on syngeneic and some other allogeneic pregnancies. Interestingly, the same CBA/J x BALB/c combination also induced an exacerbation of thyroiditis. This finding indicated that pregnancy loss and thyroiditis during pregnancy were strongly related to paternal antigens. In humans, the disparity in HLA class II antigens between mother and fetus has been reported to influence the maternal immune system in pregnancy. For example, the more different the fetal HLA class II genes, the greater the reported amelioration of rheumatoid arthritis during pregnancy (4). Although this has not been a general observation, these reports indicated that paternal HLA antigens may directly influence maternal immunity.

Although the obvious cause of pregnancy loss would logically be related to the feto-placental unit (2), there are also data that fetal cells in the maternal circulation may influence the immune response during pregnancy. Human fetal progenitor cells appeared in the maternal circulation during pregnancy and persisted for more than 20 yr after birth (17, 18). Fetal cells have also been found in the murine circulation during pregnancy (19) but appeared not to persist into the postpartum (19). It has been suggested that the presence of such fetal cells may influence the maternal immune response and may become a source of autoantigenicity. For example, persisting fetal cells in the maternal peripheral blood were more frequent in women with scleroderma than healthy controls after child birth, and fetal cells were also found within these patients skin lesions (20, 21). Furthermore, in systemic screlosis patients, fetal microchimerism among T lymphocytes was strongly associated with a specific HLA class II (DQA110501) also implicated in autoimmune thyroid disease. In our murine model, we found only one specific strain (H2d) that caused the exacerbation of maternal thyroiditis and pregnancy loss. This indicated that only certain specific MHC fetal cells might be immune modulators in the murine thyroiditis model.

In addition to the transplacental passage of fetal progenitor cells, there are other nonplacental mechanisms by which the fetus may influence the maternal immune response. Because the induction of Th1-type immunity during pregnancy has been reported as one of the important causes of fetal abortion (2), we examined the secretion of IFN-{gamma} and IL-4 by pregnant spleen cells as markers of Th1 and Th2, respectively. In our study, IL-4 secretion was suppressed in H2k (CBA/J) pregnant mice when they were mated with H2d (BALB/c) males, whereas IFN-{gamma} secretion remained unchanged, shifting the balance toward Th1. Furthermore, IFN-{gamma} secretion was increased when H2k spleen cells were stimulated by irradiated H2d spleen cells. These data indicated that a concomitant fall in Th2 cells and enhanced lymphocyte activity, especially Th1 cell reactivity to alloantigen, was associated with allogeneic BALB/c males.

In normal murine pregnancy, the mouse fetoplacental tissues have been shown to spontaneously secrete Th2 type cytokines (IL-4, IL-5, and IL-10), and the ratio of Th2 to Th1 cytokines secreted by fetoplacental cells was greater than that secreted by mitogen-stimulated spleen cells (22, 23). In contrast, Th1-type cytokines, tumor necrosis factor (TNF)-{alpha}, IFN-{gamma}, and IL-2, were significantly increased in mixed lymphocyte- placental cell reaction supernatants in the murine CBA/J x DBA/2 model of spontaneous abortion (24), suggesting a role for Th1 responses. Similarly, in humans, trophoblast antigens activated the lymphocytes of recurrent spontaneous abortion-susceptible women to produce IFN-{gamma} and TNF-ß (25, 26). Furthermore, IFN-{gamma} has been reported to inhibit trophoblast outgrowth in vitro and inhibit embryonic and fetal development as well as the proliferation of human trophoblast lines in vitro (27).These data were compatible with our observations that the immune system of allogeneic pregnant mice shifted their immune balance to Th1-type immunity and were associated with acceleration of thyroiditis and reduced fertility.

Several immune mechanisms may be associated with pregnancy loss and thyroiditis, which are influenced by this shift from Th2 to Th1 cells including MHC class II expression on placental cells. MHC class II expression is an important factor in the development of a humoral and cellular immune response, which is associated with antigen presentation to specific T cell receptors. The presence of MHC class II antigens on placental cells may have helped maternal T cells recognize fetal cells as foreign antigen and lead to the rejection of the fetus. MHC class II antigens are not usually expressed at the placenta, and when MHC class II expression on murine placenta was induced by a demethylating agent (5-azacytidine), or IFN-{gamma} treatment, it was found to lead to fetal abortion and fetal abnormalities (14, 15). Hence, Th1 dominant immunity may induce such fetal MHC class II antigen expression on placental cells and activate a maternal immune response resulting in pregnancy loss. In the present studies, we demonstrated that MHC class II expression on placental cells was induced by Tg immunization. This finding indicated that the maternal immune response to Tg also influenced the feto-placental unit and that immunity against a fetus might be more active in Tg immunized pregnant mice than control mice. However, other mechanisms may also be related to fetal loss because MHC expression was seen in not only allogeneic (x BALB/c) pregnancies which had more pregnancy loss but also in syngeneic pregnancies. Another potential mechanism may be complement related. Recently, murine complement regulator (Crry) deficient embryos were shown to have deposition of complement, which lead to an extensive invasion by polymorphonuclear inflammatory granulocytes and fetal loss (28).

In conclusion, we demonstrated that specific fetal alloantigens were associated with induction of pregnancy loss and enhanced murine thyroiditis during pregnancy. Reduced pregnancy success was concomitant with enhanced Th1 responses and the expression of MHC class II antigens on the placenta. Whether fetal progenitor cells are the primary mediators of the immune responses remains to be explored.


   Acknowledgments
 
We thank Helen Park and Thomas M. Moran, Ph.D. for their advice and their help with cytokine assays and Dr. Roy Weiss, M.D., Ph.D., University of Chicago, for mouse TSH assays. We also thank Alex Stagnaro-Green, M.D., for review of the manuscript.


   Footnotes
 
1 This work was supported in part by NIH Grants DK-52464, DK-35764, and DK-45011 (to T.F.D.), Cellular and Molecular Endocrinology Training Grant DK-07645 (to L.A.) and the David Owen Segal Endowment (to M.I. and M.K.). Back

Received August 17, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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