RESUMO
Microbes as well as immune complexes and other continuously generated inflammatory particles are efficiently removed from the human circulation by red blood cells (RBCs) through a process called immune-adherence clearance. During this process, RBCs use complement receptor 1 (CR1, CD35) to bind circulating complement-opsonized particles and transfer them to resident macrophages in the liver and spleen for removal. We here show that ligation of RBC CR1 by antibody and complement-opsonized particles induces a transient Ca(++) influx that is proportional to the RBC CR1 levels and is inhibited by T1E3 pAb, a specific inhibitor of TRPC1 channels. The CR1-elicited RBC Ca(++) influx is accompanied by an increase in RBC membrane deformability that positively correlates with the number of preexisting CR1 molecules on RBC membranes. Biochemically, ligation of RBC CR1 causes a significant increase in phosphorylation levels of ß-spectrin that is inhibited by preincubation of RBCs with DMAT, a specific casein kinase II inhibitor. We hypothesize that the CR1-dependent increase in membrane deformability could be relevant for facilitating the transfer of CR1-bound particles from the RBCs to the hepatic and splenic phagocytes.
Assuntos
Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/patologia , Macrófagos/patologia , Receptores de Complemento/metabolismo , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Contagem de Eritrócitos , Citometria de Fluxo , Humanos , Macrófagos/metabolismo , Fagocitose , Fosforilação , Espectrina/metabolismo , Canais de Cátion TRPC/metabolismoRESUMO
It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell-derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage-specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways.
Assuntos
Linfócitos B/fisiologia , Adesão Celular/fisiologia , Quimiocinas CXC/fisiologia , Linfopoese , Linhagem Celular , Quimiocina CXCL12 , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Humanos , Integrina alfa4beta1/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/metabolismo , Molécula 1 de Adesão de Célula Vascular/fisiologiaRESUMO
The primary identified function of complement receptor 1 (CR1/CD35) on primate erythrocytes is to bind complement-tagged inflammatory particles including microbes and immune complexes. When erythrocytes circulate through liver and spleen, sinusoidal phagocytes remove CR1-adherent particles and erythrocytes return to the circulation. This process of immune adherence clearance is important for host defense and prevention of autoimmunity. CR1 was previously described as clustered in the human erythrocyte membrane, which was thought to be necessary for binding complement-opsonized particles. In contrast, we demonstrate that on erythrocytes CR1 is not clustered, but dispersed, and able to bind complement-tagged particles. When fresh erythrocytes are solubilized by nonionic detergent, CR1 partitions to the cytoskeleton fraction. Using a PDZ-peptide array, CR1's cytoplasmic tail, which contains 2 PDZ-motifs, binds PDZ domains 2, 3, and 5 of Fas-associated phosphatase 1 (FAP-1), a scaffolding protein. We show that FAP-1, not previously recognized as an erythroid protein, is expressed on circulating erythrocytes. CR1 and FAP-1 coimmunoprecipitate, which confirms their molecular association. Disperse CR1 on erythrocytes may be advantageous for capturing immune-complexes, while ligation-induced CR1 clustering may prevent ingestion of the erythrocyte during the immune-complex transfer to the macrophages by keeping the opsonic stimulus localized thus preventing phagocyosis.
Assuntos
Eritrócitos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Receptores de Complemento 3b/metabolismo , Motivos de Aminoácidos , Autoimunidade , Adesão Celular , Análise por Conglomerados , Reagentes de Ligações Cruzadas/química , Citoesqueleto/metabolismo , Humanos , Sistema Imunitário , Macrófagos/metabolismo , Análise Serial de Proteínas , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
OBJECTIVES: To better understand the role of chemokines during human B-cell development in bone marrow. METHODS: Differentiation stage-specific B cells (pro-B, pre-B, immature, and mature) were analyzed for chemokine receptor expression and for migration to corresponding ligands. We also hypothesized that inflammatory conditions may cause the upregulation of certain chemokine receptors on early B cells, rendering them sensitive to extramedullary chemotactic cues. To test this hypothesis, we used human pre-B 697 cells to investigate whether various inflammatory agents could modify chemokine receptor expression and function. RESULTS: Chemotaxis to CXCL12 was observed for all B cell subsets. However, chemotactic responses to CCL19, CCL21, CXCL13, and CCL20 were limited to late-stage, IgM+ bone marrow B cells (immature B and mature B). Chemotactic responses to corresponding ligands correlated with the pattern of chemokine receptor expression. The expression of CCR7, however, was low on early (pro-B and pre-B) B cells and did not induce chemotaxis. Interestingly, both CCL19 and CCL21 could trigger ERK1/2 phosphorylation in early B cells. Exposure of pre-B 697 cells to TNF-alpha upregulated CCR7 and CXCR5 expression, whereas it had no effect on CCR6 surface expression. Correspondingly, TNF-alpha-stimulated pre-B cells chemotaxed towards CCL19 and CXCL13, in contrast to non-TNF-alpha-stimulated controls. CONCLUSION: We postulate that CXCR5, CCR7, and CCR6 participate in bone marrow trafficking and/or bone marrow egress of late-stage B cells under steady-state conditions, whereas inflammation-induced expression of CCR7 and CXCR5 may facilitate early B-cell emigration out of the bone marrow and their positioning in secondary lymphoid organs.
Assuntos
Linfócitos B/fisiologia , Hematopoese , Receptores de Quimiocinas/fisiologia , Receptores de Citocinas/fisiologia , Quimiocina CCL19 , Quimiocina CCL21 , Quimiocinas CC/fisiologia , Quimiotaxia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Regulação da Expressão Gênica , Humanos , Imunoglobulina M/biossíntese , Fosforilação , Receptores CCR6 , Receptores CCR7 , Receptores CXCR5 , Receptores de Quimiocinas/genética , Receptores de Citocinas/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Stromal cells isolated from bone marrow (BMSCs), often referred to as mesenchymal stem cells, are currently under investigation for a variety of therapeutic applications. However, limited data are available regarding receptors that can influence their homing to and positioning within the bone marrow. In the present study, we found that second passage BMSCs express a unique set of chemokine receptors: three CC chemokine receptors (CCR1, CCR7, and CCR9) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6). BMSCs cultured in serum-free medium secrete several chemokine ligands (CCL2, CCL4, CCL5, CCL20, CXCL12, CXCL8, and CX3CL1). The surface-expressed chemokine receptors were functional by several criteria. Stimulation of BMSCs with chemokine ligands triggers phosphorylation of the mitogen-activated protein kinase (e.g., extracellular signal-related kinase [ERK]-1 and ERK-2) and focal adhesion kinase signaling pathways. In addition, CXCL12 selectively activates signal transducer and activator of transcription (STAT)-5 whereas CCL5 activates STAT-1. In cell biologic assays, all of the chemokines tested stimulate chemotaxis of BMSCs, and CXCL12 induces cytoskeleton F-actin polymerization. Studies of culture-expanded BMSCs, for example, 12-16 passages, indicate loss of surface expression of all chemokine receptors and lack of chemotactic response to chemokines. The loss in chemokine receptor expression is accompanied by a decrease in expression of adhesion molecules (ICAM-1, ICAM-2, and vascular cell adhesion molecule 1) and CD157, while expression of CD90 and CD105 is maintained. The change in BMSC phenotype is associated with slowing of cell growth and increased spontaneous apoptosis. These findings suggest that several chemokine axes may operate in BMSC biology and may be important parameters in the validation of cultured BMSCs intended for cell therapy.
Assuntos
Células-Tronco Mesenquimais/imunologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Actinas/metabolismo , Apoptose , Sequência de Bases , Proliferação de Células , Células Cultivadas , Quimiocinas CC/biossíntese , Quimiocinas CXC/biossíntese , Quimiotaxia , DNA Complementar/genética , Expressão Gênica , Humanos , Técnicas In Vitro , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fenótipo , Transdução de SinaisRESUMO
CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However, the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells, focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation, phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of G(i), Src family, and the GTPase-activating protein, regulator of G protein signaling 1 (RGS1). In the bone marrow, RGS1 mRNA expression is low in progenitor B cells and high in mature B cells, implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses.
Assuntos
Subpopulações de Linfócitos B/enzimologia , Quimiocinas CXC/fisiologia , Células-Tronco Hematopoéticas/enzimologia , Sistema de Sinalização das MAP Quinases/imunologia , Microdomínios da Membrana/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas RGS/fisiologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Adesão Celular/imunologia , Diferenciação Celular/imunologia , Inibição de Migração Celular , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inibidores , Quimiocinas CXC/metabolismo , Cisteína/genética , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Ativação Enzimática/imunologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Microdomínios da Membrana/genética , Microdomínios da Membrana/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Proteínas RGS/biossíntese , Proteínas RGS/genética , Proteína p130 Retinoblastoma-Like , Tirosina/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Quinases da Família src/fisiologiaRESUMO
Although the SDF-1 (CXCL12)/CXCR4 axis is important for B-cell development, it is not yet clear to what extent CC chemokines might influence B lymphopoiesis. In the current study, we characterized CC chemokine receptor 5 (CCR5) expression and function of primary progenitor B-cell populations in human bone marrow. CCR5 was expressed on all bone marrow B cells at levels between 150 and 200 molecules per cell. Stimulation of bone marrow B cells with the CCR5-binding chemokine macrophage inflammatory protein 1beta (MIP-1beta; CCL4) did not cause chemotaxis, but CCL4 was able to trigger potent calcium mobilization responses and activation of the mitogen-activated protein kinase (MAPK) pathway in developing B cells. We also determined that CCR5-binding chemokines MIP-1alpha (CCL3), CCL4, and RANTES (CCL5), specifically by signaling through CCR5, could affect all progenitor B-cell populations through a novel mechanism involving heterologous desensitization of CXCR4. This cross-desensitization of CXCR4 was manifested by the inhibition of CXCL12-induced calcium mobilization, MAPK activation, and chemotaxis. These findings indicate that CCR5 can indeed mediate biologic responses of bone marrow B cells, even though these cell populations express low levels of CCR5 on their cell surface. Thus, by modulation of CXCR4 function, signaling through CCR5 may influence B lymphopoiesis by affecting the migration and maturation of B-cell progenitors in the bone marrow microenvironment.