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Original Studies |
Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California-Los Angeles School of Medicine, Los Angeles, California 90048
Address all correspondence and requests for reprints to: Dr. Sandra M. McLachlan, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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10-10 M), comparable with
those of human TPO autoantibodies and about 10-fold higher than those
in purified TPO plus adjuvant-immunized mice. Moreover, more than 90%
of TPO antibodies in TPO+/class II+
fibroblast-injected mice, compared with only approximately 50% in TPO
plus adjuvant-immunized mice, were to the immunodominant region
recognized by patients’ autoantibodies. Consistent with this epitopic
restriction, TPO+/class II+ fibroblast-injected
mice had TPO antibody epitopic fingerprints similar to those of human
autoantibodies. In conclusion, mice injected with TPO+/class II+ fibroblasts, but not those injected with purified TPO and adjuvant, develop antibodies closely resembling autoantibodies in human disease. These observations indicate that some animal models based on conventional immunization may not be representative of human diseases with a major humoral component.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The basis for this qualitative difference between antibodies arising spontaneously vs. antibodies induced by immunization with purified antigen and adjuvant is not known. However, the manner of antigen presentation can modulate the subsequent immune response. Evidence for this possibility is provided by a novel approach used to develop the first animal model of Graves’ hyperthyroidism. The Shimojo model was achieved by injecting mice with fibroblasts coexpressing syngeneic major histocompatibility complex (MHC) class II and the TSHR (8, 9). Aberrant MHC class II expression on thyroid cells from patients with autoimmune thyroid disease was first observed in 1983 (10). The Shimojo approach is based on these early observations, and its success supports the hypothesis that thyroid cells may function as antigen-presenting cells (APC) and initiate the autoimmune response (11).
The approach used in the Shimojo model now provides the opportunity to examine the relationship between antigen presentation to the immune system and the development of antibodies that resemble those observed in human thyroid autoimmunity. In the present study, we compared the qualitative nature of antibodies developing in the same strain of mice immunized by conventional means and by injecting fibroblasts expressing both antigen and MHC class II on their cell surface. The use of TPO, rather than the TSHR, in these studies offers a number of advantages, including the availability of large amounts of purified, native mammalian antigen (12) as well as a panel of human monoclonal autoantibodies that define the TPO-immunodominant region (13). Remarkably, although both approaches elicited TPO antibodies, only mice receiving fibroblasts coexpressing TPO and MHC class II+ fibroblasts develop antibodies that closely resemble patients’ autoantibodies in terms of their high affinity and predominant recognition of the TPO-immunodominant region.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT4.15HP fibroblasts (14) (provided by Dr. Ron Germain, NIH, Bethesda, MD), were propagated in DMEM with high glucose, 10% FCS, and antibiotics (Life Technologies,Gaithersburg, MD). Cells were stably transfected using lipofectin (Life Technologies) with complementary DNAs (cDNAs) for 1) human TPO (pECE-hTPO cotransfected with pSV2-NEO) (15), 2) human TSHR (pSV2-NEO-ECE-TSHR 5',3') (16), 3) both TPO and TSHR (as above), and, as a control, 4) vector alone (pSV2-NEO-ECE). Selection with G418 (400 mg/mL) and cloning by limiting dilution were performed using standard techniques.
Cells, detached by mild trypsinization, were monitored for cell surface
TPO expression by flow cytometry using a human monoclonal TPO
autoantibody Fab (WR1.7) (13) followed by phycoerythrin-conjugated
antihuman 
(Caltag, Burlingame, CA), as described previously (17).
Fluorescence was analyzed using the Becton Dickinson and Co. FACScan-CellQuest system (Mountain View, CA). Chinese
hamster ovary (CHO) cells overexpressing TPO (18) provided a positive
control. Negative controls were RT4.15HP cells (14) and untransfected
CHO cells (DG44; provided by Dr. Robert Schimke, Stanford University,
Palo Alto, CA). Assays included cells treated with second antibody
alone and normal mouse serum. TSHR expression in transfected RT4.15HP
monolayers was monitored by [125I]TSH binding, as
described for CHO cells (16). Clones expressing TPO alone, TSHR alone,
or TPO and TSHR or vector-transfected cells were expanded in
hypoxanthine-aminopterin-thymidine medium (HAT, Sigma Chemical Co., St. Louis, MO) to preserve MHC class II expression. Class
II expression was assessed by flow cytometry using fluorescein
isothiocyanate-conjugated anti-IA-k(aak)
(PharMingen, San Diego, CA). A line of RT4.15HP cells,
propagated without HAT selection medium, provided a negative
control.
Induction of TPO antibodies using fibroblasts coexpressing TPO and MHC class II
Six-week-old female AKR/N mice (NCI, Bethesda, MD) received six ip injections at 2-week intervals of RT4.15HP fibroblasts (107 cells/injection) expressing TPO, TSHR, or TPO and TSHR or vector-transfected fibroblasts (five mice per group). Before transfer, fibroblasts were pretreated with 50 µg/mL mitomycin C (Sigma Chemical Co.). This protocol is similar to that described for induction of TSHR antibodies using RT4.15HP-TSHR cells (8). Blood was obtained from the tail vein 2 weeks after the fifth injection and by cardiac puncture when the animals were killed 2 weeks after the sixth injection. All animal studies were performed in accordance with the highest standards of humane care.
Conventional immunization with purified TPO and adjuvant
Recombinant human TPO was prepared as previously reported (12). The membrane-bound TPO protein (933 amino acids, including signal peptide) was converted into an 848-residue molecule by introduction of a stop codon at the ectodomain/plasma membrane junction followed by transgenome amplification with a dihydrofolate reductase minigene (18). TPO secreted by CHO cells was affinity purified from culture medium. Concentration was determined by spectrophotometry at 280 nm optical density (extinction coefficient = 17.9), and purity was determined by PAGE. AKR/N female mice (6 weeks old) were injected ip with purified TPO (50 µg/mouse) in complete Freund’s adjuvant (Sigma Chemical Co.). Two weeks later, immunization was repeated with the same antigen dose in incomplete Freund’s adjuvant (Sigma Chemical Co.). A similar protocol is used to immunize mice against a variety of purified protein antigens including TPO (19). Blood was obtained from the tail vein 7 days after boosting to assess TPO antibody levels, and a final sample was collected by cardiac puncture at death 4 weeks after priming.
Flow cytometric analysis of TPO antibodies
Mouse sera (diluted 1:10) were analyzed using TPO-expressing CHO cells (18) and fluorescein isothiocyanate-conjugated, affinity-purified goat antimouse IgG (Caltag, South San Francisco, CA) as described previously (17). Assays included cells treated with second antibody alone and normal mouse serum.
TPO antibody binding of [125I]TPO and affinity for TPO
The assay was performed as previously described (20). In brief,
duplicate aliquots of mouse sera (diluted 1:20 unless otherwise
specified) were incubated with [125I]TPO (20,000 cpm;
labeled using iodogen to a specific activity of 50 µCi/µg). To
precipitate the antigen-antibody complex, protein A (Pansorbin;
Calbiochem, La Jolla, CA) or antimouse IgG coupled to a
solid phase (Sac Cel, IDS, Boldon, Tyne and Wear, U.K.) was added, and
the incubation was continued. After the addition of assay buffer [0.1
M NaCl, 10 mM Tris-HCl (pH 7.5), 0.1%
Tween-20, and 0.5% BSA], the mixture was vortexed and centrifuged,
supernatants were removed, and radiolabeled TPO remaining in the
pellets was counted. Nonspecific [125I]TPO binding by
normal mouse serum (3% of the total counts per min) was subtracted
in calculating the percentage of [125I]TPO bound by
antibodies in mouse serum. Antibody affinities were determined by
Scatchard analysis (21) from binding values obtained in the presence of
increasing concentrations of unlabeled TPO.
Interaction between mouse antibodies and the TPO-immunodominant region recognized by human autoantibodies
[125I]TPO binding by mouse serum antibodies was
examined in the absence and presence of four recombinant human
TPO-specific autoantibody Fab (SP1.4, WR1.7, TR1.8, and TR1.9) (13)
that define the TPO-immunodominant region recognized by patients’ TPO
autoantibodies (reviewed in Ref. 1). For simplification, the subdomains
recognized by these Fab were previously renamed A1, A2, B1, and B2,
respectively (22). Duplicate aliquots of serum were incubated with
[125I]TPO (20,000 cpm) alone or with the four Fab pools
(each Fab at 4 x 10-8 M). Recombinant
Fab were prepared as previously described in detail (23). After 1
h at room temperature, complexes were precipitated using protein A, and
radiolabeled TPO remaining in the pellets was counted. Fab lack the CH2
domain of the Fc region and are not precipitated by protein A.
Preliminary experiments were performed to determine the serum antibody
dilutions required to provide binding values of about 15% in the
absence of TPO Fab. Such dilution is necessary to attain maximal
inhibition of TPO binding by the addition of an excess concentration of
Fab. Nonspecific [125I]TPO binding (3% of the total
counts per minute) was subtracted to calculate the percent inhibition
by the TPO-specific Fab. Epitopic profiles of the mouse TPO antibodies
were determined using the same assay, except competition for
[125I]TPO binding was performed in the presence of the
individual human TPO-specific Fab that define the immunodominant
region.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The RT4.15HP fibroblast line expresses a recombinant MHC class II
molecule essentially identical to MHC class II (I-Ak) of AKR/N mice
(14). TPO was readily detectable on MHC class II-expressing RT4.15HP
cells transfected with the cDNA for human TPO (Fig. 1A
). However, as anticipated, the
magnitude of fluorescence for the highest expressing clone was far less
than that for CHO cells overexpressing TPO consequent to transgenome
amplification (18) (Fig. 1B). Stable transfection with TPO cDNA did not
alter class II levels compared with those in the parent line (Fig. 1, C
vs. D). Of the clones doubly transfected with TPO and TSHR
cDNA, TPO expression for the best expressing clone was slightly lower
than that for the best expressor among the clones transfected with TPO
cDNA alone (median, 15.7 vs. 20.0 fluorescent units,
respectively). Similarly, TSHR expression, assessed by
[125I]TSH binding, was lower in the optimal double
transfectant than in the optimal TSHR-only transfected clone (14.9%
vs. 16.2%, respectively; 4.3% binding by untransfected
RT4.15HP cells).
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As assessed by flow cytometry with TPO-expressing CHO cells, sera
from four of five AKR/N mice injected with
TPO+/class II+ RT4.15HP
fibroblasts produced high levels of IgG class TPO antibodies relative
to those in the five mice injected with TSHR+/class
II+ fibroblasts (t = 4.44;
P = 0.002; Fig. 2A).
These observations were confirmed by protein A precipitation of
[125I]TPO-IgG antibody complexes, an assay routinely used
to detect patients’ serum TPO autoantibodies (24). Thus, sera from all
five mice injected with TPO+/class
II+ fibroblasts bound significantly more TPO than
sera from mice injected with TSHR+/class
II+ fibroblasts (t = 7.55;
P < 0.001; Fig. 2B).
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As measured by flow cytometry and [125I]TPO binding,
AKR/N mice immunized with purified TPO and adjuvant developed higher
levels of TPO antibodies than mice injected with
TPO+/class II+ fibroblasts.
Thus, the median fluorescence was 458 ± 15 (mean ±
SEM; sera diluted 1:20) for five mice immunized with
purified TPO compared with 240 ± 42 in similarly diluted sera
from five mice injected with TPO+ fibroblasts.
Similarly, [125I]TPO binding was significantly greater in
sera from conventionally immunized mice than in sera from mice injected
with class II+ fibroblasts coexpressing TPO alone
or with the TSHR (by ANOVA: P < 0.001; F = 21.29,
24.89, and 21.72 for sera diluted 1:50, 1:100, and 1:500; Fig. 3). Coexpression of the TSHR with TPO had
no significant effect on the titers of TPO antibodies.
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). The mean
affinity for TPO antibodies in mice injected with
TPO+/class II+ cells was
1.9 ± 0.6 x 10-10 M
Kd (±SD; n = 3) vs.
1.2 ± 0.3 x 10-9 M
Kd (n = 3) in mice immunized with purified
TPO and adjuvant (t = 6.45; P = 0.003).
The higher antibody affinities in the former group of mice were
comparable with those of autoantibodies in patients’ sera, namely
Kd of approximately 10-10
M (24).
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Because of the foregoing resemblance to human autoantibodies, we
wished to determine whether the antibodies arising in response to
TPO+/class II+ fibroblasts
shared other characteristics with human TPO autoantibodies, in
particular recognition of the TPO-immunodominant region. This region of
overlapping domains on native TPO is defined by four human monoclonal
TPO autoantibodies in the form of recombinant Fab (13) (Fig. 5, upper panel). The concept
that the majority of TPO autoantibodies within an individual patient’s
serum are to the immunodominant regions has been confirmed in a
three-way collaborative comparison of the epitopes of the four original
Fab with the epitopes of 1) a panel of murine monoclonal antibodies to
TPO (2) and 2) a panel of human Fab isolated independently by a
different laboratory (20).
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, lower panel). In contrast, inhibition by the four Fab pool
was significantly lower (only 50%) for TPO antibodies in the
conventionally immunized mice (P < 0.001; F =
30.04; by ANOVA). Epitopic fingerprinting of TPO antibodies in immunized mice
When used separately, the four human autoantibodies to the
TPO-immunodominant region (Fab A1, A2, B1, and B2) can be used to
determine a quantitative epitopic fingerprint for TPO autoantibodies in
an individual patient (23; reviewed in Ref. 1). The epitopic
fingerprints of mice injected with TPO+/class
II+ fibroblasts were similar to those observed in
human autoantibodies (Fig. 6, upper and middle panels). For example, TPO
antibodies in mouse 6 were markedly inhibited by Fab A1 and A2 (>80%)
and to a lesser extent by Fab B2 and B1 (50% or less), indicating
preferential recognition of the A domain. In four mice (no. 5, 8, 9,
and 10), Fab B2 was the dominant inhibitor (>80%), whereas in three
mice (no. 5, 4, and 10) TPO binding was reduced more than 75% by Fab
B1. Comparable inhibition by A and B domain Fab was observed in three
mice (no. 2, 3, and 7). The fingerprints were unrelated to percent
inhibition by the four-Fab pool.
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, lower panels). Such low inhibition values reflect overlap
with only about 50% of epitopes in the TPO-immunodominant region (Fig. 5). Not surprisingly, we have rarely observed epitopic profiles like
those of purified TPO-immunized mice 13, 14, and 15 in human thyroid
autoimmunity. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10-9
M) are about 10-fold lower than the affinities of
autoantibodies in patients’ sera (24). Moreover, only 50% of the
epitopes recognized by antibodies in purified TPO-immunized mice
overlap with the epitopes in the immunodominant region recognized by
the majority of TPO autoantibodies in all patients’ sera (reviewed in
Ref. 1). In contrast, mice injected with
TPO+/class II+ fibroblasts
develop lower TPO antibody titers. However, these murine antibodies
closely resemble patients’ autoantibodies in terms of their high
affinities (Kd = 10-10
M), restricted epitopes (predominant recognition of the
immunodominant region), and TPO epitopic fingerprints. It is intriguing and perhaps contrary to expectation that a heterologous antigen such as human TPO (or human TSHR, see introduction) expressed on the surface of class II+ fibroblasts, but not as purified antigen combined with adjuvant, is capable of inducing antibodies in recipient mice with properties similar to those of autoantibodies in humans. It should be emphasized that we do not know the basis for the differences among the titers, affinities for TPO, and antibody epitopes that develop using these immunization protocols. However, a number of factors may be involved including 1) immunization with monomeric purified TPO vs. dimeric TPO expressed on the cell surface, 2) use of Freund’s adjuvant, 3) differences in antigen processing, and 4) differences in protein concentration. The first two parameters can probably be excluded. First, like patients’ autoantibodies (17), sera from mice immunized using either approach interact with both purified, monomeric radiolabeled TPO as well as with dimeric TPO expressed on the cell surface. Second, after immunization and screening with conformationally intact purified TPO, Ruf et al. (2) isolated a panel of mouse monoclonal antibodies that resembled serum antibodies in our adjuvant immunized mice in recognizing diverse epitopes on TPO (20) despite the use of a different adjuvant (Bordetella pertussis vs. Freund’s adjuvant in our study).
Two other factors are potentially important in eliciting the difference between the two forms of immunization. One possibility is that, unlike in purified TPO- and adjuvant-immunized mice, the class II+, TPO+ fibroblasts are the major antigen presenting cells (APC) and present a different spectrum of peptides than conventional APC in T cell activation. This concept has been suggested with respect to the Shimojo model for inducing Graves’ disease-like stimulatory TSHR antibodies (see introduction). Stimulatory TSHR antibodies have also been generated by intramuscular immunization with "naked" TSHR cDNA in mice pretreated with cardiotoxin to induce local muscle damage (25). Because human myoblasts incubated with cytokines are able to function as APC (26), it is conceivable that myoblasts are the major APC in this model.
The possibility that professional and nonprofessional APC (such as thyroid cells or TPO-expressing fibroblasts) present different epitopes is consistent with the ability of some T cell clones to recognize TPO endogenously processed by autologous thyroid cells (27) or by TPO-transfected B cells (28, 29), but not exogenous, purified TPO presented by conventional APC (29). On the other hand, evidence against a role for nonprofessional APC in our study is the development of TSHR antibodies in mice receiving fibroblasts expressing the TSHR in the absence of class II (30). However, few of these mice had TSHR antibodies with the stimulatory characteristics of Graves’ IgG, suggesting that coexpression of class II and TSHR influences induction of antibodies with an epitope(s) required for TSHR stimulation. Nevertheless, we cannot exclude the possibility that conventional APC are responsible for processing TPO in mice injected with a population of TPO+ fibroblasts that is alive but rendered incapable of dividing and destined to die in the peritoneum.
Turning to antigen concentration, mice injected with TPO-expressing
fibroblasts receive an extremely small dose of TPO. Based on the
relative fluorescence of TPO-transfected fibroblasts and CHO cells (the
latter expressing about 106 molecules/cell), the course of
six injections of the former cells delivers, at most, 0.5 µg
TPO/mouse. In contrast, we and others (2, 19, 31) have used a total of
70–200 µg (generally in two doses) of purified human TPO for
conventional immunization that has, intentionally or otherwise,
resulted in TPO antibody production. Indeed, a lower dose of purified
TPO (40 µg) was ineffective for TPO antibody induction (19). We
cannot be sure of the concentration of TPO available to the immune
system in vivo using either of the two immunization
protocols. However, the higher affinities of TPO antibodies arising in
mice receiving TPO-transfected fibroblasts (vs.
conventionally immunized mice) are consistent with a lower dose of
antigen for the following reason. The progression from a primary to a
secondary antibody response involves selection of B cells with surface
Igs of progressively higher affinity (reviewed in Ref. 32). When
antigen levels are limiting (as is likely to be the case with
TPO+/class II+
fibroblasts), only the highest affinity B cells can engage antigen and
become activated. It should be emphasized that unlike T cells, which
only recognize processed peptides, B cells usually bind
conformationally intact protein antigens. As mentioned above, TPO
antibodies arising in mice injected with TPO-expressing fibroblasts
recognize TPO on transfected cells.
In contrast to the present data on TPO antibodies, neither the epitopic repertoire nor the affinities of antibodies have been compared in mice immunized with fibroblasts expressing the TSHR and MHC class II (8, 9) vs. purified TSHR and a variety of adjuvants (reviewed in Ref. 7). Our present observations for TPO suggest that TSHR antibodies generated using the Shimojo protocol may have a restricted epitopic repertoire and higher affinities for the TSHR than antibodies in conventionally immunized mice. Moreover, it is conceivable that the unusual characteristic of TSHR antibodies that only develop in mice injected with TSHR-expressing fibroblasts, namely their ability to stimulate the thyroid, may be related to affinity and/or epitopic restriction.
In conclusion, injection of AKR/N mice with fibroblasts coexpressing TPO and class II, but not conventional immunization with purified antigen, induces antibodies that closely resemble patients’ serum TPO autoantibodies. These observations in the same strain of mice indicate that some animal models based on conventional immunization using high doses of purified antigen may not be representative of human autoimmune diseases with a major humoral component.
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Acknowledgments |
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Footnotes |
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2 Present address: Endocrinology Division, Veterans Administration
Medical Center and University of California, San Francisco, California
94121.
Received October 26, 1998.
Revised January 21, 1999.
Accepted February 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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