RESUMO
BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of glycan-binding inhibitory receptors, and among them, Siglec-8 is selectively expressed on human eosinophils, basophils, and mast cells. On eosinophils, Siglec-8 engagement induces apoptosis, but its function on mast cells is unknown. OBJECTIVE: We sought to study the effect of Siglec-8 engagement on human mast cell survival and mediator release responses. METHODS: Human mast cells were generated from CD34+ precursors. Apoptosis was studied by using flow cytometry. Mast cell mediator release or human lung airway smooth muscle contraction was initiated by FcepsilonRI cross-linking with or without preincubation with Siglec-8 or control antibodies, and release of mediators was analyzed along with Ca++ flux. RBL-2H3 cells transfected with normal and mutated forms of Siglec-8 were used to study how Siglec-8 engagement alters mediator release. RESULTS: Siglec-8 engagement failed to induce human mast cell apoptosis. However, preincubation with Siglec-8 mAbs significantly (P < .05) inhibited FcepsilonRI-dependent histamine and prostaglandin D(2) release, Ca++ flux, and anti-IgE-evoked contractions of human bronchial rings. In contrast, release of IL-8 was not inhibited. Siglec-8 ligation was also shown to inhibit beta-hexosaminidase release and Ca++ flux triggered through FcepsilonRI in RBL-2H3 cells transfected with full-length human Siglec-8 but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal immunoreceptor tyrosine-based inhibitory motif domain. CONCLUSION: These data represent the first reported inhibitory effects of Siglec engagement on human mast cells.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Cálcio/metabolismo , Lectinas/metabolismo , Mastócitos/metabolismo , Receptores de IgE/antagonistas & inibidores , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Brônquios/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Liberação de Histamina , Humanos , Interleucina-8/metabolismo , Lectinas/genética , Mastócitos/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Prostaglandina D2/metabolismo , Receptores de IgE/metabolismo , Transfecção , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Adhesion molecules and chemokines contribute to selective eosinophil recruitment in allergic inflammation. In this study, we examined the effects of eotaxin-2, a CCR3-specific chemokine, on integrin-mediated eosinophil adhesion to vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), or both using a parallel plate flow system. Tissue culture plates were coated with various combinations of VCAM-1, ICAM-1, and/or eotaxin-2. Human eosinophils were infused at physiologic shear stress (0.5 dyn/cm(2)) for 10 min, and the numbers of attached eosinophils were monitored using video microscopy. Cells accumulated efficiently on VCAM-1 and even better on surfaces co-coated with VCAM-1 and ICAM-1, but poorly on surfaces coated with ICAM-1 or bovine serum albumin alone. When eotaxin-2 was co-immobilized with adhesion proteins, fewer cells adhered to VCAM-1 and more adhered to ICAM-1, whereas levels of attachment to VCAM-1 plus ICAM-1 showed no net change. However, experiments with adhesion molecule blocking monoclonal antibody showed that the contribution of ICAM-1-mediated adhesion was always greater if eotaxin-2 was present. Pretreatment of cells with a CCR3-blocking mAb, or PD98059, a MAP-kinase inhibitor, prevented the eotaxin-2-induced changes in eosinophil attachment. These data suggest that eotaxin-2, acting via MAP kinases, may facilitate eosinophil recruitment at sites of allergic inflammation by shifting their adhesion molecule usage away from VCAM-1-dominated to ICAM-1-dominated pathways.
Assuntos
Quimiocinas CC/fisiologia , Eosinófilos/fisiologia , Integrinas/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Quimiocina CCL24 , Quimiocinas CC/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The inherent electron-capture properties of triamcinolone acetonide (TAA) fatty acid conjugates were exploited for development of a GC-MS technique for quantitation of C21 long-chain fatty esters of TAA synthesized in BEAS-2B cells, an immortalized airway epithelium cell line. TAA esters extracted from BEAS-2B cells were purified and detected via selected ion monitoring of the molecular anions generated from the TAA esters under electron-capture negative-ion mass spectrometric conditions. Standard curves were linear over a range of 0.0 to >4.5 ng/mg protein with r(2) values = 1. Levels of TAA conjugates extracted from BEAS-2B treated with 10(-5)M TAA for 24h ranged from 0.024 to 0.301 ng/mg protein. Further evidence for confirmation of the identity of TAA fatty esters formed in BEAS-2B cells was obtained via selected reaction monitoring. The transition monitored was formation of the carboxy anion generated from each of the respective molecular anions of the TAA esters during collision-induced decomposition. These findings indicate that the GC-MS analysis is suitable for studies of the kinetics of the TAA fatty acid conjugates formation in vitro and may be directly applicable to determination of the kinetics of TAA fatty acid conjugation in vivo.