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Autoimmune Disease Unit (C.-R.C., H.A., P.N.P., S.M.M., B.R.), Cedars-Sinai Research Institute and University of California, Los Angeles, School of Medicine, Los Angeles, California 90048; and Department of Medical Gene Technology (Y.N.), Nagasaki University School of Biomedical Sciences, Nagasaki 852-8523, Japan
Address all correspondence and requests for reprints to: Dr. Basil Rapoport, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: rapoportb{at}cshs.org.
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Abstract |
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Introduction |
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An early observation during the rapid evolution of induced animal models of Graves’ disease was the importance of mouse strain. For example, TSHR plasmid DNA vaccination produced TSHR antibodies in most BALB/c mice (5) but hyperthyroidism only in outbred mice (2). In our experience with plasmid TSHR-DNA vaccination, TSHR antibody generation in BALB/c mice occurred very rarely, which was quite different from C57BL/6 mice in which TSHR antibodies were readily detected (6). However, the TSHR antibodies in the C57BL/6 mice were not of a type able to activate the TSHR and cause hyperthyroidism. In the TSHR-adenovirus model of Graves’ disease, BALB/c, C57BL/6, and SJL/J mice developed similar titers of TSHR antibodies with TSH binding inhibition (TBI) activity (3). Despite this commonality, the incidence of hyperthyroidism varied widely among the strains, namely, approximately 50, 25, and 0% for BALB/c, C57BL/6, and SJL/J strains, respectively.
We were puzzled as to why, in view of the marked efficacy of plasmid DNA vaccination in generating TSHR antibodies in C57BL/6 relative to BALB/c mice, TSHR adenovirus immunization less effectively induced hyperthyroidism in C57BL/6 than in BALB/c mice. We considered that two factors could contribute to the relatively low incidence of hyperthyroidism in the TSHR adenovirus-immunized C57BL/6 mice. First was the form of TSHR antigen expressed by the virus. Nagayama et al. (3) used an adenovirus vector expressing the TSH holoreceptor. Second was the number of viral particles injected. The foregoing study involved injection of a large number (1011) of viral particles (3). More recent experience with this model revealed that immunization with adenovirus expressing the free TSHR A subunit rather than the holoreceptor (4), as well as with fewer numbers of adenovirus particles (7), alters the qualitative nature of induced TSHR antibodies. Under these conditions, the balance shifts in favor of TSAbs and away from nonfunctional, or blocking, TSHR antibodies. In the present study, we tested the hypothesis that injection of a low dose of adenovirus expressing the TSHR A subunit would enhance the incidence of hyperthyroidism in C57BL/6 mice.
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Materials and Methods |
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Immunization of mice with adenovirus expressing the TSHR-A subunit
Construction and purification of adenovirus containing TSHR amino acid residues 1–289 (A-subunit-Ad) and control adenovirus (Control-Ad) expressing ß-galactosidase have been described previously (3, 4). In brief, adenoviruses A-subunit-Ad and Control-Ad were propagated in HEK293 cells (American Type Culture Collection, Manassas, VA) and purified by CsCl density gradient centrifugation, and viral particle concentration was determined by measuring the absorbance at 260 nm (8). All viruses used in this study were from the same preparation, stored in aliquots at –80 C.
Mice (10 C57BL/6 and 10 BALB/c in each of two sequential series) were injected im with 108 A-subunit-Ad particles per injection (in 50 µl PBS) according to our optimized protocol described previously (7). Concurrently, two series of mice (total of 10 mice of each strain) received the same number of Control-Ad particles. Mice were injected three times at three weekly intervals. Blood was drawn 1 wk after the second injection, and all animals were euthanized 4 wk after the third injection to obtain blood, spleens, and thyroid glands. Thyroid histology was examined on formalin-fixed tissue sections stained with hematoxylin and eosin.
Serum T4 levels
Total T4 in mouse sera was measured in undiluted serum (25 µl) by RIA using a kit (Diagnostic Products Corp., Los Angeles, CA).
TBI, TSAb, and TSH blocking antibody (TBAb) assays
TBI was measured using a commercial kit according to the protocol of the manufacturer (Kronus, Boise, ID). In brief, duplicate 5-µl aliquots of immunized mouse serum (plus 30 µl of normal human serum as carrier protein) were incubated with detergent-solubilized TSHR; 125I-labeled TSH was added, and the TSHR-antibody complexes were precipitated with polyethylene glycol. TBI values were calculated from the following formula:
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TSAb and TBAb were measured and calculated as previously described (4). In brief, monolayers of Chinese hamster ovary (CHO) cells expressing the wild-type TSHR in 96-well plates were incubated with 3% test serum in 100 µl Hanks’ buffer without NaCl and supplemented with 20 mM HEPES (pH 7.4), 1 mM isobutylmethylxanthine, 220 mM sucrose, and 0.3% BSA. After 3 h at 37 C, total cAMP content (medium and cells) was measured by RIA. TSAb was expressed as a percentage of basal cAMP generated in the presence of serum from control adenovirus-immunized mice. Incidentally, in all TSHR antibody assays, we have observed no difference between control adenovirus-immunized mice and unimmunized mice. TBAb was calculated as follows:
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TSHR antibody IgG subclass measured by ELISA
ELISA plates coated with purified TSHR A subunits (TSHR-289; 1 µg/ml in 10 mM Tris, pH 7.4; 50 mM NaCl) (9, 10) were blocked with BSA (Sigma, St. Louis MO) before incubation with test mouse sera (1:100). Subsequently, the plates were incubated with the following biotinylated antibodies: monoclonal rat antimouse IgG1, monoclonal rat antimouse IgG2a, and goat antimouse IgG2b (all from Caltag, Burlingame, CA). After incubation with streptavidin-conjugated horseradish peroxidase (PharMingen, San Diego, CA), color was developed using o-phenylene diamine. Subclass specificity of the biotinylated antibodies was previously confirmed (11) using purified myeloma proteins (IgG1, IgG2a, IgG2b, and IgG3; Cappel, Aurora, OH).
Statistical analyses
Fisher’s exact test was used to determine the significance of differences between the number of mice in a group positive or negative for a particular parameter. Significant differences between the magnitudes of responses of mice in different groups were determined by Kruskal-Wallis ANOVA on Ranks.
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Results |
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). One week after two injections of A-subunit adenovirus, 13 of 20 BALB/c mice (65%) were hyperthyroid relative to the animals of the same strain injected with control adenovirus. In contrast, only 1 of 20 (5%) C57BL/6 mice injected in parallel with A-subunit adenovirus developed hyperthyroidism. This large difference remained 4 wk after the third and final immunization, with 10 of 20 (50%) BALB/c and 1 of 20 (5%) C57BL/6 mice being hyperthyroid. T4 levels in Control-Ad-immunized mice differed in the two strains, with mean ± SD serum T4 values of 7.29 ± 0.84 and 5.25 ± 0.90 µg/dl in the BALB/c and C57BL/6 mice, respectively. In the hyperthyroid animals with goiter, unlike in euthyroid or control mice, histological analysis revealed hyperplastic follicles with cuboidal or columnar thyroid epithelial cells, as reported previously (4) (data not shown).
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). Moreover, quantitatively, TBI activities did not differ significantly between BALB/c and C57BL/6 mice after either the second or third injections.
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). Nevertheless, it is evident that, as assayed, the dramatic interspecies difference in hyperthyroidism cannot be fully explained by the functional TSHR antibody data. Possible reasons for this observation, based on previous reports in the literature, are discussed below.
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) resembled more closely that in BALB/c than that in C57BL/6 mice (Fig. 1
). Consistent with serum TBI levels in both BALB/c and C57BL/6, TBI activity after A-subunit-Ad immunization was detectable in the great majority of F1 hybrids (Fig. 4B). Quantitatively, therefore, TBI values were not significantly different from values in the parental strains. However, as for thyrotoxicosis, the functional TSHR antibody pattern more closely resembled that in the BALB/c mice prone to hyperthyroidism. That is, whereas TSAb was detectable in the majority (8 of 10) of F1 hybrids, TBAb activity was present in only half (5 of 10) of these animals (Fig. 4C).
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). After TSHR A-subunit-Ad immunization, TSHR antibodies in BALB/c mice were predominantly within subclasses IgG1 and IgG2a, with very little IgG2b (Fig. 5B). Similar immunization of C57BL/6 mice led to a reciprocal subclass distribution. TSHR antibodies were of higher titer within the IgG2b than in the IgG1 subclass. As expected, because C57BL/6 mice lack the gene for IgG2a (15), no TSHR antibodies of this subclass were detected. In the A-subunit-Ad-immunized F1 mice, the proportions of TSHR antibody IgG1 and IgG2b resembled that in C57BL/6 rather than that in BALB/c mice (IgG2b > IgG1) (Fig. 5B).
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Discussion |
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What factor(s) could account for this marked difference between BALB/c and C57BL/6 mice in their susceptibility to developing hyperthyroidism? First, we observed a modest shift in the balance between TSAb and TBAb in these two strains after immunization with A-subunit-Ad. In humans, TBAb capable of causing hypothyroidism (16) can counterbalance, wholly or in part, the action of TSAb (17). In the hyperthyroid-resistant C57BL/6 mice, TBAb activity was more dominant than in the hyperthyroid-prone BALB/c mice. The same phenomenon was suggested as the likely explanation for the greater incidence of hyperthyroidism in the modified TSHR adenovirus model of Graves’ disease (discussed above) (7). However, in the present study, the functional TSHR antibody assays revealed a trend rather than an unequivocal distinction for the difference in disease susceptibility. Intuitively, hyperthyroidism can only occur if TSAb predominates over TBAb. How then can we explain the apparent lack of correlation between TSAb, TBAb (in vitro), and hyperthyroidism (in vivo) in the two strains? One possible explanation is the assay conditions. Of necessity, for reasons described above, mouse serum is assayed after substantial dilution. It is well established from human studies that serum dilution can alter the net balance between TSAb and TBAb activity measured in vitro (17). Furthermore, in a case of neonatal hypothyroidism caused by transplacental transmission of TBAb, delayed hyperthyroidism emerged when autoantibody levels declined over the weeks after separation from the maternal circulation (18). That is, serum dilution, as in our mice, tends to reduce the suppressive action of TBAb relative to TSAb activity, possibly because of different affinities of these two antibodies. Therefore, in undiluted serum in vivo (as opposed to the assay conditions), the TBAb activity may be relatively more potent in the C57BL/6 strain, with TSAb activity only emerging in vitro upon dilution. Nevertheless, this explanation alone may be insufficient to explain our findings. Another contributing factor may be that blocking activity measured in terms of TSH inhibition may not be identical with blocking activity against TSAb. A corollary of this explanation is that not all TBAb interact with the same epitopes. Indeed, a more diverse range of epitopes has been noted for TBAb than for TSAb in human disease (19).
The balance between TSAb and TBAb activities represents the final common pathway in the development of hyperthyroidism. The question remains as to what proximal factors influence this balance. One consideration is MHC. In genetic studies, MHC class II is clearly established as being associated with, but not necessarily linked to, Graves’ disease (reviewed in Ref. 20). In animals, induction of hyperthyroidism by injecting fibroblasts coexpressing the TSHR and MHC class II varies in mice with different background genes despite sharing the same MHC genes (H-2d) (21). On the other hand, mice transgenic for human HLA-DR3 (an allele associated with spontaneous Graves’ disease in humans) have an enhanced TSHR antibody response (22, 23), as well as a higher prevalence of hyperthyroidism (23), following TSHR plasmid DNA vaccination. Consequently, it was possible that H-2d MHC could account for the higher frequency of induced hyperthyroidism (65–70%) in BALB/c vs. the much lower (5%) incidence of hyperthyroidism in C57BL/6 mice with MHC H-2b. Indeed, consistent with these data, myelin oligodendrocyte glycoprotein DNA vaccination of H-2b-strain mice (C57BL/6, C57BL/10, and BALB/b) fails to induce antibodies to pathogenic B-cell epitopes (24).
Nevertheless, despite this circumstantial evidence, MHC is unlikely to be a major factor contributing to the different incidences of hyperthyroidism in BALB/c and C57L/6 mice observed in our study. As already described (introductory section), of five mouse strains immunized with TSHR adenovirus, hyperthyroidism was induced in 50% of BALB/c, 25% of C57BL/6, and 0% of the other three strains (3). The development of Graves’ disease in BALB/c mice injected with TSHR-expressing B cells provides additional support for the susceptibility of this strain to induced hyperthyroidism (25). Most important, in the TSHR adenovirus model of Graves’ disease, hyperthyroidism developed with the same high frequency (50%) in mice with different MHC genes but the same background genes [namely, BALB/c (H-2d) and BALB/k (H-2k)] but in a low frequency (5%) in DBA/2J mice, which have the same MHC as BALB/c mice (H2-d) but have different background genes (3). Overall, although we cannot definitively exclude MHC, in our view it is unlikely to play a major role in susceptibility (or resistance) to induced hyperthyroidism in mice.
A second possible factor contributing to the different incidence of induced hyperthyroidism between BALB/c and C57BL/6 mice is the propensity of the former, but not the latter, to develop a T helper (Th) 2-type immune response. Because human Graves’ disease is directly caused by the humoral arm of the immune response and is reported to be associated with seasonal allergy (26), elevated serum IgE levels (27), and Th2 cytokine production (IL-4, IL-5, and IL-13) (reviewed in Ref. 28), it is generally accepted to be a Th2-dominated disease (for example, see Ref. 29). Studies in some induced animal models of Graves’ disease further support the thesis of Th2 involvement. Pertussin toxin (Th2 adjuvant) and complete Freund’s adjuvant (Th1), respectively, enhanced and delayed the onset of hyperthyroidism in the Shimojo model (30). Likewise, Graves’ disease induced by injecting TSHR-expressing B cells together with cholera toxin is reduced in IL-4- but not IFN-
-null mice (31). At face value, our present data are consistent with the Th2 paradigm in Graves’ disease because BALB/c mice are typically characterized by Th2-dominant immune responses (32). Indeed, the Graves’ disease-susceptible BALB/c mice develop TSHR antibodies (as measured by ELISA) predominantly of the IgG1 subclass (Th2-associated), whereas these antibodies in the disease-resistant C57B/6 mice are predominantly IgG2b (Th1).
Nevertheless, with regard to the present concept that Graves’ disease, in mice and humans, is a Th2-associated disease, there is strong evidence to the contrary. In human Graves’ disease, TSAbs are reported to be restricted to IgG1 (33), a Th1-dependent subclass in humans. Consistent with this information, the only human monoclonal TSAb isolated to date is of the IgG1 subclass (34). No particular cytokine profile (Th1 and Th2) dominates in the thyroid-infiltrating lymphocytes in patients with Graves’ disease (reviewed in Ref. 35). In animal models of hyperthyroidism induced by injecting TSHR adenovirus (7), splenocytes challenged in vitro with TSHR antigen generate IFN- but not IL-4, which is again consistent with a Th1 response. After naked TSHR-DNA vaccination, splenocytes similarly generate IFN- upon antigen challenge (36), and immune deviation away from Th1 in IFN- knockout mice does not enhance TSHR antibody production (37). Murine monoclonal antibodies generated by TSHR plasmid DNA vaccination whose subclass has been determined are IgG2a, a Th1-determined subclass (5). Further evidence against a Th2 bias in the TSHR adenovirus mouse model of Graves’ hyperthyroidism is that IL-4 but not IL-12 decreases TSAb production and delays the onset of hyperthyroidism (38).
In the present report, we observed that TSHR-adenovirus immunization induces hyperthyroidism more efficiently in BALB/c than in C57BL/6 mice. As reported previously (38), the TSHR antibody subclass distribution in BALB/c mice was mixed, including IgG1, IgG2a, and (as measured for the first time in our study) IgG2b. In the hyperthyroid-resistant C57BL/6 mice, the TSHR antibody profile (IgG2b > IgG1) reflects a Th1 bias. These data would support a role for a Th2 immune response in murine Graves’ disease. Remarkably, TSHR adenovirus immunization of the F1 hybrids provides revealing information regarding this issue. There is a clear dissociation between susceptibility to hyperthyroidism (similar to BALB/c) and the IgG subclass profile. Thus, TSHR antibodies in these animals (IgG2b > IgG1) reflect the Th1 influence of the C57BL/6 parental strain. Therefore, our data indicate that factors other than the Th1/Th2 paradigm play an important role in the murine model of Graves’ disease.
In summary, in our view, the most important information derived from our data on the BALB/c, C57BL/6, and F1 strains is that BALB/c mice carry a dominant gene(s) for susceptibility to the induction of hyperthyroidism. Our observations suggest that this genetic influence is unrelated to the Th1/Th2 paradigm. Moreover, because previous data suggest that susceptibility to hyperthyroidism in the adenovirus Graves’ disease model is not greatly influenced by MHC genes, other presently unknown genes may play an important role. Although the genetic contribution in human Graves’ disease is highly complex and multigenic (reviewed in Ref. 20), future information on the susceptibility genes for this syndrome in mice will provide useful information on potential candidate genes in human disease.
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Footnotes |
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Abbreviations: A-subunit-Ad, Adenovirus containing TSHR amino acid residues 1–289; CHO, Chinese hamster ovary; Control-Ad, control adenovirus; IFN, interferon; MHC, major histocompatibility complex; TBAb, TSH blocking antibody; TBI, TSH binding inhibition; Th, T helper; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor.
Received June 4, 2004.
Accepted for publication July 16, 2004.
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production, rather than antibody, dominates the immune response in mice. Endocrinology 142:3530–3536This article has been cited by other articles:
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