RESUMEN

During mouse development, definitive hematopoietic stem cells (dHSCs) emerge by late E10.5 to E11 in several hematopoietic sites. Of them, the aorta-gonad-mesonephros (AGM) region drew particular attention owing to its capacity to autonomously initiate and expand dHSCs in culture, indicating its key role in HSC development. The dorsal aorta contains characteristic hematopoietic clusters and is the initial site of dHSC emergence, where they mature through vascular endothelial (VE)-cadherin(+)CD45(-)CD41(low) (type 1 pre-HSCs) and VE-cadherin(+)CD45(+) (type 2 pre-HSCs) intermediates. Although dHSCs were also found in other embryonic niches (placenta, yolk sac, and extraembryonic vessels), attempts to detect their HSC initiating potential have been unsuccessful to date. Extraembryonic arterial vessels contain hematopoietic clusters, suggesting that they develop HSCs, but functional evidence for this has been lacking. Here we show that umbilical cord and vitelline arteries (VAs), but not veins, contain pre-HSCs capable of maturing into dHSCs in the presence of exogenous interleukin 3, although in fewer numbers than the AGM region, and that pre-HSC activity in VAs increases with proximity to the embryo proper. Our functional data strongly suggest that extraembryonic arteries can actively contribute to adult hematopoiesis.

Asunto(s)

Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Arterias Umbilicales/citología , Conducto Vitelino/citología , Animales , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Arterias Umbilicales/embriología , Conducto Vitelino/irrigación sanguínea , Conducto Vitelino/embriología

RESUMEN

BACKGROUND: Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are common abnormalities in elderly men. It is considered that epithelial stem cells are involved in the etiology and development of both diseases. To distinguish aberrant from normal cells, the knowledge about primary epithelial stem/progenitor cells (ES/P) is essential. The aim of this study was to examine the role of surface markers to distinguish between different subsets of prostate basal epithelium. METHODS: The expression pattern of prostate tissue single cell suspensions was analyzed by flow cytometry using different markers. Sorted cell populations were examined for their clonogenic capacity and the resulted colonies were analyzed with flow cytometry, Western blot, and qPCR for stem cell, basal, and luminal epithelium markers. Additionally, the histological localization of the examined markers was determined using immunofluorescence. RESULTS: Using the combination of CD49f, Trop-2, and surface CD24, basal cell subsets with distinct differentiation capacities were dissected (CD49f(+) Trop-2(+) CD24(-) and CD49f(+) Trop-2(+) CD24(+) ). Although cells from the two subsets gave rise to similar basal colonies, qPCR of primary tissue revealed that higher levels of basal marker expression were detected in the CD49f(+) Trop-2(+) CD24(-) subset. Immunofluorescence analysis showed a prominent expression of CD24 by luminal and basal cells. CONCLUSIONS: Subsets with distinct differentiation capacities within the basal epithelium (CD49f(+) Trop-2(+) CD24(-) and CD49f(+) Trop-2(+) CD24(+) ) can be distinguished in human prostate. CD24 is a marker expressed on the basal transit-amplifying cells (transition cells) and may play a role in the differentiation and migration of ES/P cells to the luminal layer. The knowledge of this mechanism is of relevance for treatment of both diseases.

Asunto(s)

Antígeno CD24 , Próstata/patología , Hiperplasia Prostática , Neoplasias de la Próstata , Anciano , Antígenos de Diferenciación , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Antígeno CD24/genética , Antígeno CD24/metabolismo , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica , Células Epiteliales/metabolismo , Células Epiteliales/patología , Expresión Génica , Humanos , Integrina alfa6/metabolismo , Masculino , Próstata/metabolismo , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Células Madre/metabolismo , Células Madre/patología

RESUMEN

Several strategies have been developed to facilitate the prospective isolation of bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) based on the selective expression or absence of surface markers. Recently, we described the monoclonal antibodies W3D5 and W5C5, which selectively react with BM-MSCs, but not with hematopoietic cells. Both antibodies showed an identical reactivity pattern, indicating that they may recognize the same molecule. To identify the cognate antigen, cultured MSCs were sorted for cells expressing either very high levels of W5C5/W3D5 antigen or for cells which were negative for this antigen. Further processing of these cells for microarray analysis revealed a 20-fold enrichment of the type 1 integral membrane protein Sushi domain containing 2 (SUSD2) in the in W5C5(+) subset. To confirm the identity of the W5C5/W3D5 antigen to SUSD2, HEK293 cells were transfected with the full-length coding sequence of human SUSD2 followed by reactivity analysis of W5C5 and W3D5 antibodies with the transfected line. Flow cytometric analysis showed that both antibodies selectively recognized HEK293/huSUSD2 cells, but not the parental cell line. In line with this, SUSD2 siRNA treatment of SUSD2(+) WERI-RB-1 retinoblastoma cells reduced the expression levels of W3D5 and W5C5 antigens to ~39% and 37%, respectively. Finally, FACSorting and colony assays revealed that only SUSD2(+), but not SUSD2(-) BM cells give rise to colony-forming units-fibroblasts and are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In conclusion, we identified SUSD2 as a novel and specific marker for the prospective isolation of BM-MSCs.

Asunto(s)

Anticuerpos Monoclonales , Células de la Médula Ósea/citología , Glicoproteínas de Membrana/inmunología , Células Madre Mesenquimatosas/citología , Adipocitos/citología , Células de la Médula Ósea/inmunología , Diferenciación Celular , Linaje de la Célula , Condrocitos/citología , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Células Madre Hematopoyéticas/inmunología , Humanos , Glicoproteínas de Membrana/biosíntesis , Células Madre Mesenquimatosas/inmunología , Osteoblastos/citología

RESUMEN

Bone marrow-derived mesenchymal stromal/stem cells (MSCs) are nonhematopoietic cells that are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In addition, they are known to participate in niche formation for hematopoietic stem cells and to display immunomodulatory properties. Conventionally, these cells are functionally isolated from tissue based on their capacity to adhere to the surface of culture flasks. This isolation procedure is hampered by the unpredictable influence of secreted molecules, the interactions between cocultured hematopoietic and other unrelated cells, and by the arbitrarily selected removal time of nonadherent cells before the expansion of MSCs. Finally, functionally isolated cells do not provide biological information about the starting population. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSCs based on the selective expression, or absence, of surface markers. In this report, we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow- and amnion-derived MSCs.

Asunto(s)

Amnios/citología , Células de la Médula Ósea/clasificación , Células Madre Mesenquimatosas/clasificación , Amnios/metabolismo , Anticuerpos Monoclonales , Antígenos CD/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Separación Celular , Ensayo de Unidades Formadoras de Colonias , Femenino , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismo , Fenotipo , Embarazo , Nicho de Células Madre

RESUMEN

T-cell activation and the subsequent transformation of activated T cells into T-cell blasts require profound changes in cell volume. However, the impact of cell volume regulation for T-cell immunology has not been characterized. Here we studied the role of the cell-volume regulating osmolyte transporter Taut for T-cell activation in Taut-deficient mice. T-cell mediated recall responses were severely impaired in taut(-/-) mice as shown with B16 melanoma rejection and hapten-induced contact hypersensitivity. CD4(+) and CD8(+) T cells were unequivocally located within peripheral lymph nodes of unprimed taut(-/-) mice but significantly decreased in taut(-/-) compared with taut(+/+) mice following in vivo activation. Further analysis revealed that Taut is critical for rescuing T cells from activation-induced cell death in vitro and in vivo as shown with TCR, superantigen, and antigen-specific activation. Consequently, reduction of CD4(+) and CD8(+) T cells in taut(-/-) mice upon antigen challenge resulted in impaired in vivo generation of T-cell memory. These findings disclose for the first time that volume regulation in T cells is an element in the regulation of adaptive immune responses and that the osmolyte transporter Taut is crucial for T-cell survival and T-cell mediated immune reactions.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de Transporte de Membrana/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/inmunología , Línea Celular Tumoral , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Superantígenos/inmunología , Superantígenos/farmacología

RESUMEN

The aorta-gonad-mesonephros region plays an important role in hematopoietic stem cell (HSC) development during mouse embryogenesis. The vascular endothelial cadherin⁺ CD45⁺ (VE-cad⁺CD45⁺) population contains the major type of immature pre-HSCs capable of developing into long-term repopulating definitive HSCs. In this study, we developed a new coaggregation culture system, which supports maturation of a novel population of CD45-negative (VE-cad⁺CD45⁻CD41⁺) pre-HSCs into definitive HSCs. The appearance of these pre-HSCs precedes development of the VE-cad⁺CD45⁺ pre-HSCs (termed here type I and type II pre-HSCs, respectively), thus establishing a hierarchical directionality in the developing HSC lineage. By labeling the luminal surface of the dorsal aorta, we show that both type I and type II pre-HSCs are distributed broadly within the endothelial and subendothelial aortic layers, in contrast to mature definitive HSCs which localize to the aortic endothelial layer. In agreement with expression of CD41 in pre-HSCs, in vivo CD41-Cre-mediated genetic tagging occurs in embryonic pre-HSCs and persists in all lymphomyeloid lineages of the adult animal.

Asunto(s)

Células Madre Hematopoyéticas/citología , Antígenos Comunes de Leucocito/biosíntesis , Animales , Aorta/metabolismo , Cadherinas/metabolismo , Linaje de la Célula , Separación Celular , Endotelio Vascular/citología , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Modelos Genéticos , Fenotipo , Glicoproteína IIb de Membrana Plaquetaria/biosíntesis , Transgenes

RESUMEN

We have recently identified 2 distinct CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-) MSC subsets in primary femur-derived bone marrow (BM), which differ in their expression pattern and morphology as well as in their clonogenic and differentiation capacity. Here, we show that MSCA-1 is identical to tissue non-specific alkaline phosphatase (TNAP), an ectoenzyme known to be expressed at high levels in liver, bone, and kidney as well as in embryonic stem (ES) cells. SDS-PAGE of WERI-RB-1 cell lysate and supernatant from phosphatidylinositol-specific phospholipase C (PI-PLC)-treated WERI-RB-1 cells resulted in the appearance of a prominent 68-kDa band. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) sequence analysis revealed TNAP-specific peptides. Screening of the MSCA-1-specific antibody W8B2 on HEK-293 cells transfected with the full-length coding sequence of TNAP showed specific reactivity with transfected but not with parent cell line. In addition, TNAP-specific mRNA expression was selectively detected in the transfectant line. In agreement with these findings, enzymatic activity of TNAP was exclusively detected in sorted MSCA-1(+) BM cells but not in the MSCA-1(-) negative fraction. Surface marker analysis revealed coexpression of the embryonic marker SSEA-3 but not SSEA-4, TRA-1-60, and TRA-1-81. In endometrium, TNAP is expressed at intermediate levels on CD146(+) cells and at high levels in the luminal space of glandular epithelia. Our results demonstrate that TNAP is a selective marker for the prospective isolation of BM-derived MSC and MSC-like cells in endometrium.

Asunto(s)

Antígenos de Superficie , Proteínas Portadoras , Adulto , Fosfatasa Alcalina , Antígenos de Superficie/análisis , Antígenos de Superficie/aislamiento & purificación , Biomarcadores , Células de la Médula Ósea/citología , Proteínas Portadoras/análisis , Proteínas Portadoras/aislamiento & purificación , Endometrio/citología , Femenino , Humanos , Persona de Mediana Edad , Distribución Tisular

RESUMEN

Very recently, we identified two distinct mesenchymal stem cell (MSC) subsets in primary bone marrow (BM) that differ in their expression pattern (CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-)) and morphology as well as in their clonogenic and differentiation capacity. Here we analyzed the cell surface antigen expression in these subsets in more detail and compared the profiles with the expression pattern on cultured MSCs. Most of the tested antigens, including CD13, CD15, CD73, CD140b, CD144, CD146, and CD164, are expressed at similar levels in both primary BM populations. However, a number of markers were differentially expressed. Of these, CD166 (ALCAM), CD200, and CD106 (VCAM-1) showed an almost selective expression on either CD271(bright)MSCA-1(dim)CD56(+) (increased CD166 and CD200 expression) or CD271(bright)MSCA-1(bright)CD56(-) (increased CD106 expression) MSCs, respectively. Additional markers with elevated expression on CD56(+) MSCs include F9-3C2F1, HEK-3D3, HEK5-1B3, and W1C3 antigens, whereas CD10, CD26, CD106, 7C5G1, 9A3G2, 56A1C2, 66E2D11, HEK-3D6, HEK4-1A1, HEK4-2D6, W1D6, W4A5, W7C6, and W8B2 (MSCA-1) antigens showed increased expression in the CD56(-) population. The majority of the analyzed markers found on primary MSCs were also expressed on cultured MSCs. However, in contrast to primary MSCs, HEK7-1C4, W1C3, W1D6, and W4A5 antigens were absent on the cultured counterparts. 7G5G1 and 9A3G2 antigens showed reduced, and HEK-3D6, F9-3C2, and HEK-3D3 showed increased expression on cultured cells. The extended knowledge about the phenotype of the two subsets and the identification of novel MSC markers may result in the isolation of attractive starting populations for applications in regenerative medicine.

Asunto(s)

Células de la Médula Ósea/citología , Médula Ósea/fisiología , Separación Celular , Células Madre Mesenquimatosas/citología , Anticuerpos Monoclonales , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación/biosíntesis , Biomarcadores/análisis , Biomarcadores/metabolismo , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Adhesión Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/metabolismo , Fenotipo

RESUMEN

The PI3K pathway plays a pivotal role in the stimulation of mast cells. PI3K-dependent kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored the role of SGK1 in mast cell function. Mast cells were isolated from bone marrow (BMMC) of SGK1 knockout mice (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)). The BMMC number as well as CD117, CD34, and FcepsilonRI expression in BMCCs were similar in both genotypes. Upon Ag stimulation of the FcepsilonRI receptor, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in sgk1(-/-) BMMCs. The currents through Ca(2+)-activated K+ channels induced by Ag were significantly higher in sgk1(+/+) BMMCs than in sgk1(-/-) BMMCs. Treatment with the Ca(2+) ionophore ionomycin (1 microM) led to activation of the K+ channels in both genotypes, indicating that the Ca(2+)-activated K+ channels are similarly expressed and sensitive to activation by Ca(2+) in sgk1(+/+) and sgk1(-/-) BMMCs, and that blunted stimulation of Ca(2+)-activated K+ channels was secondary to decreased Ca(2+) entry. Ag-IgE-induced degranulation and early IL-6 secretion were also significantly blunted in sgk1(-/-) BMMCs. The decrease in body temperature following Ag treatment, which reflects an anaphylactic reaction, was substantially reduced in sgk1(-/-) mice, pointing to impaired mast cell function in vivo. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk1(-/-) than in sgk1(+/+)mice. The observations reveal a critical role for SGK1 in ion channel regulation and the function of mast cells, and thus disclose a completely novel player in the regulation of allergic reaction.

Asunto(s)

Marcación de Gen , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/genética , Mastocitos/inmunología , Mastocitos/patología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Anafilaxia/enzimología , Anafilaxia/inmunología , Anafilaxia/metabolismo , Anafilaxia/patología , Animales , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Células Cultivadas , Femenino , Proteínas Inmediatas-Precoces/fisiología , Masculino , Mastocitos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/fisiología , Canales de Potasio Calcio-Activados/biosíntesis , Canales de Potasio Calcio-Activados/genética , Canales de Potasio Calcio-Activados/fisiología , Proteínas Serina-Treonina Quinasas/fisiología

RESUMEN

Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation.

Asunto(s)

Activación del Canal Iónico , Mastocitos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Antígenos/farmacología , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Canales de Calcio/metabolismo , Degranulación de la Célula/efectos de los fármacos , Cromonas/farmacología , Femenino , Hexosaminidasas/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/fisiología , Ratones , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Canales de Potasio Calcio-Activados/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Rojo de Rutenio/farmacología

RESUMEN

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.

Asunto(s)

Inmunoglobulina E/fisiología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/deficiencia , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Mastocitos/inmunología , Mastocitos/metabolismo , Anafilaxia/genética , Anafilaxia/inmunología , Anafilaxia/metabolismo , Animales , Antígenos/inmunología , Transporte Biológico Activo/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Calcio/antagonistas & inhibidores , Calcio/fisiología , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Proliferación Celular , Tamaño de la Célula , Células Cultivadas , Dinitrobencenos/inmunología , Endotelina-1/antagonistas & inhibidores , Endotelina-1/fisiología , Femenino , Regulación de la Expresión Génica/inmunología , Inmunoglobulina E/biosíntesis , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/biosíntesis , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Masculino , Mastocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados

RESUMEN

Side effects of cyclosporine treatment include anemia. Most recent studies have found that anemia may be caused by triggering of suicidal erythrocyte death (eryptosis), i.e. activation of an erythrocyte scramblase and phosphatidylserine exposure at the erythrocyte surface. Phosphatidylserine exposing cells are rapidly cleared from circulating blood by phagocytosis. Stimulators of erythrocyte membrane scrambling include cytosolic Ca(2+) and ceramide, which are increased by entry through Ca2+-permeable cation channels and by activation of a sphingomyelinase, respectively. The present study has been performed to test for an effect of cyclosporine on eryptosis. Erythrocytes from healthy volunteers were exposed to cyclosporine, and phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3-dependent fluorescence), ceramide formation (anti-ceramide-FITC antibody), and 45Ca2+ uptake were determined by flow cytometry and tracer flux measurements, respectively. Exposure of erythrocytes to cyclosporine triggered annexin V binding and significantly enhanced the increased annexin V binding both following glucose depletion and after hyperosmotic or isotonic cell shrinkage. However, cyclosporine significantly decreased cytosolic Ca2+ activity and did not stimulate 45Ca2+ uptake. Instead, cyclosporine transiently stimulated ceramide formation, decreased the cytosolic ATP concentration and potentiated the decline of cytosolic ATP concentration following glucose depletion. Elevated ceramide levels and ATP depletion, in turn, sensitize the erythrocytes for the eryptotic effects of Ca2+. The present observations may provide a mechanistic explanation for the anemia following treatment with this important immunosuppressive drug.

Asunto(s)

Apoptosis/efectos de los fármacos , Ciclosporina/toxicidad , Eritrocitos/efectos de los fármacos , Inmunosupresores/toxicidad , Adenosina Trifosfato/análisis , Anexina A5/metabolismo , Calcio/metabolismo , Ceramidas/biosíntesis , Eritrocitos/citología , Humanos , Fosfatidilserinas/metabolismo

RESUMEN

Side effects of cytostatic treatment include development of anemia resulting from either decreased generation or accelerated clearance of circulating erythrocytes. Recent experiments revealed a novel kind of stress-induced erythrocyte death, i.e. eryptosis, which is characterized by enhanced cytosolic Ca(2+) levels, increased ceramide formation and exposure of phosphatidylserine at the cell surface. The present study explored whether cytostatic treatment with paclitaxel (Taxol) triggers eryptosis. Blood was drawn from cancer patients before and after infusion of 175 mg/m2 Taxol. The treatment significantly decreased the hematocrit and significantly increased the percentage of annexin-V-binding erythrocytes in vivo (by 37%). In vitro incubation of human erythrocytes with 10 microM paclitaxel again significantly increased annexin-V-binding (by 129%) and augmented the increase of annexin-V-binding following cellular stress. The enhanced phosphatidylserine exposure was not dependent on caspase-activity but paralleled by erythrocyte shrinkage, increase of cytosolic Ca(2+) activity, ceramide formation and activation of calpain. Phosphatidylserine exposure was similarly induced by docetaxel but not by carboplatin or doxorubicin. Moreover, eryptosis was triggered by the Ca(2+) ionophore ionomycin (10 microM). In mice, ionomycin-treated eryptotic erythrocytes were rapidly cleared from circulating blood and sequestrated into the spleen. In conclusion, our data strongly suggest that paclitaxel-induced anemia is at least partially due to induction of eryptosis.

Asunto(s)

Eritrocitos/metabolismo , Paclitaxel/farmacología , Fosfatidilserinas/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Recuento de Células Sanguíneas , Calcio/metabolismo , Carboplatino/administración & dosificación , Tamaño de la Célula/efectos de los fármacos , Ceramidas/metabolismo , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Neoplasias Endometriales/sangre , Neoplasias Endometriales/tratamiento farmacológico , Eritrocitos/efectos de los fármacos , Femenino , Hematócrito , Humanos , Ratones , Ratones Endogámicos C57BL , Presión Osmótica , Neoplasias Ováricas/sangre , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/administración & dosificación , Fosfatidilserinas/sangre , Unión Proteica/efectos de los fármacos , Esfingomielina Fosfodiesterasa/metabolismo , Factores de Tiempo , Gemcitabina

RESUMEN

Glucose depletion of erythrocytes leads to activation of Ca2+-permeable cation channels, Ca2+ entry, activation of a Ca2+-sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca2+ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKCalpha, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 microM staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 microM) and the phosphatase inhibitor okadaic acid (1-10 microM) mimicked the effect of glucose depletion and similarly led to translocation of PKCalpha and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 microM). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca2+ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion.

Asunto(s)

Autofagia/fisiología , Eritrocitos/citología , Eritrocitos/enzimología , Glucosa/farmacología , Proteína Quinasa C-alfa/metabolismo , Adenosina Trifosfato/metabolismo , Antimetabolitos/farmacología , Desoxiglucosa/farmacología , Inhibidores Enzimáticos/farmacología , Membrana Eritrocítica/enzimología , Eritrocitos/efectos de los fármacos , Humanos , Ácido Ocadaico/farmacología , Fosforilación