This Article Full Text Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guo, J. Articles by McLachlan, S. M. Search for Related Content PubMed PubMed Citation Articles by Guo, J. Articles by McLachlan, S. M.

Recombinant Thyroid Peroxidase-Specific Fab Converted to Immunoglobulin G (IgG) Molecules: Evidence for Thyroid Cell Damage by IgG1, but Not IgG4, Autoantibodies¹

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: Dr. Sandra McLachlan, Veterans Administration Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement Street, San Francisco, California 94121.

A recombinant autoantibody Fab (SP1.4) to thyroid peroxidase (TPO), cloned from intrathyroidal B cell immunoglobulin genes, interacts with an epitope on TPO recognized by all patients with autoimmune thyroid disease. To compare the biological properties of IgG1 and IgG4 TPO autoantibodies, we converted Fab SP1.4 to full-length immunoglobulins. The SP1.4 heavy and light chain variable region genes, spliced by overlap PCR to a mammalian signal peptide, were transferred to expression vectors for human IgG1, IgG4, and L chains. Plasmids containing the IgG1 (or IgG4) heavy chain and the L chain were cotransfected into SP2/0 mouse myeloma cells. Cells secreting TPO autoantibodies were cloned, and IgG1-SP and IgG4-SP were affinity purified from medium using protein G. Their subclass specificities were confirmed by enzyme-linked immunosorbent assay and fluorometry after binding to Chinese hamster ovary cells expressing cell surface TPO. Further confirmation of SP1.4 Fab conversion to full-length molecules was the ability of protein A to precipitate IgG1-SP and IgG4-SP complexed to [¹²⁵I]TPO. IgG1-SP1.4, IgG4-SP1.4, and Fab SP1.4 had similar high affinities for TPO (K_d = 2 x 10^-10 mol/L). Complexes of [¹²⁵I]TPO and IgG1-SP (but not IgG4-SP) bound to peripheral blood mononuclear cells (PBMC), but not to a B cell line. Flow cytometry demonstrated Fc receptors FcRI, FcRII, and FcRIII on PBMC, but only FcRII on the B cell line. Together, these data indicate that IgG1-SP/TPO complexes bind to either FcRI on monocytes or RIII on natural killer cells. In assays for antibody-dependent cytotoxicity using PBMC, ⁵¹Cr release was higher for thyroid cells preincubated with IgG1-SP (13.4%) than with IgG4-SP (2.5%) or with culture medium alone (-0.7%). No specific ⁵¹Cr release was observed when either fibroblasts or Chinese hamster ovary cells expressing cell surface TPO were used as target cells.

In conclusion, a human TPO-specific Fab converted to IgG1, but not IgG4, can mediate cytotoxic effects on human thyroid cells in vitro. These observations support the clinical relevance of TPO autoantibody subclass distribution and emphasize the likelihood that, as opposed to being simple markers of thyroid damage, TPO autoantibodies may play a role in the induction of thyroid dysfunction in vivo.

This article has been cited by other articles:

				S. A. Rebuffat, B. Nguyen, B. Robert, F. Castex, and S. Peraldi-Roux Antithyroperoxidase Antibody-Dependent Cytotoxicity in Autoimmune Thyroid Disease J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 929 - 934. [Abstract] [Full Text] [PDF]

				C.-R. Chen, G. D. Chazenbalk, K. A. Wawrowsky, S. M. McLachlan, and B. Rapoport Evidence that Human Thyroid Cells Express Uncleaved, Single-Chain Thyrotropin Receptors on Their Surface Endocrinology, June 1, 2006; 147(6): 3107 - 3113. [Abstract] [Full Text] [PDF]

				H. P. Ng and A. W. C. Kung Induction of Autoimmune Thyroiditis and Hypothyroidism by Immunization of Immunoactive T Cell Epitope of Thyroid Peroxidase Endocrinology, June 1, 2006; 147(6): 3085 - 3092. [Abstract] [Full Text] [PDF]

				J. Guo, S. M. McLachlan, P. N. Pichurin, C.-R. Chen, N. Pham, H. A. Aliesky, C. S. David, and B. Rapoport Relationship between Thyroid Peroxidase T Cell Epitope Restriction and Antibody Recognition of the Autoantibody Immunodominant Region in Human Leukocyte Antigen DR3 Transgenic Mice Endocrinology, November 1, 2005; 146(11): 4961 - 4967. [Abstract] [Full Text] [PDF]

				D. Bresson, M. Pugniere, F. Roquet, S. A. Rebuffat, B. N-Guyen, M. Cerutti, J. Guo, S. M. McLachlan, B. Rapoport, V. Estienne, et al. Directed Mutagenesis in Region 713-720 of Human Thyroperoxidase Assigns 713KFPED717 Residues as Being Involved in the B Domain of the Discontinuous Immunodominant Region Recognized by Human Autoantibodies J. Biol. Chem., September 10, 2004; 279(37): 39058 - 39067. [Abstract] [Full Text] [PDF]

				H. P. Ng, J. Paul Banga, and A. W. C. Kung Development of a Murine Model of Autoimmune Thyroiditis Induced with Homologous Mouse Thyroid Peroxidase Endocrinology, February 1, 2004; 145(2): 809 - 816. [Abstract] [Full Text] [PDF]

				D. Bresson, M. Cerutti, G. Devauchelle, M. Pugniere, F. Roquet, C. Bes, C. Bossard, T. Chardes, and S. Peraldi-Roux Localization of the Discontinuous Immunodominant Region Recognized by Human Anti-thyroperoxidase Autoantibodies in Autoimmune Thyroid Diseases J. Biol. Chem., March 7, 2003; 278(11): 9560 - 9569. [Abstract] [Full Text] [PDF]

				J. Guo, S. M. McLachlan, and B. Rapoport Localization of the Thyroid Peroxidase Autoantibody Immunodominant Region to a Junctional Region Containing Portions of the Domains Homologous to Complement Control Protein and Myeloperoxidase J. Biol. Chem., October 18, 2002; 277(43): 40189 - 40195. [Abstract] [Full Text] [PDF]

				J. Guo, X.-M. Yan, S. M. McLachlan, and B. Rapoport Search for the Autoantibody Immunodominant Region on Thyroid Peroxidase: Epitopic Footprinting with a Human Monoclonal Autoantibody Locates a Facet on the Native Antigen Containing a Highly Conformational Epitope J. Immunol., January 15, 2001; 166(2): 1327 - 1333. [Abstract] [Full Text] [PDF]

				R. S. McIntosh, M. S. Asghar, E. H. Kemp, P. F. Watson, A. Gardas, J. P. Banga, and A. P. Weetman Analysis of Immunoglobulin G{kappa} Antithyroid Peroxidase Antibodies from Different Tissues in Hashimoto's Thyroiditis J. Clin. Endocrinol. Metab., November 1, 1997; 82(11): 3818 - 3825. [Abstract] [Full Text] [PDF]