Isolation and characterization of proinsulin-producing medullary thymic epithelial cell clones.
Proinsulin, like many tissue-specific antigens, is expressed by rare (1-3%) cells of the thymus medullary stroma, presumably for the purpose of self-tolerance. Levels of this expression are associated with type 1 diabetes susceptibility in humans and in the NOD mouse. To further understand the mechanism of central tolerance induction by these rare cells, we have isolated and cultured two proinsulin-producing epithelial cell clones from murine thymus. These cells have a typical epithelial morphology and, by flow cytometry, a surface phenotype representative of mature thymic medullary epithelial cells (G8.8(+)/UEA-1(+)/DEC205(-)/CD45(-)/MHC II(+)). By RT-PCR, they express predominantly Ins2 as opposed to Ins1, as does whole thymus. Expression of the transcription factor Aire, implicated in enhancing promiscuous thymic expression of tissue-specific antigens, fell to very low levels after a few passages but increased 20-fold upon exposure to an agonistic anti-lymphotoxin B antibody, concurrent with 2.5-fold enhanced insulin expression. RNA of Pdx-1, Glut-2, and Gck was detectable by RT-PCR in whole thymus but not in the clones, suggesting thymic proinsulin expression is Pdx-1 independent and that Pdx-1, Glut-2, and Gck are likely expressed in the thymus as antigens, not as regulatory molecules.
Palumbo
Michael O
MO
McGill University Health Center, Montreal Children's Hospital, Research Institute, Montreal, Quebec, Canada H3H 1P3.
Levi
Dina
D
Chentoufi
Aziz Alami
AA
Polychronakos
Constantin
C
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002469:Cell Separation; D002478:Cells, Cultured; D002999:Clone Cells; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D005941:Glucokinase / Q000096:biosynthesis; D051273:Glucose Transporter Type 2 / Q000096:biosynthesis; D018398:Homeodomain Proteins / Q000096:biosynthesis; D007328:Insulin / Q000096:biosynthesis*; D008233:Lymphotoxin-alpha / Q000276:immunology; D053308:Lymphotoxin-beta; D008565:Membrane Proteins / Q000276:immunology; D051379:Mice; D011384:Proinsulin / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013950:Thymus Gland / Q000166:cytology* / Q000378:metabolism; D015534:Trans-Activators / Q000096:biosynthesis; D014157:Transcription Factors / Q000096:biosynthesis; D000096927:AIRE Protein
D000906:Antibodies; D005941:Glucokinase; D051273:Glucose Transporter Type 2; D018398:Homeodomain Proteins; D007328:Insulin; D008233:Lymphotoxin-alpha; D053308:Lymphotoxin-beta; D008565:Membrane Proteins; D011384:Proinsulin; D015534:Trans-Activators; D014157:Transcription Factors; D000096927:AIRE Protein; C505887:Ltb protein, mouse; C495682:Slc2a2 protein, mouse; C084010:pancreatic and duodenal homeobox 1 protein