Biomedical subjects

Gregorio D Chazenbalk

Publications and source records attributed to Gregorio D Chazenbalk.

12 recordsLinked to original sources

PubMed2006

Evidence that human thyroid cells express uncleaved, single-chain thyrotropin receptors on their surface.

The prevailing concept is that, in human thyroid tissue in vivo, all cell-surface TSH receptors (TSHR) cleave into disulfide linked A and B subunits. Because this viewpoint is based on studies using homogenized thyroid tissue and because of TSHR fragility, we studied TSHR subunit structure in intact thyroid cells, primary human thyrocyte cultures, FRTL-5 rat thyroid cells, and WRO (follicular) and NPA (papillary) thyroid cancer cell lines. To overcome the handicap of very low TSHR expression in thyroid cells, we generated a TSHR-expressing adenovirus (TSHR-Ad-RGD) with an integrin-binding RGD motif enabling efficient entry into cells lacking the coxsackie-adenovirus receptor. Two days after TSHR-Ad-RGD infection, [125I]TSH cross-linking to intact cells revealed uncleaved, single-chain TSHR as well as cleaved TSHR A subunits on the surface of all four thyroid cell types. The extent of TSHR cleavage, which is independent of the level of TSHR expression, was consistently lower in the human thyroid cancer cell lines than in the other cell lines. In flow cytometry studies after TSHR-Ad-RGD infection, strong signals were detected in all four thyroid cell types using a monoclonal antibody that primarily recognizes the uncleaved TSHR. Finally, using the same monoclonal antibody, confocal microscopy confirmed the presence of single-chain TSHR on TSHR-Ad-RGD-infected thyroid cells. In summary, TSH covalent cross-linking, flow cytometry, and confocal microscopy demonstrate the presence of uncleaved TSHR on the human thyrocyte surface. These data provide stronger evidence for this alternative than the contrary view based on the finding of only cleaved TSHR in homogenized thyroid cells.

Animals↗

PubMed2006

Targeted expression of the human thyrotropin receptor A-subunit to the mouse thyroid: insight into overcoming the lack of response to A-subunit adenovirus immunization.

The thyrotropin receptor (TSHR), the major autoantigen in Graves' disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves' hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, "non-self" regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.

Adenoviridae↗

PubMed2004

"Hijacking" the thyrotropin receptor: A chimeric receptor-lysosome associated membrane protein enhances deoxyribonucleic acid vaccination and induces Graves' hyperthyroidism.

Naked DNA vaccination with the TSH receptor (TSHR) does not, in most studies, induce TSHR antibodies and never induces hyperthyroidism in BALB/c mice. Proteins expressed endogenously by vaccination are preferentially presented by major histocompatibility complex class I, but optimal T cell help for antibody production requires lysosomal processing and major histocompatibility complex class II presentation. To divert protein expression to lysosomes, we constructed a plasmid with the TSHR ectodomain spliced between the signal peptide and transmembrane-intracellular region of lysosome-associated membrane protein (LAMP)-1, a lysosome-associated membrane protein. BALB/c mice pretreated with cardiotoxin were primed intramuscularly using this LAMP-TSHR chimera and boosted twice with DNA encoding wild-type TSHR, TSHR A-subunit, or LAMP-TSHR. With each protocol, spleen cells responded to TSHR antigen by secreting interferon-gamma, and 60% or more mice had TSHR antibodies detectable by ELISA. TSH binding inhibitory activity was present in seven, four, and two of 10 mice boosted with TSHR A-subunit, LAMP-TSHR, or wild-type TSHR, respectively. Importantly, six of 30 mice had elevated T4 levels and goiter (5 of 6 with detectable thyroid-stimulating antibodies). Injecting LAMP-TSHR intradermally without cardiotoxin pretreatment induced TSHR antibodies detectable by ELISA but not by TSH binding inhibitory activity, and none became hyperthyroid. These findings are consistent with a role for cardiotoxin-recruited macrophages in which (unlike in fibroblasts) LAMP-TSHR can be expressed intracellularly and on the cell surface. In conclusion, hijacking the TSHR to lysosomes enhances T cell responses and TSHR antibody generation and induces Graves'-like hyperthyroidism in BALB/c mice by intramuscular naked DNA vaccination.

Animals↗

PubMed2004

Evidence that the thyrotropin receptor protease is membrane-associated and is not within lipid rafts.

The thyrotropin receptor (TSHR) cleaves to a variable extent within the ectodomain into a ligand-binding A subunit linked by disulfide bonds to the largely transmembrane B subunit. To obtain insight into this variability, we examined the extent of cleavage of TSHR ectodomains tethered to the plasma membrane by different means: (1) the wild-type, serpentine region, (2) a glycosylphosphatidylinositol (GPI) anchor, and (3) a single CD8alpha transmembrane region. For this purpose, we covalently cross-linked(125)I-TSH to the TSHR ectodomain expressed on the surface of intact cell monolayers. The extent of cleavage of the CD8alpha-tethered ectodomain was similar to the wild-type TSHR (approximately 50%) whereas the same ectodomain with a GPI anchor remained almost entirely (approximately 90%) uncleaved. These findings have three possible implications. First, differential cleavage of the TSHR ectodomain depending on its attachment to the plasma membrane suggests that the TSHR protease is membrane-associated and is not a soluble (secreted or shed) protease. Second, because GPI-anchored proteins (unlike CD8alpha) segregate in membrane lipid rafts, the TSHR protease appears not to be associated with lipid rafts. Finally, the similar extent of cleavage of the wild-type TSHR and the CD8alpha (not the GPI) tethered ectodomain supports the concept that the wild-type TSHR resides largely outside lipid rafts.

Animals↗

PubMed2004

Thyroid stimulation does not require antibodies with identical epitopes but does involve recognition of a critical conformation at the N terminus of the thyrotropin receptor A-subunit.

Whether monoclonal antibodies with thyroid-stimulating activity [thyroid-stimulating antibody/antibodies (TSAb)] from immunized animals are identical to human autoantibodies in Graves' disease is unknown. Here, we compared properties of a monoclonal hamster TSAb (MS-1) with human autoantibodies. The epitopes of neither MS-1 nor human autoantibodies can be determined by peptide scanning, indicating their conformational nature. A property of human TSAb is that their epitope is partially obscured on the TSH holoreceptor on the cell surface relative to the TSH receptor (TSHR) ectodomain tethered to the membrane by a glycosylphosphatidyl inositol anchor. On flow cytometry, as for human autoantibodies, MS-1 preferentially recognized the glycosylphosphatidyl inositol-anchored ectodomain vs. the TSH holoreceptor on Chinese hamster ovary cells. Also, as with human autoantibodies, only A-subunits with the active (but not the inactive) conformation adsorbed MS-1 binding activity. This difference localizes antibody binding to a cysteine-rich region at the TSHR N terminus. Remarkably, active TSHR A-subunit more effectively ( approximately 40-fold) neutralized human autoantibodies than it did MS-1. Therefore, MS-1 interacts less well than autoantibodies with the free A-subunit. In summary, we provide evidence that TSAb need not have identical epitopes. However, the TSAb epitope does appear to require involvement of the highly conformational N terminus of the A-subunit.

Animals↗

PubMed2004

Affinity-enrichment of thyrotropin receptor autoantibodies from Graves' patients and normal individuals provides insight into their properties and possible origin from natural antibodies.

We used purified recombinant TSH receptor (TSHR) antigen prepared in mammalian cells to affinity-enrich TSHR autoantibodies from Graves' patients' IgG. Autoantibody enrichment, assayed by TSH binding inhibitory activity, was 20- to 1000-fold. Thyroid-stimulating antibody activity enrichment, although more difficult to quantitate, was comparable. TSHR-autoantibody approximate affinities for the holoreceptor assessed indirectly by TSH binding inhibition were 4-27 x 10(-9) m, an underestimate because 100% TSHR autoantibody purity was not attained. Consistent with previous data for serum, highly enriched TSHR autoantibodies in three of four patients showed lambda light chain bias. However, in contrast to expectations, antigen-enriched IgG was skewed primarily toward IgG2 and IgG3, subclasses associated with polysaccharides and microorganisms, respectively. Subclass depletion studies on antigen-enriched IgG indicated that TSHR autoantibodies were predominantly IgG1 and, surprisingly, IgG4. As controls, we affinity-enriched pooled IgG from normal individuals on TSHR antigen. This enriched IgG had detectable TSH binding inhibitory activity, although with lower specific activity than, and lacking the thyroid stimulatory activity of, Graves' IgG. Moreover, these natural IgG class autoantibodies largely recognized the same conformational variation in the TSHR N-terminal region as disease-associated TSHR autoantibodies. These studies suggest that TSHR autoantibodies may arise from natural autoantibodies, possibly by class switching from cross-reacting antibodies to microorganisms.

Antibodies↗

PubMed2003

Low-dose immunization with adenovirus expressing the thyroid-stimulating hormone receptor A-subunit deviates the antibody response toward that of autoantibodies in human Graves' disease.

Immunization with adenovirus expressing the TSH receptor (TSHR) induces hyperthyroidism in 25-50% of mice. Even more effective is immunization with a TSHR A-subunit adenovirus (65-84% hyperthyroidism). Nevertheless, TSHR antibody characteristics in these mice do not mimic accurately those of autoantibodies in typical Graves' patients, with a marked TSH-blocking antibody response. We hypothesized that this suboptimal antibody response was consequent to the standard dose of TSHR-adenovirus providing too great an immune stimulus. To test this hypothesis, we compared BALB/c mice immunized with the usual number (10(11)) and with far fewer viral particles (10(9) and 10(7)). Regardless of viral dose, hyperthyroidism developed in a similar proportion (68-80%) of mice. We then examined the qualitative nature of TSHR antibodies in each group. Sera from all mice had TSH binding-inhibitory (TBI) activity after the second immunization, with TBI values in proportion to the viral dose. After the third injection, all groups had near-maximal TBI values. Remarkably, in confirmation of our hypothesis, immunization with progressively lower viral doses generated TSHR antibodies approaching the characteristics of autoantibodies in human Graves' disease as follows: 1) lower TSHR antibody titers on ELISA and 2) lower TSH-blocking antibody activity without decrease in thyroid-stimulating antibody activity. In summary, low-dose immunization with adenovirus expressing the free TSHR A-subunit provides an induced animal model with a high prevalence of hyperthyroidism as well as TSHR antibodies more closely resembling autoantibodies in Graves' disease.

Adenoviridae↗

PubMed2003

Does thyrotropin cleave its cognate receptor?

A recent report of major pathophysiological significance, and opposed to present concepts, is that TSH (but not MS-1, a hamster monoclonal thyroid-stimulating antibody), cleaves the single-chain TSH receptor (TSHR) on the cell surface into its two-subunit form. We reassessed the issue using two approaches. First we wished to confirm the flow-cytometric assay previously used to quantitate TSHR cleavage. We used CHO cell lines expressing large (TSHR-10,000 cells) or conventional (TSHR-0 cells) numbers of TSHR. Cells were preincubated (16 h) in either control medium or medium supplemented with TSH (5 x 10(-8) m) or MS-1 (10 microg/ml). After stringent washing to maximize removal of residual ligand, we performed flow cytometry with two antibodies, one recognizing only the single-chain TSHR, the other recognizing all (cleaved and uncleaved) TSHRs. TSH pretreatment did not appear to increase TSHR cleavage. Instead we observed ligand occupancy of the TSHR (with MS-1) or fewer receptors on the cell surface (down-regulation), particularly with the TSHR-0 cells. Second, we covalently cross-linked [125I]TSH to monolayers of these cells, an unequivocal method to determine directly the proportion of single-chain and two-subunit TSHR forms. Pretreatment of TSHR-10,000 and TSHR-0 cells with TSH had no effect on the degree of TSHR cleavage. MS-1 slightly reduced spontaneous cleavage. In conclusion, in contrast to a recent report, we show that TSH does not alter the subunit structure of its cognate receptor, and we provide insight into the difficulties associated with the flow-cytometric assay for TSHR cleavage.

Animals↗

PubMed2003

Targeted restoration of cleavage in a noncleaving thyrotropin receptor demonstrates that cleavage is insufficient to enhance ligand-independent activity.

Two unusual features of the TSH receptor (TSHR) ectodomain are its intramolecular cleavage at the cell surface into disulfide-linked subunits and its constraint of ligand-independent (constitutive) activity inherent to the serpentine region. Whether ectodomain cleavage alters the level of TSHR constitutive activity is an important unanswered question. To address this issue, we used a TSHR engineered so as not to undergo spontaneous cleavage into subunits (deletion of amino acid residues 317-366 and GQE(367-369)NET substitution). Into this noncleaving TSHR (termed TSHR-D1-NET), we introduced thrombin recognition motifs (termed Thr 6 and Thr 18) at the site of spontaneous cleavage. Treatment of intact Chinese hamster ovary cells expressing TSHR-D1-NET-Thr 6 and -Thr 18 with thrombin induced cleavage into A and B subunits, as determined by (125)I-TSH covalent cross-linking. Nevertheless, constitutive activity of the thrombin-cleaved TSHR was unaltered. The level of TSHR constitutive activity was, therefore, fully dissociated from intramolecular cleavage into subunits. Trypsin treatment of the same cells expressing the noncleaving TSHR also generated disulfide-linked A and B subunits but, in contrast to thrombin, enhanced TSHR constitutive activity. Therefore, the activating effect of trypsin appears to involve clipping at an additional, as-yet unidentified, site. In summary, our data demonstrate that TSHR cleavage is, by itself, insufficient to reduce TSHR ectodomain constraint on ligand-independent constitutive activity. These data are consistent with other evidence that A subunit shedding consequent to TSHR cleavage is a critical factor in enhancing TSHR constitutive activity.

Animals↗

PubMed2003

Evidence that the C terminus of the A subunit suppresses thyrotropin receptor constitutive activity.

The TSH receptor (TSHR), unlike the LH receptor (LHR), has considerable ligand-independent adenylyl cyclase activity, a feature of pathophysiological importance. The TSHR ectodomain partially suppresses constitutive activity, an effect reversed by trypsin treatment of intact cells. Localizing the functional site of trypsin action would provide insight into how the TSHR ectodomain exerts its constraint. For this purpose, we examined the effect of trypsin on intact cells expressing a series of modified TSHR. Trypsin did not increase cAMP production by a chimeric TSH-LH receptor involving substitution of TSHR residues 261-418 (the ectodomain C terminus). In contrast, with the wild-type TSHR, trypsin enhanced constitutive activity despite mutation of the following potential tryptic cleavage sites [arginine (R) and lysine (K) residues]: 1) K565, K651, K660 in the extracellular loops of the serpentine region; 2) B subunit juxtamembrane residues K371, K401, K415; 3) A subunit residues R310, R312, K313. We previously excluded K337 and K339 from being implicated in TSHR tryptic activation. By exclusion, only one R/K cluster remains as a possible target for the functional effect of trypsin, namely K287, K290, K291, and R293. Mutation of this cluster is incompatible with TSHR cell surface expression. However, tryptic clipping at this locus would reproduce a previously demonstrated structural effect of trypsin on the TSHR, removal of about a 2-kDa polypeptide fragment extending downstream from the locus to the C terminus of the A subunit. Taken together, these data suggest that the C terminus of the A subunit functions as a suppressor of TSHR constitutive activity.

Animals↗

PubMed2002

Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor.

Graves disease is directly caused by thyroid-stimulating autoantibodies (TSAb's) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb's]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb's, but not for TBAb's, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals.

Animals↗

PubMed2002

Immune deviation away from Th1 in interferon-gamma knockout mice does not enhance TSH receptor antibody production after naked DNA vaccination.

TSH receptor (TSHR) DNA vaccination induces high TSHR antibody levels in BALB/c mice housed in a conventional facility. However, under pathogen-free conditions, we observed a Th1 cellular response to TSHR antigen characterized by interferon-gamma (IFN gamma) production. In the present study we investigated the effect on TSHR DNA vaccination of diverting the cytokine milieu away from Th1 using 1) IFN gamma knockout BALB/c mice, and 2) wild-type mice covaccinated with DNA for the TSHR and for IFN gamma/receptor-Fc protein that prevents IFN gamma from binding to its receptor. Neither approach enhanced TSHR antibody levels, although splenocyte IFN gamma production in response to TSHR antigen was absent (IFN gamma knockouts) or reduced (IFN gamma 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 gamma 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.

Animals↗