1 Center for Autoimmune Diseases, Department of Medicine B, Sheba Medical Center, Tel Hashomer & Sackler Faculty of Medicine, Tel-Aviv, 2 Department of Anatomy, Hadassah Faculty of Medicine, Hebrew University, Jerusalem, 3 Department of Pathology, Rambam Medical Center, Technion University, Haifa, Israel, 4 Departments of Pathology and of Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, 5 Department of Medicine, and 6 Department of Pediatrics, University of Chicago, Chicago, Illinois, USA
7 To whom correspondence should be addressed at: Department of Medicine B, Sheba Medical Center, Tel Hashomer, 52621, Israel. e-mail: shoenfel{at}post.tau.ac.il
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Abstract |
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Key words: animal model/autoimmunity/fetal loss/thyroglobulin/thyroid
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Introduction |
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Materials and methods |
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Screening of mice sera for autoantibodies
Anti-Tg
ELISA plates were coated with human Tg (10 µg/ml in carbonate buffer) and blocked with 5% BSA. Following extensive washing, mouse sera [diluted 1:200 in tris-buffered saline (TBS)] were added to the wells. Bound antibodies were detected using anti-mouse IgG rabbit serum conjugated to alkaline phosphatase (Jackson Immunoresearch 115055146) and the intensity of the colour in the plates was read in a Titertrek ELISA reader using a 405 nm filter and a 620 nm reference filter.
Anti-dsDNA, anti-ssDNA and anti-cardiolipin
ELISA plates were coated with poly-L-lysin, calf thymus DNA (250 ng/ml in TBS) and cardiolipin (50 µg/ml in ethanol), for the detection of double stranded DNA (dsDNA), single stranded DNA (ssDNA) and cardiolipin (Cl) respectively, as previously described (Blank et al., 2002). Mouse sera (diluted 1:200 in TBS) were added to the wells and bound antibodies were detected as described above.
Anti-thyroxine (T4)
T4 antibodies were tested prior to pregnancy in 10 mice injected with CFA and 12 mice immunized with Tg, and during pregnancy in 18 mice injected with CFA and 19 immunized with Tg (all of which were randomly chosen). The percentage of T4 bound to IgG was measured. Serum (20 µl) was incubated with 15 000 cpm of 125I-thyroxine in 30 µl of barbital buffer for 6 h at 20°C. Bound 125I-thyroxine was precipitated with 450 µl of 20% PEG 6000 at 4°C for 30 min. The amount of labelled thyroxine in the pellet was quantified and is expressed as the percentage of the total.
Thyroid function tests
T4 levels were determined 3 weeks after injection/immunization and also immediately prior to killing. Total T4 levels were measured in each mouse from blood spotted on filter paper using the neonatal kit (Diagnostic Products Corporation, Los Angeles, USA) according to the manufacturers instructions. Free T4 was measured by equilibrium dialysis using a kit (Quest Diagnostics, Nichols Institute, San Juan Capistrano, USA). Pools of sera with antibodies to T4 >15% (range 15.855.0) and <2% (range 0.61.8) were prepared. Serum TSH was measured in 50 µl of serum using a sensitive, heterologous, disequilibrium, double antibody precipitation RIA as described elsewhere (Pohlenz et al., 1999). The sensitivity of this assay was 510 mIU/l. The intra- and inter-assay coefficients of variations were 16 and 27% at 20 mIU/l, 6.3 and 8.2% at 200 mIU/l, 5.4 and 9.8% at 850 mIU/l and 10 and 24% at 2000 mIU/l respectively. Thyroid-stimulating hormone (TSH) levels were tested prior to pregnancy in 12 mice injected with CFA and 14 immunized with Tg, and also in 18 randomly selected pregnant mice from both groups.
Histology of the thyroid gland
Thyroid glands of 10 Tg-immunized and 10 CFA-injected mice were excised after killing. The thyroids were fixed in 4% paraffin blocks, cut into 5 µm thick slices and stained with haematoxylin and eosin. Three slices were taken from each gland, the slices contained three or more sections separated by >50 µm apart, and these were evaluated by an observer unfamiliar with the study protocol and the different mouse groups.
Competition of binding to anti-Tg antibodies
Sera of three Tg immunized mice were pooled, and the dilution of the sera giving 50% of maximal binding to Tg-coated plates (prepared as described above) was employed for the competition assay. Different concentrations of Tg (100, 50, 25, 12, 6 and 3 µg/ml) were preincubated with the diluted sera for 1 h at room temperature, or overnight at 4°C. The mixtures of Tg and sera were further incubated for 2 h on the Tg-coated plates. The assay was continued as described for Tg ELISA.
Evaluation of pregnancy outcome
The uterine horns were examined to determine the number of live fetuses and number of resorbed pregnancies in order to calculate the percentage index of fetal resorption. Fetal resorption was identified as growth arrest and regression into an oval-shaped mass smaller than expected for the 14th day of pregnancy (with a size similar to 78 days pregnancy). Placental and fetal weights of randomly chosen mice (15 Tg and 15 CFA immunized mice) were determined. The person who selected the mice from both groups was blinded regarding the treatment groups.
Elution of antibodies from the placentae and the embryos
Six placentae and embryos were separated, hand-homogenized and washed five times with 50 ml phosphate buffered saline (PBS). The last wash was retained in order to confirm that no IgG was found in them. Immunoglobulins were eluted from the washed homogenates with glycine HCl buffer (0.1 mol/l pH 2.7, 15 min, room temperature) that were later neutralized with TBS. Following centrifugation, the supernatants were separated and dialyzed against maltose. The supernatants were analysed for the presence of antibodies to Tg and Cl.
Statistical analysis
Data were analysed by Students t-test and the 2-test. P < 0.05 was considered statistically significant.
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Results |
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Discussion |
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Some reports support an association between Tg antibodies and fetal loss (Stagnaro-Green et al., 1990; Glinoer et al., 1991
; Pratt et al., 1993b
; Takashi et al., 1997
; Kutteh et al., 1999a
; Abramson and Stagnaro-Green, 2001
; Matalon-Tartakover et al., 2001
); however, a causative role has not been established. Fetal loss among patients with thyroid antibodies could be induced by several putative mechanisms. The most obvious one is thyroid dysfunction, as is commonly seen in Hashimotos thyroiditis. However, the increase in miscarriages cannot always be explained by thyroid dysfunction alone (Dendrinos et al., 2000
). This suggests that the higher rate of miscarriages observed in women with autoimmune thyroid disturbances reflect primarily an autoimmune phenomenon, rather than, or in addition to, a consequence of overt thyroid hormone abnormalities. Increased pregnancy loss is found among patients suffering from different autoimmune diseases, and organ specific antibodies to the thyroid have been found in parallel to non-organ specific autoantibodies (Magaro et al., 1992
). Therefore, the presence of antibodies to the thyroid could represent a secondary marker of a predisposition for an autoimmune disease rather than the actual cause of fetal loss (Coulman et al., 1999
; Sherer and Shoenfeld, 1999
). However, we failed to identify other non-organ specific autoantibodies among the mice that developed anti-Tg and increased fetal resorption rates. Other studies have also reported discordance between the presence of thyroid autoantibodies and non-organ specific autoantibodies (Pratt et al., 1993
; Bussen and Steck, 1997
; Mecacci et al., 2000
), thus excluding polyclonal activation as the cause for thyroid autoantibody production.
Immunization with Tg was associated with lower placental and embryonic weights. The finding of anti-Tg binding to the placenta and not to the embryo-body might suggest that these autoantibodies have a direct effect on the placenta. It is well known that autoantibodies can pass into the amniotic fluids (Cohen et al., 2000) and interact with the syncytiotrophoblast and cytotrophoblast (Kutteh et al., 1999b
). However, it is possible that anti-Tg bound passively to the placenta but exhibited no harmful effect, as many other antibodies and immune complexes do. Moreover, a direct pathogenic effect of anti-Tg antibodies is only a possible explanation for the reproductive failure, as other possibilities which interfere with reproduction can occur following immunization, such as a switch to Th1. Further studies will clarify the precise effect that anti-Tg may exert on placental function, and whether these antibodies have a pathogenic role on pregnancy. These should include, for example, passive transfer of anti-Tg antibodies, and transfer of sera from mice immunized with Tg into naive mice.
In conclusion, mice immunized with Tg developed high titres of Tg antibodies without thyroid dysfunction, had a higher incidence of fetal resorptions and reduced placental and embryo weights. Hence, anti-Tg might exert a direct pathogenic effect on pregnancy outcome in the absence of thyroid dysfunction, but more studies are required in order to confirm this assumption.
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Acknowledgements |
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References |
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Submitted on July 26, 2002; resubmitted on November 28, 2002; accepted on January 20, 2003.