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Experimental Studies |
Thyroid Molecular Biology Unit, Veterans Administration Medical Center, and the University of California, San Francisco, California 94121
Address all correspondence and requests for reprints to: Juan Carlos Jaume, M.D., Veterans Administration Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement Street, San Francisco, California 94121.
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Abstract |
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250 U) for TPO
autoantibodies and TSHR autoantibodies at dilutions of 1:1000 and 1:10,
respectively. Thus, by flow cytometry, the titer of TPO autoantibodies
in the BBl serum is about 100-fold higher than that for TSHR
autoantibodies in the same serum. In conclusion, the present data provide the strongest support for the idea that TSHR autoantibodies in the sera of patients with autoimmune thyroid disease are present at much lower levels than are TPO autoantibodies. This finding has important implications for the diagnostic detection of TSHR autoantibodies and for understanding the pathogenesis of Graves’ disease.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of factors may contribute to the difficulty in detecting TSHR autoantibody binding by direct means, including 1) a low TSHR concentration, 2) the requirement for conformational integrity of the antigen, 3) the high background observed with polyclonal antibodies in human sera; and 4) a low autoantibody titer. Autoantibodies to thyroid peroxidase (TPO) may be present at very high concentrations (14). An early study (15) suggested that TSHR autoantibodies are present at very high concentrations in serum (2–3% of the total IgG). More recently, consistent with the small number of TSHR autoantibody-specific B cells in Graves’ patients (16), immunofluorescence data suggest that TSHR autoantibody concentrations may be low (4, 17). The use of different assays to detect TPO and TSHR autoantibodies has precluded a direct comparison of their concentrations in the same serum.
In the present study, we used a new TSHR-expressing mammalian cell line that overcomes two of these handicaps, namely TSHR concentration and structural integrity. Thus, TSHR-10,000 Chinese hamster ovary (CHO) cells express high numbers of mature TSHR on their surface in vivo (18). Using these cells, we now report the ability to detect directly, by flow cytometry, binding of autoantibodies in a few patients’ sera to the native TSHR. However, flow cytometric analysis of TPO autoantibodies in the same sera indicates that TSHR autoantibodies are present at much lower levels than TPO autoantibodies. This finding has important implications for the diagnostic detection of TSHR autoantibodies and for understanding the pathogenesis of Graves’ disease.
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Materials and Methods |
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Serum BBl, kindly provided by Dr. Stephanie Lee, New England Medical Center Hospitals (Boston, MA), was from a patient with Graves’ disease who subsequently became hypothyroid with the development of blocking antibodies. Thirty sera were generously provided by Mr. Juan Tercero, Corning-Nichols Institute (San Juan Capistrano, CA). These sera were submitted without clinical information for the assay of autoantibodies to the TSHR (see below), presumably for suspicion of Graves’ disease. Ten of the 30 sera were selected for the absence of TSHR autoantibodies, 10 for the presence of moderate TSH binding inhibitory (TBI) activity, and an additional 10 for their high (>50%) TBI activities. Four laboratory personnel were the source of sera from individuals clearly without autoimmune thyroid disease, and 4 sera were from patients with systemic lupus erythematosus with anti-DNA and/or anti-cardiolipin antibodies.
All sera were tested in our laboratory for TBI activity using a commercially available diagnostic kit (Kronus, San Clemente, CA). The 30 sera obtained from Corning-Nichols had been tested previously using the same assay with very similar results. TSHR autoantibody titers of selected sera were determined by dilution of these sera in serum from a normal individual without a history of autoimmune thyroid disease and with undetectable TSHR autoantibody activity. Sera were also tested for TPO autoantibodies using [125I]TPO (recombinant) as described previously (19).
Cells expressing TPO and TSHR
Construction of plasmids and CHO stable transfections for overexpression of human TSHR and TPO have been described previously (18, 20). These cells as well as untransfected CHO cells (DG44, provided by Dr. Robert Schimke, Stanford University, Palo Alto, CA) were propagated in Ham’s F-12 medium supplemented with 10% FCS, penicillin (100 U/mL), gentamicin (50 µg/mL), and amphotericin B (2.5 µg/mL).
Flow cytometric analysis [fluorescein-activated cell sorting (FACS)]
For analysis of patients’ sera, cells were processed as described previously for TPO expression on CHO cells using patients’ sera and human monoclonal autoantibodies (21, 22), with modifications. In brief, after detachment by mild trypsinization, cells were pelleted and rinsed (5 min at 100 x g) in Ham’s F-12 medium supplemented with 10% dialyzed FCS and antibiotics as described above. For preadsorption, untransfected CHO cells were resuspended in 0.18 mL buffer A [phosphate-buffered saline, 10 mmol/L HEPES (pH 7.4), 0.05% sodium azide, and 2% FCS heat-inactivated at 56 C for 30 min]. Sera (20 µL) were added (final concentration of 1:10 or as specified in the text) and gently mixed for 60 min at 4 C. After removal of the cells by centrifugation, the sera were divided into two 0.1-mL aliquots. One aliquot was added to CHO cells expressing either TSHR or TPO; the other aliquot was added to untransfected CHO cells. After incubation for 60 min at 4 C, the cells were rinsed three times in buffer A and resuspended in 0.1 mL of same buffer. Affinity-purified goat antihuman IgG (1.5 µL; Fc specific, fluorescein isothiocyanate-conjugated; Caltag, South San Francisco, CA) was added to the cells for 45 min at 4 C. After three washes in buffer A, the cells were analyzed using the Becton Dickinson FACScan-CELLQuest system (Mountain View, CA). Three parameters (forward scatter, 90° side scatter, and FL1 detector) were use for the analysis. All assays included cells treated with second antibody alone and sera from normal individuals. All sera were analyzed by flow cytometry at least twice.
Detection of TSHR on TSHR-10,000 cells using mouse monoclonal antibodies (A9 and A10; 1:100 final concentration) (13) and rabbit antiserum (1:60 final concentration; R8) (23) to the TSHR (all provided by Dr. Paul Banga, London, UK) was performed according to the protocol described for human sera, except for the use of the following second antibodies: 1) affinity-purified goat antimouse IgG (0.8 µL; fluorescein isothiocyanate-conjugated), and 2) affinity-purified goat antirabbit IgG (0.5 µL; fluorescein isothiocyanate-conjugated; both from Caltag).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously been unable to detect the TSHR stably expressed
on the surface of CHO cells using Graves’ sera. Recently, however, two
reagents became available to us: CHO cells expressing very high numbers
of TSHR (nearly 2 x 106) on their surface (18), and a
Graves’ serum (BBl) particularly potent in the indirect TBI assay.
Using this serum, a very small specific signal (relative to that in
control untransfected cells) was detected with our original line (24)
expressing about 15,000 TSHR/cell (Fig. 1A
). With the
same serum, a stronger signal was observed in a line expressing about
150,000 TSHR/cell (TSHR-0; previously termed 5'3'TR-ECE; Fig. 1B) (25).
Progressively greater specific fluorescence was evident with TSHR-800
and TSHR-10,000 cells expressing approximately 106 and
1.9 x 106 receptors/cell, respectively (Fig. 1, C and
D) (18). Expression of TSHR antigen on the surface of the transfected
CHO cells, previously determined by radiolabeled TSH binding and
TSH-mediated signal transduction (18, 24, 25), was confirmed using a
rabbit polyclonal antiserum (R8) to the TSHR (Fig. 2C). Murine
monoclonal antibodies (A9 and A10) (13) were less effective in
recognizing the native TSHR on the cell surface (Fig. 2, A and B). TSHR-10,000 cells were, therefore, used in subsequent
attempts to detect TSHR autoantibodies in the sera of other patients by
direct binding to the native antigen.
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In preliminary studies we observed that some sera, regardless of
whether they contained TSHR autoantibodies, elicited high fluorescence
on flow cytometry with TSHR-10,000 cells. We observed similar high
fluorescence when these sera were incubated with untransfected CHO
cells not expressing the TSHR (data not shown). Therefore, some sera
contained antibodies against unknown antigens on the surface of CHO
cells. For this reason, we instituted a preadsorption step, in which
sera were preincubated with untransfected CHO cells before addition to
the TSHR-10,000 cells. Preadsorption was effective in eliminating or
greatly reducing this nonspecific background. For example, without
preadsorption, a serum (7H) with a high TBI value (81.7%) induced a
strong signal on TSHR-10,000 cells relative to that of a serum without
TSHR autoantibodies (Fig. 3A). However, after
preadsorption, the activity in this apparently positive serum was
clearly nonspecific (Fig. 3B). In contrast, with sera such as BBl (see
above), preadsorption actually enhanced the specificity of the signal
(Fig. 3, C and D).
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). In addition to serum BBl, this
panel included 10 sera (1L–10L) without TBI activity (1–4.2%
inhibition), 10 sera (1M–10M) with moderately high TBI values
(17.3–39.4% inhibition), 10 sera (1H–10H) with high TBI levels
(52–95.1% inhibition), sera from four normal individuals without
autoimmune thyroid disease, and sera from four patients with systemic
lupus erythematosus with anti-DNA and/or anticardiolipin antibodies.
None of the sera from normal individuals, individuals with negative TBI
values, or patients with systemic autoimmunity generated a positive
signal on flow cytometry. Remarkably, including BBl (selected for its
very high TBI activity) only 4 of the 21 sera positive for TBI (10M,
3H, and 10H) unequivocally recognized the TSHR on flow cytometry using
cells expressing large numbers of receptors (Table 1).
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).
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).
Dilution of these sera indicated that BBl and 10H had similar high TSHR
autoantibody titers, consistent with their strong fluorescence signals
on flow cytometry. The lower TBI titer of serum 3H was also consistent
with its relatively low fluorescence signal. Surprisingly, serum 10M,
with the lowest TSHR autoantibody titer, generated a strong signal on
flow cytometry, raising the possibility of the presence in this serum
of neutral autoantibodies to the TSHR that do not inhibit TSH
binding.
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In view of the small number of sera that could unequivocally
recognize TSHR by flow cytometry, we studied TPO autoantibodies in the
same sera by the same approach, using CHO cells overexpressing TPO on
their surface (20). TPO autoantibodies commonly coexist with TSHR
autoantibodies. Indeed, of the 20 TBI-positive sera (1M–10M and
1H–10H), 19 bound more than 13% [125I]TPO, well above
the upper limit detected in the sera of normal individuals (2.6%
binding; Table 1
). In addition, 2 of the TBI-negative sera (7L and 10L)
were TPO autoantibody positive by this method. Strikingly, all 20 sera
with detectable TPO autoantibodies were clearly positive on flow
cytometry with CHO-TPO cells. Not only were more sera positive for TPO
than for TSHR by flow cytometry, but the net fluorescence (after
subtraction of fluorescence with nontransfected cells) was far higher
with TPO-expressing cells than with TSHR-expressing cells (Table 1).
Like the TSHR-10,000 cells, the cells overexpressing TPO (TPO-10,000)
had about 2 x 106 TPO molecules on their surface (our
unpublished data).
We studied the serum (BBl) with the highest TSHR autoantibody level by
flow cytometry to determine the extent to which the serum could be
diluted before loss of a fluorescence signal (Fig. 5). Serum from a
normal individual was diluted in a similar manner (open
histograms). Both sera, at all dilutions tested, were preadsorbed
on untransfected CHO cells before incubation with TSHR-10,000 cells.
The fluorescence signal with BBl (Fig. 5, left
panels, shaded histograms) progressively diminished upon dilution.
At a 1:10 dilution, the net signal (BBl - normal serum) was 276.3
fluorescence units. At a 1:1000 dilution, BBl elicited only a very
small signal (net fluorescence of 7.3 U). When the same adsorbed serum
was incubated with TPO-10,000 cells, the net fluorescence diminished
from 1240.9 to 243.6 fluorescence units (Fig. 5, right panels,
shaded histograms). Similar fluorescence (243.6 and 276.3 U) was
evident for TPO autoantibodies and TSHR autoantibodies at dilutions of
1:1000 and 1:10, respectively. Thus, by flow cytometry, the titer of
TPO autoantibodies in the BBl serum was approximately 100-fold higher
than that for TSHR autoantibodies in the same serum.
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). This greater fluorescence elicited by
some sera with control cells could not be eliminated by more stringent
adsorption (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several lines of evidence suggest that the factor responsible for the
low frequency and amplitude of TSHR autoantibody detection by flow
cytometry relative to TPO autoantibodies is the very low level in serum
of the former. First, the availability of cells expressing very large
numbers of TSHR in native conformation eliminates the limiting factor
of insufficient antigen for adequate detection. Thus, both the
TSHR-10,000 cells and the TPO-overexpressing CHO cell line have similar
numbers (2 x 106) of specific antigen molecules on
their surface. Second, the weak signal observed with TSHR
autoantibodies cannot be attributed to a low affinity for antigen.
Thus, even though their affinities have not been directly determined,
they are capable of competing for high affinity (10-10
mol/L) TSH binding to the TSHR. TPO autoantibodies are known to have
similar affinities for their antigen (reviewed in Ref.26). Finally, a
1:1000 dilution of a potent serum for TSHR autoantibodies nearly
eliminated its signal on flow cytometry, whereas at the same dilution,
TPO autoantibodies in this serum continued to produce a very strong
signal.
The reason for the net negative fluorescence for TSHR-10,000 cells observed with some sera is unknown. One possible explanation for this phenomenon is that the high level of TSHR or TPO expression diminishes the expression of another, unknown antigen in CHO cells recognized by these sera (21). Expression of this antigen could, for example, be diminished by disruption of its gene by one of the many transgenome copies produced by the amplification process. Presumably, the very high fluorescence observed with TPO autoantibody-positive sera masks the phenomenon of a negative net fluorescence.
The present findings confirm our previous hypothesis (17) of low TSHR autoantibody levels in serum based on the inability to detect TSHR autoantibodies by indirect immunofluorescence on CHO cells stably expressing the TSHR complementary DNA (cDNA). Our data are in accordance with the detection of an immunofluorescence signal on cultured thyroid cells by only two exceptionally potent Graves’ sera (4). Of interest, detection of TSHR autoantibodies by flow cytometry in a serum (10M) with only moderate TBI activity provides support for the concept that in addition to autoantibodies to the TSH-binding site, some TSHR autoantibodies recognize epitopes outside of this region.
The very low concentration of TSHR autoantibodies in serum carries a number of implications. First, our findings explain the previous difficulties in using Graves’ sera to detect the TSHR by immunoblotting or immunoprecipitation. Second, the data also explain the lesser ability, relative to that of TPO and thyroglobulin autoantibodies, to detect TSHR autoantibody synthesis and secretion by patients’ lymphocytes in vitro (27, 28). This low frequency of TSHR-specific B lymphocytes and plasma cells is also likely to contribute to the great difficulty in obtaining IgG class human monoclonal autoantibodies to the TSHR (reviewed in Ref.29). Third, flow cytometry cannot be used as an alternative to the currently used clinical assays to detect TSHR autoantibodies. Fourth, and of greater importance, it is likely to be difficult to establish direct binding assays for TSHR autoantibodies of clinical relevance, even with purified antigen.
In conclusion, the present data provide the strongest support for the
idea that TSHR autoantibodies in the sera of patients with autoimmune
thyroid disease are present at much lower levels than are TPO
autoantibodies. This finding carries important implications for future
studies to understand the pathogenesis of Graves’ disease. Thus, as
hypothesized previously (17), the low concentration in serum of TSHR
autoantibodies suggests that although often present for many years,
they arise at an early stage of the autoimmune process. Support for
this concept is provided by restricted 
- or 
-light chain usage
(30, 31, 32) and relative restriction to the IgG1 subclass (33) of TSHR
autoantibodies in some patients.
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Acknowledgments |
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Footnotes |
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2 Supported in part by the Molecular Medicine program, University of
California, San Francisco.
Received July 23, 1996.
Revised September 12, 1996.
Revised October 18, 1996.
Accepted October 28, 1996.
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G binding protein. Biochim Biophys Acta. 229:649–662.[Medline]
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