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Self-reactive B cells are not eliminated or inactivated by autoantigen expressed on thyroid epithelial cells.

Akkaraju S, Canaan K, Goodnow CC - J. Exp. Med. (1997)

Bottom Line: Nature. 353:765-769), selective expression of mHEL autoantigen on thyroid cells did not trigger elimination or inactivation of circulating HEL-reactive B cells.These results provide evidence that tolerance is not actively acquired to organ-specific antigens in the preimmune B cell repertoire, underscoring the importance of maintaining tolerance to such antigens by other mechanisms.The role of an intact endothelial barrier in sequestering organ-specific antigens from circulating preimmune B cells is discussed.

View Article: PubMed Central - PubMed

Affiliation: Program in Immunology, Department of Microbiology and Immunology, and The Howard Hughes Medical Institute, Beckman Center, Stanford University School of Medicine, Stanford, California 94305-5428, USA.

ABSTRACT
Graves' Disease results from the production of autoantibodies against receptors for thyroid stimulating hormone (TSH) on thyroid epithelial cells, and represents the prototype for numerous autoimmune diseases caused by autoantibodies that bind to organ-specific cell membrane antigens. To study how humoral tolerance is normally maintained to organ-specific membrane antigens, transgenic mice were generated selectively expressing membrane-bound hen egg lysozyme (mHEL) on the thyroid epithelium. In contrast to the deletion of autoreactive B cells triggered by systemic mHEL (Hartley, S.B., J. Crosbie, R. Brink, A.B. Kantor, A. Basten, and C.C. Goodnow. 1991. Nature. 353:765-769), selective expression of mHEL autoantigen on thyroid cells did not trigger elimination or inactivation of circulating HEL-reactive B cells. These results provide evidence that tolerance is not actively acquired to organ-specific antigens in the preimmune B cell repertoire, underscoring the importance of maintaining tolerance to such antigens by other mechanisms. The role of an intact endothelial barrier in sequestering organ-specific antigens from circulating preimmune B cells is discussed.

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C57BL/6 transgenic mice expressing mHEL on  thyroid epithelium. (A) Structure  of microinjected hybrid gene  containing 3.3 kb of the rTg promoter attached to the HEL gene,  to which the H-2Kb transmembrane domain (Kb TM) has been  added. X, XbaI; H, HindIII; C,  ClaI; K, KpnI. (B) RT/PCR  analysis of various tissues from  nontransgenic, TLK-2 transgenic, and KLK-4 transgenic  mice. KLK-4 mice express  mHEL in all tissues under the Kb  MHC class I promoter. Oligo-dT–primed cDNA was made  from the following tissue RNA:  1, spleen; 2, liver; 3, thymus; 4,  heart; 5, kidney; 6, thyroid. PCR  was performed using cDNA template and specific primers for  both HEL and actin in one reaction. (C) Immunohistochemical  staining for HEL in TLK-1 and  TLK-2 thyroids. 10-μm frozen sections of one thyroid lobe from each line was stained using a biotinylated anti-lysozyme mAb, streptavidin-alkaline phosphatase secondary stage, and Fast red substrate (red). Hematoxylin (blue) was used for counter-staining. (D) Flow cytometric analysis of thyrocytes from  nontransgenic (thin line) or TLK-2 transgenic (thick line) mice stained for cell surface HEL. The percent positive cells is indicated.
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Figure 1: C57BL/6 transgenic mice expressing mHEL on thyroid epithelium. (A) Structure of microinjected hybrid gene containing 3.3 kb of the rTg promoter attached to the HEL gene, to which the H-2Kb transmembrane domain (Kb TM) has been added. X, XbaI; H, HindIII; C, ClaI; K, KpnI. (B) RT/PCR analysis of various tissues from nontransgenic, TLK-2 transgenic, and KLK-4 transgenic mice. KLK-4 mice express mHEL in all tissues under the Kb MHC class I promoter. Oligo-dT–primed cDNA was made from the following tissue RNA: 1, spleen; 2, liver; 3, thymus; 4, heart; 5, kidney; 6, thyroid. PCR was performed using cDNA template and specific primers for both HEL and actin in one reaction. (C) Immunohistochemical staining for HEL in TLK-1 and TLK-2 thyroids. 10-μm frozen sections of one thyroid lobe from each line was stained using a biotinylated anti-lysozyme mAb, streptavidin-alkaline phosphatase secondary stage, and Fast red substrate (red). Hematoxylin (blue) was used for counter-staining. (D) Flow cytometric analysis of thyrocytes from nontransgenic (thin line) or TLK-2 transgenic (thick line) mice stained for cell surface HEL. The percent positive cells is indicated.

Mentions: Transgenic mice expressing membrane-bound HEL on the thyroid epithelium were produced by microinjecting C57BL/6 eggs with a gene construct containing 3.3 kb of the rat thyroglobulin promoter (24) linked to the membrane HEL gene (Fig. 1 a). Two transgenic lines, TLK-1 and TLK-2, were established, and mice from either line exhibited normal size, body weight, and fecundity, indicating that the transgene did not induce overt thyroid insufficiency. RT-PCR analysis of various tissues revealed that most or all mHEL RNA expression is restricted to the thyroid (Fig. 1 b). Immunohistochemical analysis of thyroid gland from both lines revealed abundant HEL expression on many follicular epithelial cells (Fig. 1 c). Western blotting showed that the membrane-bound form of HEL was made in large quantities in the TLK-2 thyroid (25), and FACS® staining of thyrocytes confirmed surface expression of HEL (Fig. 1 d). There was no evidence of inflammation in any of the mice, and follicular architecture was normal except in a fraction of older mice from the TLK-2 line (data not shown). The latter animals displayed moderately enlarged thyroids, with follicles that appeared hyperplastic and consisted of cuboidal epithelium, although there was no evidence for inflammation. Because the TLK-2 line expresses higher levels of HEL than TLK-1, the cytopathology present in a fraction of older TLK-2 animals may be a direct effect of transgene expression, as described previously (26–28), but appears not to affect acquisition of immune tolerance (see below).


Self-reactive B cells are not eliminated or inactivated by autoantigen expressed on thyroid epithelial cells.

Akkaraju S, Canaan K, Goodnow CC - J. Exp. Med. (1997)

C57BL/6 transgenic mice expressing mHEL on  thyroid epithelium. (A) Structure  of microinjected hybrid gene  containing 3.3 kb of the rTg promoter attached to the HEL gene,  to which the H-2Kb transmembrane domain (Kb TM) has been  added. X, XbaI; H, HindIII; C,  ClaI; K, KpnI. (B) RT/PCR  analysis of various tissues from  nontransgenic, TLK-2 transgenic, and KLK-4 transgenic  mice. KLK-4 mice express  mHEL in all tissues under the Kb  MHC class I promoter. Oligo-dT–primed cDNA was made  from the following tissue RNA:  1, spleen; 2, liver; 3, thymus; 4,  heart; 5, kidney; 6, thyroid. PCR  was performed using cDNA template and specific primers for  both HEL and actin in one reaction. (C) Immunohistochemical  staining for HEL in TLK-1 and  TLK-2 thyroids. 10-μm frozen sections of one thyroid lobe from each line was stained using a biotinylated anti-lysozyme mAb, streptavidin-alkaline phosphatase secondary stage, and Fast red substrate (red). Hematoxylin (blue) was used for counter-staining. (D) Flow cytometric analysis of thyrocytes from  nontransgenic (thin line) or TLK-2 transgenic (thick line) mice stained for cell surface HEL. The percent positive cells is indicated.
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Figure 1: C57BL/6 transgenic mice expressing mHEL on thyroid epithelium. (A) Structure of microinjected hybrid gene containing 3.3 kb of the rTg promoter attached to the HEL gene, to which the H-2Kb transmembrane domain (Kb TM) has been added. X, XbaI; H, HindIII; C, ClaI; K, KpnI. (B) RT/PCR analysis of various tissues from nontransgenic, TLK-2 transgenic, and KLK-4 transgenic mice. KLK-4 mice express mHEL in all tissues under the Kb MHC class I promoter. Oligo-dT–primed cDNA was made from the following tissue RNA: 1, spleen; 2, liver; 3, thymus; 4, heart; 5, kidney; 6, thyroid. PCR was performed using cDNA template and specific primers for both HEL and actin in one reaction. (C) Immunohistochemical staining for HEL in TLK-1 and TLK-2 thyroids. 10-μm frozen sections of one thyroid lobe from each line was stained using a biotinylated anti-lysozyme mAb, streptavidin-alkaline phosphatase secondary stage, and Fast red substrate (red). Hematoxylin (blue) was used for counter-staining. (D) Flow cytometric analysis of thyrocytes from nontransgenic (thin line) or TLK-2 transgenic (thick line) mice stained for cell surface HEL. The percent positive cells is indicated.
Mentions: Transgenic mice expressing membrane-bound HEL on the thyroid epithelium were produced by microinjecting C57BL/6 eggs with a gene construct containing 3.3 kb of the rat thyroglobulin promoter (24) linked to the membrane HEL gene (Fig. 1 a). Two transgenic lines, TLK-1 and TLK-2, were established, and mice from either line exhibited normal size, body weight, and fecundity, indicating that the transgene did not induce overt thyroid insufficiency. RT-PCR analysis of various tissues revealed that most or all mHEL RNA expression is restricted to the thyroid (Fig. 1 b). Immunohistochemical analysis of thyroid gland from both lines revealed abundant HEL expression on many follicular epithelial cells (Fig. 1 c). Western blotting showed that the membrane-bound form of HEL was made in large quantities in the TLK-2 thyroid (25), and FACS® staining of thyrocytes confirmed surface expression of HEL (Fig. 1 d). There was no evidence of inflammation in any of the mice, and follicular architecture was normal except in a fraction of older mice from the TLK-2 line (data not shown). The latter animals displayed moderately enlarged thyroids, with follicles that appeared hyperplastic and consisted of cuboidal epithelium, although there was no evidence for inflammation. Because the TLK-2 line expresses higher levels of HEL than TLK-1, the cytopathology present in a fraction of older TLK-2 animals may be a direct effect of transgene expression, as described previously (26–28), but appears not to affect acquisition of immune tolerance (see below).

Bottom Line: Nature. 353:765-769), selective expression of mHEL autoantigen on thyroid cells did not trigger elimination or inactivation of circulating HEL-reactive B cells.These results provide evidence that tolerance is not actively acquired to organ-specific antigens in the preimmune B cell repertoire, underscoring the importance of maintaining tolerance to such antigens by other mechanisms.The role of an intact endothelial barrier in sequestering organ-specific antigens from circulating preimmune B cells is discussed.

View Article: PubMed Central - PubMed

Affiliation: Program in Immunology, Department of Microbiology and Immunology, and The Howard Hughes Medical Institute, Beckman Center, Stanford University School of Medicine, Stanford, California 94305-5428, USA.

ABSTRACT
Graves' Disease results from the production of autoantibodies against receptors for thyroid stimulating hormone (TSH) on thyroid epithelial cells, and represents the prototype for numerous autoimmune diseases caused by autoantibodies that bind to organ-specific cell membrane antigens. To study how humoral tolerance is normally maintained to organ-specific membrane antigens, transgenic mice were generated selectively expressing membrane-bound hen egg lysozyme (mHEL) on the thyroid epithelium. In contrast to the deletion of autoreactive B cells triggered by systemic mHEL (Hartley, S.B., J. Crosbie, R. Brink, A.B. Kantor, A. Basten, and C.C. Goodnow. 1991. Nature. 353:765-769), selective expression of mHEL autoantigen on thyroid cells did not trigger elimination or inactivation of circulating HEL-reactive B cells. These results provide evidence that tolerance is not actively acquired to organ-specific antigens in the preimmune B cell repertoire, underscoring the importance of maintaining tolerance to such antigens by other mechanisms. The role of an intact endothelial barrier in sequestering organ-specific antigens from circulating preimmune B cells is discussed.

Show MeSH
Related in: MedlinePlus