BIOCHEMICAL AND MORPHOLOGICAL CHANGES IN HUMAN MAST CELLS

VERA ME; ROJAS RUDOLPH G; YEFI R; MARIANI ML; DE ROSAS JC; FOGAL TH; TONN CE; PIEZZI RS; PENISSI AB

ACTA MICROSCOPICA

CIASEM

Lugar: Caracas; Año: 2009 vol. 18 p. 591 - 592

0798-4545

Mast cells are highly specialized secretory cells that play a central role in innate and adaptative immune responses through the release of a variety of biologically active mediators [1]. LAD 2 is a cell line derived from a patient with mast cell sarcoma/leukemia. These mast cells closely resemble primary cultures of CD34+-derived human mast cells, respond to recombinant human Stem Cell Factor (rhSCF) and have functional Fc£`RI and Fcƒ×RI receptors [2]. Compound 48/80 is known to be one of the most potent mast cell secretagogues which triggers mast cell exocytosis by directly activating the pertussis toxin-sensitive G-proteins [1]. A23187 is a calcium ionophore which activates mast cell degranulation by rising intracellular calcium level. In previous work, we have demonstrated that dehydroleucodine (DhL), a sesquiterpene lactone£`RI and Fcƒ×RI receptors [2]. Compound 48/80 is known to be one of the most potent mast cell secretagogues which triggers mast cell exocytosis by directly activating the pertussis toxin-sensitive G-proteins [1]. A23187 is a calcium ionophore which activates mast cell degranulation by rising intracellular calcium level. In previous work, we have demonstrated that dehydroleucodine (DhL), a sesquiterpene lactone isolated from Artemisia douglasiana Besser (popularly known as ¡§matico¡¨), and xanthatin (Xt), a xanthanolide sesquiterpene isolated from Xanthium cavanillesii Schouw (popularly known as, and xanthatin (Xt), a xanthanolide sesquiterpene isolated from Xanthium cavanillesii Schouw (popularly known as ¡§cadillo¡¨), prevent gastrointestinal damage induced by necrosis-inducing agents. We have also shown that these £\,£]-unsaturated lactones inhibit compound 48/80- and A23187-induced rat peritoneal mast cell degranulation. The present study was designed in order to test the hypothesis that DhL and Xt inhibit LAD 2 mast cell degranulation induced by compound 48/80 or calcium ionophore A23187 [1]. The human LAD 2 cell line was incubated with: 1) Buffer (control group) or 2) compound 48/80 or 3) A23187 or 4) DhL+48/80 or 5) DhL+A23187 or 6) Xt+48/80 or 7) Xt+A23187. LAD-2 £]- hexosaminidase release studies by colorimetric assay, evaluation of mast cell morphology by light microscopy (toluidine blue stain), study of mast cell ultrastructure by transmission and scanning electron microscopy, dose-response (10-100 ƒÝM) and time-response (10, 30 and 60 min) curves, cell viability evaluation (tripan blue dye exclusion test), and comparative studies with sodium cromoglicate (a classical mast cell stabilizer), were carried out. Compound 48/80 and A23187 increased £]-hexosaminidase release from LAD-2 cells and elicited evident granule ultraestructural changes. DhL and Xt inhibited compound 48/80-induced degranulation in a dose- and time-dependent manner. Xt inhibited the A23187 effects in a dose- and time-dependent manner. No changes were observed in DhL+A23187-treated cells. The inhibitory effects of DhL and Xt on ƒÒ-hexosaminidase release were higher than those obtained with the reference compound sodium cromoglicate Crgl when LAD 2 degranulation was induced with compound 48/80. In all cases cell viability was higher than 60%. The present study demonstrates that DhL and Xt inhibit compound 48/80-induced mast cell activation and Xt inhibit A23187-incuced mast cell activation, acting thus as mast cell stabilizers in a human mast cell line., prevent gastrointestinal damage induced by necrosis-inducing agents. We have also shown that these £\,£]-unsaturated lactones inhibit compound 48/80- and A23187-induced rat peritoneal mast cell degranulation. The present study was designed in order to test the hypothesis that DhL and Xt inhibit LAD 2 mast cell degranulation induced by compound 48/80 or calcium ionophore A23187 [1]. The human LAD 2 cell line was incubated with: 1) Buffer (control group) or 2) compound 48/80 or 3) A23187 or 4) DhL+48/80 or 5) DhL+A23187 or 6) Xt+48/80 or 7) Xt+A23187. LAD-2 £]- hexosaminidase release studies by colorimetric assay, evaluation of mast cell morphology by light microscopy (toluidine blue stain), study of mast cell ultrastructure by transmission and scanning electron microscopy, dose-response (10-100 ƒÝM) and time-response (10, 30 and 60 min) curves, cell viability evaluation (tripan blue dye exclusion test), and comparative studies with sodium cromoglicate (a classical mast cell stabilizer), were carried out. Compound 48/80 and A23187 increased £]-hexosaminidase release from LAD-2 cells and elicited evident granule ultraestructural changes. DhL and Xt inhibited compound 48/80-induced degranulation in a dose- and time-dependent manner. Xt inhibited the A23187 effects in a dose- and time-dependent manner. No changes were observed in DhL+A23187-treated cells. The inhibitory effects of DhL and Xt on ƒÒ-hexosaminidase release were higher than those obtained with the reference compound sodium cromoglicate Crgl when LAD 2 degranulation was induced with compound 48/80. In all cases cell viability was higher than 60%. The present study demonstrates that DhL and Xt inhibit compound 48/80-induced mast cell activation and Xt inhibit A23187-incuced mast cell activation, acting thus as mast cell stabilizers in a human mast cell line.£\,£]-unsaturated lactones inhibit compound 48/80- and A23187-induced rat peritoneal mast cell degranulation. The present study was designed in order to test the hypothesis that DhL and Xt inhibit LAD 2 mast cell degranulation induced by compound 48/80 or calcium ionophore A23187 [1]. The human LAD 2 cell line was incubated with: 1) Buffer (control group) or 2) compound 48/80 or 3) A23187 or 4) DhL+48/80 or 5) DhL+A23187 or 6) Xt+48/80 or 7) Xt+A23187. LAD-2 £]- hexosaminidase release studies by colorimetric assay, evaluation of mast cell morphology by light microscopy (toluidine blue stain), study of mast cell ultrastructure by transmission and scanning electron microscopy, dose-response (10-100 ƒÝM) and time-response (10, 30 and 60 min) curves, cell viability evaluation (tripan blue dye exclusion test), and comparative studies with sodium cromoglicate (a classical mast cell stabilizer), were carried out. Compound 48/80 and A23187 increased £]-hexosaminidase release from LAD-2 cells and elicited evident granule ultraestructural changes. DhL and Xt inhibited compound 48/80-induced degranulation in a dose- and time-dependent manner. Xt inhibited the A23187 effects in a dose- and time-dependent manner. No changes were observed in DhL+A23187-treated cells. The inhibitory effects of DhL and Xt on ƒÒ-hexosaminidase release were higher than those obtained with the reference compound sodium cromoglicate Crgl when LAD 2 degranulation was induced with compound 48/80. In all cases cell viability was higher than 60%. The present study demonstrates that DhL and Xt inhibit compound 48/80-induced mast cell activation and Xt inhibit A23187-incuced mast cell activation, acting thus as mast cell stabilizers in a human mast cell line.£]- hexosaminidase release studies by colorimetric assay, evaluation of mast cell morphology by light microscopy (toluidine blue stain), study of mast cell ultrastructure by transmission and scanning electron microscopy, dose-response (10-100 ƒÝM) and time-response (10, 30 and 60 min) curves, cell viability evaluation (tripan blue dye exclusion test), and comparative studies with sodium cromoglicate (a classical mast cell stabilizer), were carried out. Compound 48/80 and A23187 increased £]-hexosaminidase release from LAD-2 cells and elicited evident granule ultraestructural changes. DhL and Xt inhibited compound 48/80-induced degranulation in a dose- and time-dependent manner. Xt inhibited the A23187 effects in a dose- and time-dependent manner. No changes were observed in DhL+A23187-treated cells. The inhibitory effects of DhL and Xt on ƒÒ-hexosaminidase release were higher than those obtained with the reference compound sodium cromoglicate Crgl when LAD 2 degranulation was induced with compound 48/80. In all cases cell viability was higher than 60%. The present study demonstrates that DhL and Xt inhibit compound 48/80-induced mast cell activation and Xt inhibit A23187-incuced mast cell activation, acting thus as mast cell stabilizers in a human mast cell line.ƒÝM) and time-response (10, 30 and 60 min) curves, cell viability evaluation (tripan blue dye exclusion test), and comparative studies with sodium cromoglicate (a classical mast cell stabilizer), were carried out. Compound 48/80 and A23187 increased £]-hexosaminidase release from LAD-2 cells and elicited evident granule ultraestructural changes. DhL and Xt inhibited compound 48/80-induced degranulation in a dose- and time-dependent manner. Xt inhibited the A23187 effects in a dose- and time-dependent manner. No changes were observed in DhL+A23187-treated cells. The inhibitory effects of DhL and Xt on ƒÒ-hexosaminidase release were higher than those obtained with the reference compound sodium cromoglicate Crgl when LAD 2 degranulation was induced with compound 48/80. In all cases cell viability was higher than 60%. The present study demonstrates that DhL and Xt inhibit compound 48/80-induced mast cell activation and Xt inhibit A23187-incuced mast cell activation, acting thus as mast cell stabilizers in a human mast cell line.£]-hexosaminidase release from LAD-2 cells and elicited evident granule ultraestructural changes. DhL and Xt inhibited compound 48/80-induced degranulation in a dose- and time-dependent manner. Xt inhibited the A23187 effects in a dose- and time-dependent manner. No changes were observed in DhL+A23187-treated cells. The inhibitory effects of DhL and Xt on ƒÒ-hexosaminidase release were higher than those obtained with the reference compound sodium cromoglicate Crgl when LAD 2 degranulation was induced with compound 48/80. In all cases cell viability was higher than 60%. The present study demonstrates that DhL and Xt inhibit compound 48/80-induced mast cell activation and Xt inhibit A23187-incuced mast cell activation, acting thus as mast cell stabilizers in a human mast cell line.ƒÒ-hexosaminidase release were higher than those obtained with the reference compound sodium cromoglicate Crgl when LAD 2 degranulation was induced with compound 48/80. In all cases cell viability was higher than 60%. The present study demonstrates that DhL and Xt inhibit compound 48/80-induced mast cell activation and Xt inhibit A23187-incuced mast cell activation, acting thus as mast cell stabilizers in a human mast cell line.