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TRH-TSH-THYROID |
Knockout Mice Does Not Enhance TSH Receptor Antibody Production after Naked DNA Vaccination
Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California School of Medicine, Los Angeles, California 90048
Address all correspondence and requests for reprints to: Sandra M. McLachlan, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: . mclachlans{at}cshs.org
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Abstract |
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(IFN) production. In the present study we investigated the effect on TSHR DNA vaccination of diverting the cytokine milieu away from Th1 using 1) IFN knockout BALB/c mice, and 2) wild-type mice covaccinated with DNA for the TSHR and for IFN/receptor-Fc protein that prevents IFN from binding to its receptor. Neither approach enhanced TSHR antibody levels, although splenocyte IFN production in response to TSHR antigen was absent (IFN knockouts) or reduced (IFN receptor-Fc). Moreover, production of IL-2, another Th1 cytokine, but not Th2 cytokines, indicated that neither strategy overcame the Th1 bias of im DNA vaccination. Importantly, splenocyte production of IFN and IL-2 provides a sensitive detection system for TSHR-specific T cells. Unexpectedly, higher TSHR antibody levels developed in rare mice. High titer animals had TSHR-specific responses of both Th2 and Th1 types, whereas low titer animals had Th1-restricted TSHR responses. The heterogeneity of responses induced by TSHR DNA vaccination in mice may provide insight into the titers and IgG subclasses of spontaneous autoantibodies in humans. |
Introduction |
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(IFN; but not Th2 cytokine IL-4, IL-5, or IL-10) in response to TSHR antigen (3). Moreover, our observations of Th1-type responses accompanying intramuscular (im) DNA vaccination are consistent with findings of others using cDNAs encoding a variety of antigens (4, 5, 6) as well as with the subclass (IgG2a) of three TSHR-specific monoclonal antibodies isolated by Costagliola and colleagues (1). One difference between the two studies of TSHR DNA vaccination concerns the mouse housing conditions, which were pathogen free in our case but not in the studies by Costagliola et al. (1). Skewing toward Th2 and away from Th1 responses was observed for experimentally induced autoimmune encephalitis in conventionally housed mice compared with mice maintained in a pathogen-free facility (7). Assuming that these findings are generally applicable, it is possible that modulation of the cytokine bias away from Th1 and toward Th2 would enhance TSHR antibody levels.
A second difference between the study by Costagliola et al. (1) and ours involves the TSHR itself. The TSHR DNA used in the earlier investigation codes for a tyrosine at residue 601 (TSHR-Y601), whereas that cloned by our laboratory (8) encodes histidine (TSHR-H601), a rare polymorphism subsequently found to be associated with low constitutive cAMP production (9). Because of its intracellular location, it is unlikely that histidine at TSHR position 601 would influence DNA vaccination. However, this possibility could not be excluded.
The present study was performed to determine whether the TSHR antibody response to DNA vaccination could be increased by either 1) modulating the cytokine environment away from Th1, or 2) vaccination with TSHR-Y601 rather than TSHR-H601. We used two different approaches to induce a bias away from Th1: 1) vaccinating mice in which the IFN gene had been disrupted (IFN knockout mice), and 2) covaccinating wild-type mice with a construct encoding an IFN receptor-Fc fusion protein, shown to ameliorate murine lupus (10), which acts as a decoy by diverting the binding of IFN to its membrane-bound receptor.
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Materials and Methods |
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receptor-fusion protein in the VR1255-C vector (10), which we refer to as an IFN decoy, was provided by Dr. A. Theofilopoulos (Scripps Research Institute, San Diego, CA). DNA vaccination was performed as previously described (1, 3). In brief, female mice (see below), aged 6–7 wk, were pretreated in the anterior tibialis muscle with cardiotoxin (100 µl/injection,10 µM Naja nigricollis; Calbiochem, La Jolla, CA). Five to 7 d later, the mice were injected in the same muscle with DNA (50 or 100 µg; see below). The vaccination protocol was repeated 3 and 6 wk later (total of three vaccinations). Four to 5 wk after completing the DNA vaccinations, mice were killed to obtain blood, thyroid glands, and spleens. The thyroid glands were fixed in 4% paraformaldehyde (Sigma, St. Louis, MO; pH 7.5). Serial sections were prepared from paraffin sections, stained with hematoxylin and eosin, and examined for lymphocytic infiltrates by Dr. Helen Braley-Mullen (Medicine, Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO).
Two types of experiment were performed. First, we compared TSHR DNA vaccination of wild-type and IFN knockout (IFN-/-) BALB/c mice (both from The Jackson Laboratory, Bar Harbor, ME). For these studies we used TSHR-H601 DNA. Second, BALB/c mice were vaccinated with DNA for 1) TSHR-Y601(100 µg), 2) TSHR-Y601 (50 µg), 3) IFN receptor-Fc fusion protein (IFN decoy; 50 µg) and TSHR-Y601 (50 µg), or 4) IFN decoy alone (50 µg). As controls for some studies we used previously described sera from wild-type BALB/c mice conventionally immunized with TSHR protein (50 µg TSHR-289; see below) and adjuvant (3). All animal studies were approved by the institutional animal care and use committee and were performed in accordance with the highest standards of humane care in a pathogen-free facility.
Purified TSHR antigen
TSHR-289 is a TSHR variant corresponding approximately to the extracellular A subunit (11). This protein, expressed in Chinese hamster ovary (CHO) cells, contains two conformationally different forms: active and inactive, with respect to their recognition by human TSHR autoantibodies (12). We showed previously that splenocytes from TSHR DNA-vaccinated mice responded equally to both forms of TSHR-289 protein (3). Consequently, we only used inactive TSHR-289 in the present study. As previously described (13), TSHR-289 was isolated from culture medium by affinity chromatography using mouse mAb 3BD10 and was analyzed by SDS-PAGE to determine its purity and concentration. Before use in ELISA, in lymphocyte cultures (below), or as an immunogen (above), TSHR-289 was dialyzed against 10 mM Tris, pH 7.4, and 50 mM NaCl.
ELISA for TSHR antibodies
ELISA wells coated with TSHR-289 (1 µg/ml in 10 mM Tris, pH 7.4, and 50 mM NaCl) were incubated with test sera (diluted 1:100 for DNA-vaccinated mice and 1:10,000 for purified TSHR- and adjuvant-immunized mice), as previously described (3). Antibody binding was detected with horseradish peroxidase-conjugated mouse anti-IgG (Sigma), the signal was developed with o-phenylenediamine and H2O2, and OD was read at 490 nm. The same approach was followed to determine TSHR antibody IgG subclasses using the following biotinylated antibodies: monoclonal rat antimouse IgG1, monoclonal rat antimouse IgG2a, goat antimouse IgG2b, and goat antimouse IgG3 (all from Caltag Laboratories, Inc., Burlingame, CA), followed by streptavidin-conjugated horseradish peroxidase (BD PharMingen, San Diego, CA). The subclass specificity of the biotinylated antibodies was confirmed using purified myeloma proteins (MOPC 21 for IgG1, UPC10 for IgG2a, MOPC 195 for IgG2b and FLOPC21 for IgG3; Cappel, Aurora, OH) (14).
Flow cytometry for serum binding to TSHR-expressing cells
Mouse sera (diluted 1:50) were examined for antibody binding to CHO cells expressing high numbers of TSH receptors (
2 x 106 TSHR/cell) (15) and detection with fluorescein isothiocyanate-conjugated, affinity-purified goat antimouse IgG (Caltag Laboratories, Inc.) as previously described (14). All assays included cells incubated with second antibody alone and with normal mouse serum. Flow cytometry was performed (10,000 events) using a FACScan with CellQuest software (Becton Dickinson and Co., San Jose, CA).
Inhibition of [125I]TSH binding to its receptor (TBI)
The ability of TSHR antibodies to inhibit TSH binding to the TSHR (TBI) (16) was measured using a kit (Kronus, Boise, ID). Duplicate serum aliquots (50 µl; undiluted) were incubated with detergent-solubilized porcine TSHR; [125I]TSH (bovine) was added, and the TSHR-antibody complexes were precipitated with polyethylene glycol. The TBI values are calculated from the formula:
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Thyroid-stimulating antibody (TSAb) assay
TSAb activity was assayed following the approach of Costagliola et al. (1, 2). CHO cells expressing approximately 150,000 TSHR/cell (17) in 96-well plates were incubated (4 h at 37 C) with test sera diluted 1:20 in hypotonic buffer (18) containing 10 mM HEPES (pH 7.4), 1 mM isobutylmethylxanthine, and 0.3% BSA. Without aspirating the medium, the plates were then frozen (1 h, -80 C) and thawed. Supernatants (diluted 1:400) were acetylated (20 µl triethylamine and 10 µl acetic anhydride/ml), and cAMP levels were measured by RIA using 2'-O-succinyl-[125I]iodotyrosine methyl ester (NEN Life Science Products, Boston, MA) and a rabbit anti-cAMP antibody (Fitzgerald, Concord, MA). Results are expressed as the percentage of basal cAMP released in the presence of normal mouse serum.
Cytokine response to incubation with TSHR antigen
Splenocytes (quadruplicate aliquots; 6 x 105 cells; 200 µl) were incubated in round-bottomed 96-well plates in the presence or absence of TSHR 289 (see above; 10 µg/ml). We used unfractionated splenocytes because isolating T cells inevitably leads to some cell loss, and our goal was to examine responses in individual animals rather than in pooled spleen samples. Moreover, we previously observed that, unlike proliferative responses ([3H]thymidine uptake) that had a substantial background, IFN production in response to TSHR-289 provided a specific indicator of prior sensitization to the TSHR in BALB/c mice) (3). The culture medium was RPMI 1640, 10% heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamicin, 50 µM ß-mercaptoethanol, and 100 U/ml penicillin. After 5–6 d (37 C, 5% CO2), supernatants from quadruplicate wells were pooled, centrifuged to remove cell debris, and stored (-80 C). Duplicate aliquots (100 µl) were assayed for IFN and IL-2 (and in some cases IL-4, IL-5, and IL-10) by ELISA using capture and biotinylated detection antibodies from BD PharMingen. Cytokine production is reported as picograms per milliliter, estimated using recombinant cytokine standards (PharMingen).
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Results |
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modulation as an approach to enhance TSHR antibody levels knockout (-/-) mice vs. wild-type mice (both on the BALB/c background). TSHR antibodies measured by ELISA were undetectable in vaccinated IFN-/- mice and were slightly higher for wild-type mice (Fig. 1A
; sera diluted 1:100). However, because of variability between individual mice (see later), there were no statistically significant differences between control or TSHR DNA-vaccinated mice. Moreover, even the slightly elevated levels were much lower than those in sera (diluted 1:10,000) from wild-type BALB/c mice conventionally immunized with TSHR-289 protein and adjuvant as previously reported (3).
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receptor-Fc fusion protein (50 µg of each). The soluble IFN receptor acts as a decoy by diverting IFN from binding to its membrane-bound receptor. As positive controls, mice were vaccinated with TSHR DNA alone, either 50 µg (low dose) or 100 µg (regular dose). Vaccination with IFN decoy DNA alone (50 µg) provided a negative control. Again, however, this strategy for modulating levels of the Th1 cytokine IFN had no enhancing effect on TSHR antibody levels. Thus, TSHR antibodies were undetectable in mice covaccinated with TSHR DNA and IFN decoy DNA; the highest levels (on the average) were observed in the group of mice vaccinated with the low dose of TSHR DNA (50 µg), but none of the differences between groups was statistically significant (Fig. 1B
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The TSHR DNA used in the first set of studies encodes histidine at amino acid reside 601(H-601), whereas the DNA used in the second set of studies encodes Y-601. Importantly, there were no major differences between TSHR antibody levels induced using TSHR-H601 DNA or TSHR-Y601 DNA as determined by ELISA (Fig. 1, A vs. B). Furthermore, flow cytometric analysis of serum antibody binding to TSHR-expressing CHO cells confirmed the lack of an enhancing effect on TSHR antibody levels by 1) deviation away from IFN, or 2) using DNA encoding TSHR-Y601 vs. TSHR-H601 (data not shown). The observation that the H/Y601 polymorphism does not influence the outcome of DNA vaccination is consistent with our previous studies of the Shimojo model for Graves’ disease (14, 19). In particular, we observed that AKR/N mice injected with fibroblasts coexpressing TSHR-H601 and major histocompatibility complex class II develop TSHR antibodies and hyperthyroidism to the same extent as reported by other groups using fibroblasts expressing TSHR-Y601 (20, 21, 22).
Response of splenocytes to TSHR antigen in vitro
The low or undetectable levels of TSHR antibodies in some mice could have arisen from inadequacies in our vaccination protocol. To exclude this possibility, we analyzed the cytokine response of spleen lymphocytes incubated with TSHR antigen (TSHR-289). Splenocytes from TSHR DNA vaccinated wild-type mice produced IFN when cultured with TSHR-289 (Fig. 2A), in agreement with our previous observations (3). As expected, IFN knockout mice were unable to produce this cytokine. However, the efficacy of the TSHR DNA vaccination in knockout mice was confirmed by the ability of their splenocytes to produce IL-2 (Fig. 2B). Indeed, IL-2 production in response to TSHR antigen was higher (on the average) for splenocytes from IFN knockout mice than in those from WT mice (Fig. 2B). IFN knockout mice have multiple defects of immune function, including uncontrolled splenocyte proliferation in response to mitogen (23), which may account for enhanced IL-2 production. However, the difference between the amounts of IL-2 produced by WT and knockout mice was not statistically significant. Neither IFN nor IL-2 was produced by splenocytes from TSHR-vaccinated mice cultured without TSHR-289 (Fig. 2, A and B), and Th2 cytokines (IL-4, IL-5, and IL-10) were undetectable.
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decoy. Production of IFN in response to TSHR-289 was reduced in splenocytes from mice covaccinated with the TSHR DNA plus IFN decoy DNA compared with the amounts produced by splenocytes from TSHR DNA only-vaccinated mice (Fig. 3A). The difference was statistically significant for mice vaccinated with 100 µg TSHR DNA (t = 2.684; P = 0.028), but not for mice vaccinated with 50 µg TSHR DNA. All three groups of TSHR DNA-vaccinated mice, but not mice vaccinated with IFN decoy DNA alone, produced IL-2 when cultured with TSHR-289. As in the studies reported above, Th2 cytokines were undetectable.
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). These high antibody values were responsible for the increased mean OD levels in TSHR-vaccinated WT mice (Fig. 1A; mouse 1) and mice vaccinated with 50 µg TSHR DNA (Fig. 1B; mouse 2). TSHR antibodies quantified by flow cytometry using CHO cells expressing the TSHR on their cell surface gave more variable results than ELISA. Thus, sera from a number of mice exceeded the mean background fluorescence values observed for vector vaccinated controls (Fig. 4B). Serum from mouse 2 (with the highest OD in ELISA) was among this group. Mouse 1 (which gave the second highest ELISA signal) had the highest serum binding to the TSHR expressed on intact cells.
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). Neither mouse 1 nor mouse 2 had detectable TSAb activity (Fig. 5B) despite the ability of their serum antibodies to recognize the native TSHR on the cell surface. Insufficient material was available to allow us to test sera from our high titer mice for their ability to block stimulation by TSH in a bioassay.
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(left panel), TSHR antibodies in mouse 1 (wild-type; TSHR-H601 DNA-vaccinated) were restricted to IgG1, a Th2 subclass in mice (reviewed in Ref. 24). Sera from all IFN knockout mice were negative (like serum from a normal mouse). However, wild-type mouse 3 vaccinated with TSHR-H601 DNA had low, but clearly detectable, levels of both IgG3 (Th1-type) as well as IgG1 (Th2-type). Also shown are data from two positive controls. First, mouse monoclonal TSHR antibody 3BD10 (12) was, as anticipated, restricted to a single subclass (IgG1). Second, serum antibodies from a mouse conventionally immunized with TSHR-289 and Freund’s adjuvant (complete, followed by incomplete, abbreviated to CFA/IFA) were IgG1, IgG2a, and IgG2b. Th2 and Th1 responses would be anticipated after immunization with CFA and IFA (25). Moreover, these data are in agreement with the subclass distribution we observed for antibodies in mice immunized with thyroid peroxidase and CFA/IFA (14).
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, right panel). As noted, a feature of some low antibody titer mice was the presence of detectable IgG3 TSHR antibodies in the absence of other subclasses (for example, mouse 4).
Overall, mice with high TSHR antibody titers have immune responses reflecting Th1 cytokines (IFN and IL-2 production induced by TSHR antigen; IgG2a antibodies) and Th2 cytokines (IgG1 antibodies). In contrast, in mice with low levels of TSHR antibodies, the immune response is usually restricted to Th1 (production of IFN and IL-2; IgG3 antibodies).
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Discussion |
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(IFN knockout mice), and 2) covaccination with DNA encoding an IFNR/Fc fusion protein that diverts IFN from binding to its receptor. Neither of these approaches enhanced TSHR antibody levels, although splenocyte production of IFN in response to TSHR antigen was absent (knockout mice) or reduced (covaccination with IFNR/Fc). However, production of IL-2 (another Th1 cytokine) in response to TSHR antigen was robust in the IFN knockout mice. This observation together with the absence of Th2 cytokines indicate that neither strategy could overcome the Th1 bias of im DNA vaccination toward Th2.
Our finding that IFN is not required for successful DNA vaccination (at least in terms of T cell responses) is consistent with observations for humoral and cellular responses in IFN-deficient mice vaccinated with DNA encoding a nucleoprotein gene from lymphocytic choriomeningitis virus (26). Moreover, the flexibility of the immune system in overcoming the lack of an apparently critical cytokine, presumably by employing an alternative molecule, should not be underestimated (reviewed in Ref. 27). For example, there is abundant evidence that TNF
and lymphotoxin- are critical for the development of experimentally induced autoimmune encephalitis. However, disease typical of that induced in normal mice occurs in the double TNF/lymphotoxin- knockout mice (27).
Unexpectedly, among 53 BALB/c and AKR/N mice in the current and a previous study (3), two animals had relatively high levels of TSHR antibodies measured by ELISA. Variation in antibody levels is more unexpected among inbred than outbred animals. Nevertheless, Costagliola et al. (1) observed antibody variability (assessed by ELISA) among BALB/c mice in response to TSHR DNA vaccination with sucrose. In addition, substantial variation of antibody responses was characteristic of CBA/Ca mice pretreated with cardiotoxin and vaccinated with the human immunodeficiency envelope glycoprotein gp120 (28).
Returning to the two mice with high TSHR antibodies by ELISA, sera from both animals recognized the native TSHR expressed on mammalian cells. Neither had detectable TSAb activities, although one had low, but clearly detectable, TBI activity. We have no explanation for the development of high TSHR antibody levels in these individual mice that were injected on the same day with the same amount and the same DNA preparation as other mice within their respective groups. Thyroid histology was normal in both mice, and there was no lymphocytic infiltrate. However, the TSHR-specific cytokine profiles of these rare mice were substantially different from those of low titer mice. In particular, high TSHR antibody levels were associated with both Th1 and Th2 responses, whereas low antibody levels were associated with Th1 responses.
Substantial evidence implicates immune responses to the TSHR in the pathogenesis of Graves’ ophthalmopathy (reviewed in Refs. 29 and 30). An animal model of Graves’ thyroiditis and ophthalmopathy, induced by transferring TSHR-specific T cells to naive BALB/c mice, is characterized by Th2 responses (31). We did not examine eye tissue in our mice. However, neither of the animals with high TSHR antibody titers (and Th2-type responses) had thyroiditis. It is worth noting that the cytokine profile of human T cells cloned from Graves’ orbital tissue was related to disease duration, namely Th1 in early-onset disease and Th2 in long-standing disease (32).
In human autoimmunity there is evidence for both Th1- and Th2-type responses, and it may be simplistic to characterize Hashimoto’s or Graves’ disease as representing opposite poles of the cytokine spectrum. Focusing on antibodies (rather than disease), it is interesting to speculate on a parallel between concentrations and IgG subclasses of TSHR antibodies induced in mice by DNA vaccination and spontaneously arising autoantibodies in humans. TSHR autoantibodies in the majority of patients (Graves’ disease) are present at low concentrations (12, 33, 34) and are restricted to IgG1 (35), a Th1-type subclass in humans (reviewed in Ref. 24). Very rarely, patients develop TSHR antibodies that block the stimulatory effects of TSH. As shown by dilution studies, these patients have high titers of TSHR autoantibodies (36). In addition, such TSHR antibodies are distributed among all four human IgG subclasses (37), reflecting the influence of both Th1 and Th2 cytokines. It must be emphasized that neither the high titer nor low titer TSHR antibodies induced by DNA vaccination of BALB/c mice under pathogen-free conditions had detectable stimulating antibodies, and only one had TBI activity. However, it is striking that in both mice and humans, higher antibody titers are associated with Th1 and Th2 responses, whereas lower titers appear to be restricted to a TSHR-specific Th1 profile.
Although TSHR autoantibodies that stimulate the thyroid or block TSH activation produce diametrically opposite clinical phenotypes, the immune response in both patient groups is directed toward the same autoantigen, the TSHR. Moreover, there is evidence for similar genetic susceptibilities in these two clinically different patient groups, at least in terms of their human leukocyte antigen associations (38, 39). If similar genetic backgrounds can give rise to either TSHR-stimulating or TSH-blocking antibodies, other factors must play a major role in fashioning the epitopes recognized as well as limiting or expanding the magnitude of the autoimmune response to generate stimulators or blockers, respectively.
In conclusion, IFN is not required for successful TSHR DNA vaccination of BALB/c mice, and the absence of this Th1 cytokine neither modulates toward Th2 nor enhances TSHR antibody production. Importantly, splenocyte production of IFN and IL-2 provides a sensitive detection system for TSHR-specific T cells induced in vivo without adjuvant. Finally, the association between Th1 responses and low TSHR antibody levels (most mice) vs. both Th1 and Th2 responses and high TSHR antibody levels (rare mice) may have implications for the development of stimulating and blocking autoantibodies in humans.
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Acknowledgments |
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R/Fc fusion protein. We also thank Dr. Helen Braley-Mullen (Medicine, Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO) for preparing thyroid sections and characterizing the thyroid histology of mice for this study. |
Footnotes |
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Abbreviations: CFA/IFA, Freund’s adjuvant (complete, followed by incomplete); IFN, interferon-; TBI, inhibition of [125I]TSH binding to its receptor; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor.
Received October 25, 2001.
Accepted for publication December 20, 2001.
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References |
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production, rather than antibody, dominates the immune response in mice. Endocrinology 142:3530–3536R/Fc. J Clin Invest 106:207–215[Medline]
genes. Science 259:1739–1742. Virology 263:175–183[CrossRef][Medline]
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