RESUMO
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
Chemokines orchestrate the organization of leucocyte recruitment during inflammation and homeostasis. Despite growing knowledge of chemokine receptors, some orphan chemokine receptors are still not characterized. The gene CCRL2 encodes such a receptor that exists in two splice variants, CRAM-A and CRAM-B. Here, we report that CRAM is expressed by human peripheral blood and bone marrow B cells, and by different B-cell lines dependent on the B-cell maturation stage. Intriguingly, CRAM surface expression on the pre-B-cell lines Nalm6 and G2 is specifically upregulated in response to the inflammatory chemokine CCL5 (RANTES), a chemokine that is well known to play an important role in modulating immune responses. Although Nalm6 cells do not express any of the known CCL5 binding receptors, extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated upon CCL5 stimulation, suggesting a direct effect of CCL5 through the CRAM receptor. However, no calcium mobilization or migratory responses upon CCL5 stimulation are induced in B-cell lines or in transfected cells. Also, ERK1/2 phosphorylation cannot be inhibited by pertussis toxin, suggesting that CRAM does not couple to Gi proteins. Our results describe the expression of a novel, non-classical chemokine receptor on B cells that is potentially involved in immunomodulatory functions together with CCL5.
Assuntos
Linfócitos B/imunologia , Quimiocina CCL5/imunologia , Receptores CCR/metabolismo , Linfócitos B/ultraestrutura , Sinalização do Cálcio/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Quimiotaxia de Leucócito/imunologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Fosforilação , Isoformas de Proteínas/imunologia , Transdução de Sinais/imunologia , Fibras de Estresse/imunologiaRESUMO
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
We report pharmacokinetics, pharmacodynamics, and safety of a novel anti-CD28 domain antibody antagonist (lulizumab pegol) in healthy subjects following single- or multiple-dose administration. A minimal anticipated biological effect level approach was used to select a 0.01 mg starting dose for a single-ascending-dose (SAD), double-blind, first-in-human study. Part 1 included 9 intravenous (IV; 0.01-100 mg) and 3 subcutaneous (SC; 9-50 mg) doses or placebo. In part 2, a keyhole limpet hemocyanin (KLH) immunization was performed in 16 subjects/panel, who received 1 of 3 IV doses (9-100 mg) or placebo. In a double-blind, multiple-ascending-dose (MAD) study, subjects received SC lulizumab 6.25 mg every 2 weeks, 12.5 mg weekly, 37.5 mg weekly, or placebo. Among 180 treated subjects, 169 completed the studies. Peak concentrations and areas under the curve from time 0 to infinity increased dose proportionally. Estimated SC bioavailability was 68.2%. Receptor occupancy of approximately ≥80% was maintained for ≥2 weeks at ≥9-mg doses (SAD) and throughout the dosing interval (MAD). IV doses ≥9 mg inhibited antibody production against KLH for 2 weeks. No significant cytokine or immune cell changes were observed. No immunogenicity responses persisted, and there was no correlation to adverse events. Headache occurred in 21 SAD and 4 MAD subjects receiving lulizumab; in the MAD study 5 lulizumab subjects experienced infections. Lulizumab IV or SC was safe at all doses studied, without evidence of cytokine release.
Assuntos
Anticorpos Bloqueadores/metabolismo , Antígenos CD28/imunologia , Polietilenoglicóis/farmacocinética , Administração Intravenosa , Adolescente , Adulto , Anticorpos/efeitos adversos , Anticorpos Bloqueadores/efeitos adversos , Disponibilidade Biológica , Citocinas/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Cefaleia/induzido quimicamente , Voluntários Saudáveis , Hemocianinas/imunologia , Humanos , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Polietilenoglicóis/efeitos adversos , Receptores Imunológicos/efeitos dos fármacos , Adulto JovemRESUMO
OBJECTIVE: Molecular medicine raised expectations for strategically targeted biologic agents in systemic lupus erythematosus (SLE), but clinical trial results have been disappointing and difficult to interpret. Most studies add investigational agents to various, often effective, standard therapy immunosuppressants used at baseline, with unknown treatment interactions. Eliminating polypharmacy in trials of active lupus remains controversial. We undertook the Biomarkers of Lupus Disease study to test withdrawal of immunosuppressants as a novel approach to rendering SLE trials interpretable. METHODS: In 41 patients with active, non-organ-threatening SLE flare (group A), temporary steroids were given while background immunosuppressants were withdrawn. Time to loss of disease suppression (time to disease flare) and safety were evaluated; standard therapy was immediately resumed when symptoms recurred. Immunologic impacts of standard therapy were studied at baseline by multiplex assay, enzyme-linked immunosorbent assay, and messenger RNA array in group A patients plus 62 additional patients donating a single sample (group B). RESULTS: Patients with lower or higher baseline disease activity had median times to flare of 71 or 45 days, respectively; 40 of 41 patients (98%) had disease flares by 6 months. All flares were treated and resolved within 6 weeks. No serious adverse events occurred from flare or infection. Type I interferon (IFN), Th17, and B lymphocyte stimulator pathways tracked together. Baseline immunosuppressants had distinct impacts on Th17 and B lymphocyte stimulator, depending on IFN signature. CONCLUSION: Trials in active, non-organ-threatening SLE can safely withdraw background treatments if patients who have disease flares are designated nonresponders and returned to standard therapy. Immunologic effects of standard therapy vary between IFN-defined subsets. These findings provide a strategy for minimizing or optimizing treatment combinations in lupus trials and clinical care.
Assuntos
Imunossupressores/administração & dosagem , Lúpus Eritematoso Sistêmico/sangue , Esteroides/administração & dosagem , Suspensão de Tratamento , Adulto , Fator Ativador de Células B/sangue , Biomarcadores/sangue , Estudos Transversais , Feminino , Humanos , Interferon Tipo I/sangue , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Mensageiro/sangue , Exacerbação dos Sintomas , Células Th17/efeitos dos fármacos , Fatores de TempoRESUMO
BACKGROUND AND OBJECTIVES: Oxidant damage is an important contributor to the premature destruction of erythrocytes and anemia in thalassemias. To assess the extent of oxidant damage of circulating erythrocytes and the effects of antioxidant therapy on erythrocyte characteristics and anemia, we used a mouse model of human beta-thalassemia intermedia (b1/b2 deletion). DESIGN AND METHODS: Several parameters indicative of oxidant damage were measured at baseline and following administration of the semi-synthetic flavonoid antioxidant, 7-monohydroxyethylrutoside (monoHER), to beta-thalassemic mice at a dose of either 500 mg/kg i.p. once a day (n=6) or 250 mg/kg i.p. twice a day (n=6) for 21 days. RESULTS: Significant erythrocyte oxidant damage at baseline was indicated by: (i) dehydration, reduced cell K content, and up-regulated K-Cl co-transport; (ii) marked membrane externalization of phosphatidylserine; (iii) reduced plasma and membrane content of vitamin E; and (iv) increased membrane bound IgG. MonoHER treatment increased erythrocyte K content, and markedly improved all cellular indicators of oxidant stress and of lipid membrane peroxidation. While anemia did not improve, monoHER therapy reduced reticulocyte counts, improved survival of a fraction of red cells, and reduced ineffective erythropoiesis with decreased total bilirubin, lactate dehydrogenase and plasma iron. INTERPRETATION AND CONCLUSIONS: Antioxidant therapy reverses several indicators of oxidant damage in vivo. These promising antioxidant effects of monoHER should be investigated further.
Assuntos
Antioxidantes/uso terapêutico , Eritrócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Talassemia beta/sangue , Talassemia beta/tratamento farmacológico , Animais , Anexinas/sangue , Antioxidantes/metabolismo , Cloretos/sangue , Modelos Animais de Doenças , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Feminino , Hidroxietilrutosídeo/análogos & derivados , Hidroxietilrutosídeo/sangue , Hidroxietilrutosídeo/uso terapêutico , Imunoglobulina G/sangue , Transporte de Íons , Masculino , Camundongos , Potássio/sangue , Vitamina E/sangue , Talassemia beta/patologiaRESUMO
Homing to secondary lymphoid organs and bone marrow (BM) is a central aspect of leukemic pathophysiology. We investigated the roles of the two major lymphocyte integrins LFA-1 and VLA-4 on B-cell chronic lymphocytic leukemia (CLL) cells in these processes. We found that the majority of CLL cells expressed significantly reduced LFA-1 due to low beta2 integrin transcripts. VLA-4 expression was heterogeneous but underwent rapid activation by the BM chemokine CXCL12. CLL cells failed to transmigrate across VCAM-1-expressing, ICAM-1-expressing, and CXCL12-expressing endothelium, whereas when LFA-1 expression was regained in subsets of CLL cells, these lymphocytes rapidly transmigrated the endothelium. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LN) in a LFA-1-dependent manner, whereas CLL cells did not. Nevertheless, only residual CLL subsets could reenter BM, whereas both normal and CLL cells homed to the mice spleen in an LFA-1-independent and VLA-4-independent manner. Our results suggest that CLL cells have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than spleen. Integrin blocking could thus be an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM but not in spleen.
Assuntos
Medula Óssea/imunologia , Movimento Celular/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfonodos/imunologia , Células Neoplásicas Circulantes/imunologia , Animais , Medula Óssea/patologia , Quimiocinas/imunologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Humanos , Integrina alfa4beta1/imunologia , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , Antígeno-1 Associado à Função Linfocitária/biossíntese , Antígeno-1 Associado à Função Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Neoplásicas Circulantes/patologia , Baço/imunologiaRESUMO
TR1.3 is a Friend murine leukemia virus (MLV) that induces selective syncytium induction (SI) of brain capillary endothelial cells (BCEC), intracerebral hemorrhage, and death. Syncytium induction by TR1.3 has been mapped to a single tryptophan-to-glycine conversion at position 102 of the envelope glycoprotein (Env102). The mechanism of SI by TR1.3 was examined here in comparison to the non-syncytium-inducing, nonpathogenic MLV FB29, which displays an identical BCEC tropism. Envelope protein expression and stability on both infected cells and viral particles were not statistically different for TR1.3 and FB29. However, affinity measurements derived using purified envelope receptor binding domain (RBD) revealed a reduction of >1 log in the K(D) of TR1.3 RBD relative to FB29 RBD. Whole-virus particles pseudotyped with TR1.3 Env similarly displayed a markedly reduced binding avidity compared to FB29-pseudotyped viral particles. Lastly, decreased receptor affinity of TR1.3 Env correlated with the failure to block superinfection following acute and chronic infection by TR1.3. These results definitively show that acquisition of a SI phenotype can be directly linked to amino acid changes in retroviral Env that decrease receptor affinity, thereby emphasizing the importance of events downstream of receptor binding in the cell fusion process and pathology.
Assuntos
Vírus da Leucemia Murina/metabolismo , Vírus da Leucemia Murina/patogenicidade , Receptores Virais/metabolismo , Superinfecção/virologia , Proteínas do Envelope Viral/metabolismo , Animais , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Linhagem Celular , Regulação Viral da Expressão Gênica , Humanos , Vírus da Leucemia Murina/classificação , Fusão de Membrana , Camundongos , Vírion/metabolismoRESUMO
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
Neutrophil spontaneous death plays essential roles in neutrophil homeostasis and resolution of inflammation, whereas the underlying molecular mechanisms are still ill-defined. Neutrophils die because of programmed cell death or apoptosis. However, treatment with inhibitor of caspases, which are responsible for the majority of apoptotic cell deaths, does not prevent the spontaneous death of neutrophils. PKB/Akt possesses prosurvival and antiapoptotic activities in a variety of cells. In this study, we show that Akt activity decreases dramatically during the course of neutrophil death. Both phosphatidylinositol 3-kinase and Akt inhibitors enhance neutrophil death. Conditions delaying neutrophil death, such as treatment with granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, or IFN-gamma, restore Akt activity. Finally, we demonstrate that neutrophils depleted of PTEN, a phosphatidylinositol 3'-phosphatase that negatively regulates Akt activity, live much longer than WT neutrophils. Thus, we establish Akt deactivation as a causal mediator of neutrophil spontaneous death.
Assuntos
Neutrófilos/citologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Inibidores de Caspase , Morte Celular , Regulação para Baixo/genética , Ativação Enzimática , Humanos , Camundongos , PTEN Fosfo-Hidrolase/deficiênciaRESUMO
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
Complement C3a promotes CXCL12-induced migration and engraftment of human and murine hemopoietic progenitor cells, suggesting a cross-influence between anaphylatoxin and chemokine axes. Here we have explored the underlying mechanism(s) of complement anaphylatoxin and chemokine cooperation. In addition to C3a, C3a-desArg and C4a but not C5a, are potent enhancers of CXCL12-induced chemotaxis of human and murine bone marrow (BM) stem/progenitor cells and B lineage cells. C3a enhancement of chemotaxis is chemokine specific because it is also observed for chemotaxis to CCL19 but not to CXCL13. The potentiating effect of C3a on CXCL12 is independent of the classical C3a receptor (C3aR). First, human BM CD34(+) and B lineage cells do not express C3aR by flow cytometry. Second, the competitive C3aR inhibitor SB290157 does not affect C3a-mediated enhancement of CXCL12-induced chemotaxis. Third, enhancement of chemotaxis of hemopoietic cells is also mediated by C3a-desArg, which does not bind to C3aR. Finally, C3a enhances CXCL12-induced chemotaxis of BM cells from C3aR knockout mice similar to BM cells from wild-type mice. Subsequent studies revealed that C3a increased the binding affinity of CXCL12 to human CXCR4(+)/C3aR(-), REH pro-B cells, which is compatible with a direct interaction between C3a and CXCL12. BM stromal cells were able to generate C3a, C3a-desArg, C4a, as well as CXCL12, suggesting that this pathway could function in vivo. Taken together, we demonstrate a C3a-CXCL12 interaction independent of the C3aR, which may provide a mechanism to modulate the function of CXCL12 in the BM microenvironment.
Assuntos
Quimiocinas CXC/fisiologia , Quimiotaxia , Complemento C3a/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Membrana/fisiologia , Receptores de Complemento/fisiologia , Anafilatoxinas/metabolismo , Anafilatoxinas/fisiologia , Animais , Linfócitos B/metabolismo , Medula Óssea , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas/metabolismo , Quimiocinas/fisiologia , Quimiocinas CXC/metabolismo , Complemento C3a/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células Estromais/metabolismoRESUMO
It has been shown that mice with complete deficiency of all 4.1R protein isoforms (4.1-/-) exhibit moderate hemolytic anemia, with abnormal erythrocyte morphology (spherocytosis) and decreased membrane stability. Here, we characterized the Gardos channel function in vitro and in vivo in erythrocytes of 4.1-/- mice. Compared with wild-type, the Gardos channel of 4.1-/- erythrocytes showed an increase in Vmax (9.75 +/- 1.06 vs 6.08 +/- 0.09 mM cell x minute; P < .04) and a decrease in Km (1.01 +/- 0.06 vs 1.47 +/- 1.02 microM; P < .03), indicating an increased sensitivity to activation by intracellular calcium. In vivo function of the Gardos channel was assessed by the oral administration of clotrimazole, a well-characterized Gardos channel blocker. Clotrimazole treatment resulted in worsening of anemia and hemolysis, with decreased red cell survival and increased numbers of circulating hyperchromic spherocytes and microspherocytes. Clotrimazole induced similar changes in 4.2-/- and band 3+/- mice, indicating that these effects of the Gardos channel are shared in different models of murine spherocytosis. Thus, potassium and water loss through the Gardos channel may play an important protective role in compensating for the reduced surface-membrane area of hereditary spherocytosis (HS) erythrocytes and reducing hemolysis in erythrocytes with cytoskeletal impairments.
Assuntos
Anemia Hemolítica/patologia , Hemólise , Canais de Potássio Cálcio-Ativados/fisiologia , Esferócitos/patologia , Animais , Cálcio/farmacologia , Clotrimazol/administração & dosagem , Clotrimazol/farmacologia , Proteínas do Citoesqueleto/deficiência , Hemólise/efeitos dos fármacos , Cinética , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Potássio/metabolismo , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Esferócitos/efeitos dos fármacos , Água/metabolismoRESUMO
We found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte-colony-stimulating factor (G-CSF) or the various components of SLPs (fibrinogen, fibronectin, soluble vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and urokinase plasminogen activator receptor [uPAR]) increase the chemotactic responses of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). However, alone they do not chemoattract HSPCs, but they do increase or prime the cells' chemotactic responses to a low or threshold dose of SDF-1. We observed that SLPs increased calcium flux, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, secretion of matrix metalloproteinases, and adhesion to endothelium in CD34+ cells. Furthermore, SLPs increased SDF-dependent actin polymerization and significantly enhanced the homing of human cord blood (CB)- and bone marrow (BM)-derived CD34+ cells in a NOD/SCID mouse transplantation model. Moreover, the sensitization or priming of cell chemotaxis to an SDF-1 gradient was dependent on cholesterol content in the cell membrane and on the incorporation of the SDF-1 binding receptor CXCR4 and the small GTPase Rac-1 into membrane lipid rafts. This colocalization of CXCR4 and Rac-1 in lipid rafts facilitated guanosine triphosphate (GTP) binding/activation of Rac-1. Hence, we postulate that CXCR4 could be primed by various factors related to leukapheresis and mobilization that increase its association with membrane lipid rafts, allowing the HSPCs to better sense the SDF-1 gradient. This may partially explain why HSPCs from mobilized peripheral blood leukapheresis products engraft more quickly in patients than do those from BM or CB. Based on our findings, we suggest that the homing of HSPCs is optimal when CXCR4 is incorporated in membrane lipid rafts and that ex vivo priming of HSPCs with some of the SLP-related molecules before transplantation could increase their engraftment.
Assuntos
Quimiocinas CXC/farmacologia , Quimiotaxia/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Receptores CXCR4/metabolismo , Actinas/metabolismo , Animais , Antígenos CD34/metabolismo , Medula Óssea/metabolismo , Cálcio/metabolismo , Adesão Celular , Células Cultivadas , Quimiocina CXCL12 , Colágeno , Combinação de Medicamentos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Laminina , Leucaférese , Metaloproteinases da Matriz/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteoglicanas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Normal bone marrow (BM) contains T cells whose function and origin are poorly understood. We observed that CD8+ T cells in BM consist chiefly of CCR7+ L-selectin+ central memory cells (TCMs). Adoptively transferred TCMs accumulated more efficiently in the BM than naive and effector T cells. Intravital microscopy (IVM) showed that TCMs roll efficiently in BM microvessels via L-, P-, and E-selectin, whereas firm arrest required the VCAM-1/alpha4beta1 pathway. alpha4beta1 integrin activation did not depend on pertussis toxin (PTX)-sensitive Galphai proteins but was reduced by anti-CXCL12. In contrast, TCM diapedesis did not require CXCL12 but was blocked by PTX. After extravasation, TCMs displayed agile movement within BM cavities, remained viable, and mounted potent antigen-specific recall responses for at least two months. Thus, the BM functions as a major reservoir for TCMs by providing specific recruitment signals that act in sequence to mediate the constitutive recruitment of TCMs from the blood.
Assuntos
Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Transferência Adotiva , Animais , Medula Óssea/irrigação sanguínea , Medula Óssea/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Adesão Celular , Movimento Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Integrina alfa4/metabolismo , Integrina alfa4beta1/metabolismo , Camundongos , Selectinas/metabolismo , Transdução de Sinais , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The CCL4 chemokine is secreted by a variety of cells following stimulation. CCL4 affects several different types of cells that are important for acute inflammatory responses and are critical for the development of specific immune responses to foreign antigens. The human genome contains two genes for the CCL4 chemokine. Although highly homologous, the two genes encode slightly different proteins. We analyzed the mRNA expressed in monocytes and B lymphocytes and found that while monocytes express predominantly one CCL4 gene, known as ACT-2, peripheral blood B lymphocytes express a mixture of ACT-2 and the second CCL4 gene, lymphocyte activating gene-1 ( LAG-1). Although peripheral blood B cells, CD27(-) B cells, and CD27(+) B cells all express a mixture of LAG-1 and ACT-2, the B-cell lines that were studied regulate the two genes independently. RL, SU-DHL-6, and REH cells predominantly express LAG-1. These studies demonstrate that monocytes and B cells utilize different mechanisms to regulate expression of the two CCL4 genes and suggest that the two genes may not have identical activities.
Assuntos
Linfócitos B/metabolismo , Regulação da Expressão Gênica , Monócitos/metabolismo , Proteínas/genética , Células Cultivadas/metabolismo , Quimiocina CCL4 , Quimiocinas CC , Genes , Humanos , Proteínas Inflamatórias de Macrófagos , Especificidade de Órgãos , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Quimiocinas CXC/imunologia , Células-Tronco Hematopoéticas/imunologia , Receptores CCR5/imunologia , Receptores CXCR4/imunologia , Adulto , Células da Medula Óssea/citologia , Membrana Celular/imunologia , Quimiocina CXCL12 , Quimiocinas CXC/fisiologia , Quimiotaxia/fisiologia , Células-Tronco Hematopoéticas/citologia , Humanos , Receptores CCR5/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The murine leukemia virus (MLV) TR1.3 provides an excellent model to study the wide range of retrovirus-induced central nervous system (CNS) pathology and disease. TR1.3 rapidly induces thrombotic events in brain microvessels and causes cell-specific syncytium formation of brain capillary endothelial cells (BCEC). A single amino acid substitution, W102G, in the MLV envelope protein (Env) regulates the pathogenic effects. The role of Env in determining this disease phenotype compared to the induction of spongiform encephalomyelitis with a longer latency, as seen in several other MLV and in human retroviruses, was determined by studying in vitro-attenuated TR1.3. Virus cloned from this selection, termed TRM, induced progressive neurological disease characterized by ataxia and paralysis and the appearance of spongiform neurodegeneration throughout the brain stem and spinal cord. This disease was associated with virus replication in both BCEC and highly ramified glial cells. TRM did not induce syncytium formation, either in vivo or in vitro. Sequence and mutational analyses demonstrated that TRM contained a reversion of Env G102W but that neurological disease mapped to the single amino acid substitution Env S159P. The results demonstrate that single nucleotide changes within disparate regions of Env control dramatically different CNS disease patterns.
Assuntos
Doenças do Sistema Nervoso Central/etiologia , Vírus da Leucemia Murina/patogenicidade , Proteínas do Envelope Viral/química , Animais , Linhagem Celular , Doenças do Sistema Nervoso Central/patologia , Doenças do Sistema Nervoso Central/virologia , Coturnix , Feminino , Fusão de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Tropismo , Proteínas do Envelope Viral/fisiologiaRESUMO
Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34(+) cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell-derived factor 1 (SDF-1)-dependent chemotaxis of human CD34(+) cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1-dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)-mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1(+) cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF-CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.