ABSTRACT
TSH receptor (R) binding and cAMP production by bovine (b) TSH-bound to a monoclonal antibody (MoAb) or polyclonal antibody (Ab) to bTSH were examined, using TSH receptor (R) coating tube and porcine thyroid cells. (125) I-bTSH bound-to MoAbs to bTSH(alpha) or discontinuous type MoAb showed TSHR binding (10%) similar to intact (125) I-bTSH. TSHR binding was completely decreased (<2%) when (125) I-bTSH was bound by polyclonal Abs to bTSH(alpha) in Graves' patient or rabbit polyclonal Abs to bTSH. When either of the two MoAb (No. 1 and 2) to bTSH(beta) was bound to (125) I-bTSH, TSHR binding was 4 times higher (40%) compared to intact (125) I-bTSH. Binding of another MoAb (No. 3) caused no increased binding. TSHR binding of intact (125) I-bTSH was decreased from 10% to 2% by excess amounts of bTSH. Binding of (125) I-bTSH bound to MoAb to bTSH(beta) (No. 1 and 2) decreased from 40% to 30% by excess amounts of bTSH. When (125) I-bTSH bound-Fab of MoAb was used, the binding was reduced from 30 to 10% (No. 1) and from 25 to 6% (No. 2), respectively. In contrast, cAMP production by bTSH was decreased by pre-binding of all MoAbs and polyclonal Abs. Binding of (125) I-MoAb to bTSH (beta) to a synthetic peptide array of bTSH (beta) sequence was examined by the radioautography. The epitope of MoAb to bTSH(beta) was suggested to be LPK (beta 42-44) for No. 1, KLF (beta 39-41) for No. 2 and PKYA (beta 43-46) for No. 3, respectively, although the existence of discontinuous epitope could not be ruled out. The increased TSHR binding and the decreased cAMP production by bTSH bound to MoAbs may be due to the conformational change of TSH molecule or TSHR by binding of both bTSH and MoAb.
Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Receptors, Thyrotropin/metabolism , Thyrotropin, beta Subunit/immunology , Thyrotropin/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/isolation & purification , Cattle , Cyclic AMP/metabolism , Epitope Mapping/methods , Graves Disease/blood , Graves Disease/immunology , Humans , Iodine Radioisotopes/pharmacokinetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Array Analysis , Protein Binding/drug effects , Protein Conformation , Receptors, Thyrotropin/chemistryABSTRACT
Immunofluorescent staining of thyroid tissues was done using monoclonal antibodies to dendritic cell (DC), lymphocyte, macrophage and granulocyte markers. Despite the presence of occasional CD11c+ cells, CD11b+ cells, morphologically characteristic of DCs, were abundant in thyroid of normal mice, at a density of approximately 2.0 cells per thyroid follicle, and were >tenfold more frequent than CD11c+ cells. Thyroid tissues were non-reactive with antibodies to F4/80, CD8alpha, CD40, CD80, Gr-1, CD3, or CD19, indicating that the CD11b+ cells were not macrophages, activated DCs, granulocytes, plasmacytoid DCs, T cells or B cells. Following systemic immune activation, DCs in secondary lymphoid tissues but not in the thyroid, upregulated CD80 expression. Using radiation chimeras made from bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice, EGFP+ DC-like cells were present in the thyroid from 1-20 weeks after bone marrow transfer, but were rare in the kidney and liver, although EGFP+ cells were present in secondary lymphoid tissues. Additionally, DCs generated from EGFP+ bone marrow cells localized in the thyroid of EGFP- mice following adoptive transfer. Double staining of thyroid tissue sections with antibodies to the thyroid stimulating hormone (TSH)-beta molecule and to CD11b revealed co-expression of TSHbeta and CD11b among intrathyroidal DCs. Moreover, RT-PCR analyses indicated expression of the TSHbeta gene in thyroid tissues. These findings define a novel bone marrow-derived hematopoietic cell population that resides in the thyroid of normal mice, which may have a unique role in the microregulation of thyroid physiology and homeostasis.
