RESUMEN
Microparticles (MPs) are membrane-bound vesicles shed normally or as a result of various (pathological) stimuli. MPs contain a wealth of bio-active macromolecules. Aminophospholipid phosphatidylserine (PS) is present on the surface of many MPs. As PS and phosphatidylethanolamine (PE) are related, yet distinct aminophospholipids, the purpose of this study was to systematically and directly assess PE exposure on MPs. We examined MPs from various human cellular sources (human breast cancer, endothelial, red and white blood cells) by flow cytometry using a PE-specific probe, duramycin, and two PS-specific probes, annexin V and lactadherin. PS and PE exposure percentage was comparable on vascular and blood cell-derived MPs (80-90% of MP-gated events). However, the percentage of malignant breast cancer MPs exposing PE (~90%) was significantly higher than PS (~50%). Thus, while PS and PE exposure can result from a general loss of membrane asymmetry, there may also be distinct mechanisms of PE and PS exposure on MPs that vary by cellular source.
Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Células Endoteliales/metabolismo , Eritrocitos/metabolismo , Fosfatidiletanolaminas/metabolismo , Bacteriocinas/química , Bacteriocinas/metabolismo , Bacteriocinas/farmacología , Biotina/química , Coagulación Sanguínea/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Microscopía Confocal , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Fosfatidiletanolaminas/químicaRESUMEN
Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.
Asunto(s)
Antineoplásicos/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Endocannabinoides , Glicéridos/farmacología , Neoplasias de la Próstata/metabolismo , Anilidas/farmacología , Línea Celular Tumoral , Proliferación Celular , Ciclina D1/metabolismo , Ciclina E/metabolismo , Humanos , Masculino , FN-kappa B/metabolismo , PPAR gamma/antagonistas & inhibidores , PPAR gamma/metabolismo , Próstata/patología , Neoplasias de la Próstata/patologíaRESUMEN
CD 200 is a widely expressed transmembrane glycoprotein that transmits an inhibitory signal after ligation of the structurally homologous CD 200-receptor-1 (CD 200 R1). Recently, we showed that CD 200 is expressed on keratinocytes and plays a role in protecting hair follicles from autoimmune attack. Here, we report the characterization of cell surface and mRNA expression of CD 200 R1 by cells of the murine epidermis. In addition, we report mRNA expression for other members of the CD 200 R-family (R2-R4) by quantitative real-time RT-PCR. Variable levels of CD 200 R1, R2, R3, and R4 mRNA were detected in bulk epidermal cell suspensions. Freshly isolated Langerhans cells (LC) preferentially expressed CD 200 R1. Consistent with an inhibitory role for CD 200:CD 200 R1 interaction, LC obtained from mice deficient in CD 200 (CD 200(-/-)) were in a heightened state of activation as compared with wild-type (CD 200(+/+)) cells. Freshly isolated dendritic epidermal T cells (DETC) expressed low levels of CD 200 R1, R2, and R3 mRNA, but they preferentially increased cell surface and mRNA expression of CD 200 R1 upon activation in vitro. In functional assays using sub-optimal CD3 signaling, immobilized CD 200 inhibited DETC proliferation and cytokine secretion. Collectively, these results suggest that CD 200:CD 200 R interactions may play a role in regulating both LC and DETC in cutaneous immune reactions.
Asunto(s)
Antígenos de Superficie/genética , Epidermis/fisiología , Glicoproteínas de Membrana/genética , Receptores de Superficie Celular/genética , Animales , Línea Celular , Células Dendríticas/inmunología , Oído , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Orexina , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Proteínas Recombinantes de Fusión , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
CD200 (OX-2) is a transmembrane glycoprotein that transmits an immunoregulatory signal through the CD200 receptor (CD200R) to attenuate inflammatory reactions and promote immune tolerance. CD200 expression in the skin has not been described previously. We now report that freshly isolated cells of the murine epidermis contain a subpopulation of major histocompatibility complex (MHC) class II-negative, CD3-negative keratinocytes that are CD200-positive. CD200 expression was accentuated in keratinocytes comprising the outer root sheath of the murine hair follicle (HF). When syngeneic skin grafts were exchanged between gender-matched wild-type (WT) and CD200-deficient C57BL/6 mice, significant perifollicular and intrafollicular inflammation was observed, eventually leading to the destruction of virtually all HF (alopecia) without significant loss of the CD200-negative grafts. Minimal and transient inflammation was observed in WT grafts, which persisted long term with hair. There was a 2-fold increase in graft-infiltrating T cells in CD200-deficient skin at 14 d. Alopecia and skin lesions were induced in CD200-deficient hosts by adoptive transfer of splenocytes from WT mice previously grafted with CD200-negative skin, but not from mice grafted with WT skin. Collectively, these results suggest that the expression of CD200 in follicular epithelium attenuates inflammatory reactions and may play a role in maintaining immune tolerance to HF-associated autoantigens.
Asunto(s)
Alopecia/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Folículo Piloso/inmunología , Tolerancia Inmunológica/fisiología , Traslado Adoptivo , Alopecia/genética , Alopecia/fisiopatología , Animales , Antígenos CD , Trasplante de Médula Ósea , Células Cultivadas , Dermatitis/genética , Dermatitis/inmunología , Dermatitis/fisiopatología , Femenino , Folículo Piloso/citología , Queratinocitos/citología , Queratinocitos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Fenotipo , Trasplante de Piel , Bazo/citología , Linfocitos T/inmunología , Quimera por TrasplanteRESUMEN
Mechanisms associated with type 1 diabetes (T1D) development remain incompletely defined. Using a sensitive array-based bioassay where patient plasma is used to induce transcriptional responses in healthy leukocytes, we previously reported disease-specific, partially interleukin (IL)-1-dependent signatures associated with preonset and recent onset (RO) T1D relative to unrelated healthy control subjects (uHC). To better understand inherited susceptibility in T1D families, we conducted cross-sectional and longitudinal analyses of healthy autoantibody-negative (AA(-)) high HLA-risk siblings (HRS) (DR3 and/or DR4) and AA(-) low HLA-risk siblings (LRS) (non-DR3/non-DR4). Signatures, scored with a novel ontology-based algorithm, and confirmatory studies differentiated the RO T1D, uHC, HRS, and LRS plasma milieus. Relative to uHC, T1D family members exhibited an elevated inflammatory state, consistent with innate receptor ligation that was independent of HLA, AA, or disease status and included elevated plasma IL-1α, IL-12p40, CCL2, CCL3, and CCL4 levels. Longitudinally, signatures of T1D progressors exhibited increasing inflammatory bias. Conversely, HRS possessing decreasing AA titers revealed emergence of an IL-10/transforming growth factor-ß-mediated regulatory state that paralleled temporal increases in peripheral activated CD4(+)/CD45RA(-)/FoxP3(high) regulatory T-cell frequencies. In AA(-) HRS, the familial innate inflammatory state also was temporally supplanted by immunoregulatory processes, suggesting a mechanism underlying the decline in T1D susceptibility with age.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Adolescente , Adulto , Quimiocina CCL2/sangre , Quimiocina CCL3/sangre , Quimiocina CCL4/sangre , Niño , Estudios Transversales , Femenino , Humanos , Interleucina-1/sangre , Interleucina-10/sangre , Subunidad p40 de la Interleucina-12/sangre , Estudios Longitudinales , Masculino , Linfocitos T Reguladores/metabolismo , Adulto JovenRESUMEN
We have been investigating whether alloantigen-specific CD4(+)25+ regulatory T cells can be identified for use in treating graft-versus-host disease. CD150, which is upregulated on the surface of all activated T lymphocytes, was identified as a candidate marker for alloantigen-activated CD4(+)25+ regulatory T cells by gene chip analysis. Freshly isolated CD4(+)25+ cells had only low cell-surface expression of CD150, comparable to that of CD4(+)25- T cells. Increased CD150 expression was observed on all T cells after coculture with allogeneic stimulator cells. When purified CD4(+)25+ cells were precultured with allogeneic stimulator cells, then sorted into CD150+ and CD150- subsets, allosuppressive activity was contained primarily in the CD150+ fraction. These cells also suppressed the proliferation of alloantigen-activated autologous T cells, and they could be expanded in vitro without loss of their suppressive capacity. These results suggest that CD150 can be used as a marker for the identification of purified alloantigen-activated CD4(+)25+ regulatory T cells.