RESUMO
pl6l is a membrane glycoprotein expressed on mast cells and on activated macrophages but on few if any other cells of hematopoietic lineages. Its lack of expression on basophils makes it useful to distinguish mast cells from basophils and aids in the analysis of mast cells and their precursors. p161 was purified from the mast cell line CFTL-12 by affinity chromatography and subjected to limited proteolysis. The sequences of the resultant peptides indicated that p161 is homologous with rat and human CD13/aminopeptidase N. Using oligonucleotide primers derived from rat CD13 cDNA, a mouse cDNA was obtained. Its deduced amino acid sequence displays 87% identity with rat CD13 and 76 % identity with human CD13. Expression of the mouse cDNA in M12 cells, which are p161 negative, renders these cells positive for staining with the monoclonal anti-p161 Ab, K-1. Furthermore, a mAb raised against partially purified mouse intestinal aminopeptidase N specifically blocked the binding of K-1 to both CFTL-12 cells and the transfected M12 cells. These results strongly indicate that mouse p161 is CD13/aminopeptidase N. Northern blot analysis shows that p161 mRNA is most abundantly expressed in the intestinal tract and kidney and is present in liver, lymph node, spleen, and brain.
Assuntos
Antígenos CD13/biossíntese , Antígenos CD13/isolamento & purificação , Macrófagos/enzimologia , Macrófagos/imunologia , Mastócitos/enzimologia , Mastócitos/imunologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígenos CD13/genética , Humanos , Macrófagos/metabolismo , Mastócitos/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Ratos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
A population of cells that express mast cell markers, including the membrane protein p161, but that lack expression of the high affinity IgE receptor, Fc epsilon RI, can be routinely grown from bone marrow. Ionomycin, but not IgE immune complexes, causes these cells to release serotonin and to express IL-3 and IL-13 mRNA, consistent with their being FC epsilon RI-deficient mast cells. These p161+/Fc epsilon RI- mast cells expressed normal amounts of Fc epsilon RI alpha and beta chain mRNA, but extremely low levels of Fc epsilon RI gamma chain mRNA. In addition, this novel mast cell population expressed CD3 zeta chain mRNA, which p161+/Fc epsilon RI+ mast cells did not. CD3 zeta stable transfectants of Abelson-murine leukemia virus-transformed p161+/Fc epsilon RI+ mast cells continued to express Fc epsilon RI. This strongly suggests that the failure of p161+/Fc epsilon RI- mast cells to express IgE receptors was not caused by the presence of CD3 zeta chain. Transfection of human Fc epsilon RI gamma cDNA into p161+/Fc epsilon RI- mast cells rescued IgE binding. These stable transfectants released serotonin in response to cross-linkage of Fc epsilon RI, demonstrating that the molecular defect of p161+/Fc epsilon RI- mast cells is indeed the loss of Fc epsilon RI gamma expression.
Assuntos
Imunoglobulina E/metabolismo , Mastócitos/fisiologia , Receptores de IgE/genética , Animais , Sequência de Bases , Células da Medula Óssea , Complexo CD3/genética , Primers do DNA/química , Expressão Gênica , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro/genética , Transdução de Sinais , TransfecçãoRESUMO
A monoclonal hamster antibody (K-1) specific for a 161-kD mast cell surface glycoprotein was derived. p161 is expressed on normal and cultured mast cells and on some macrophages, but not on basophils or other hematopoietic cells. A population of Fc epsilon Rneg cells expressing p161 was found in short term cultures of bone marrow cells grown in interleukin (IL)-3. These cells were purified and propagated for extended periods in IL-3. They express c-kit and Fc gamma RII/III, contain alcian blue-positive granules and histamine, and secrete IL-3 in response to ionomycin treatment. Their morphology is consistent with that of mast cells. We propose that they represent Fc epsilon RIneg mast cells that can be detected and purified because of their p161 expression.
Assuntos
Anticorpos Monoclonais/imunologia , Mastócitos/imunologia , Receptores de IgE/metabolismo , Animais , Antígenos de Superfície/análise , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Imunofenotipagem , Técnicas In Vitro , Macrófagos Peritoneais/imunologia , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBARESUMO
Previous studies demonstrated a direct action of interleukin-1 (IL-1) on release of hormones from rat anterior pituitary cells in monolayer culture. To rule out any possibility of a paracrine effect from the elevated hormones in the static monolayer system, and to examine further the dynamics of hormone release elicited by IL-1, studies were conducted with rat anterior pituitary tissue in a computer-controlled automated perifusion system. In experiments performed on the same day as sacrifice, IL-1 stimulated the release of adrenocorticotrophic hormone (ACTH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), growth hormone (GH) and prolactin (PRL) in a dose-related manner. Peak levels were achieved within 6 minutes of exposure to IL-1. However, PRL was not increased over the baseline fluctuations when pituitaries were perifused with IL-1 after 72 hours of incubation. Hormone release did not appear to undergo desensitization after multiple short pulses of IL-1. Heat-denatured IL-1 had no effect on hormone release. The rapid response suggests that IL-1 acts acutely to release preformed hormone stores.
