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1.
Curr Allergy Asthma Rep ; 14(2): 402, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24408534

RESUMO

Recent investigations have expanded our knowledge of the regulatory bone marrow (BM) niche, which is critical in maintaining and directing hematopoietic stem cell (HSC) self-renewal and differentiation. Osteoblasts, mesenchymal stem cells (MSCs), and CXCL12-abundant reticular (CAR) cells are niche components in close association with HSCs and have been more clearly defined in immune cell function and homeostasis. Importantly, cellular inhabitants of the BM niche signal through G protein-coupled surface receptors (GPCRs) for various appropriate immune functions. In this article, recent literature on BM niche inhabitants (HSCs, osteoblasts, MSCs, CAR cells) and their GPCR mechanistic interactions are reviewed for better understanding of the BM cells involved in immune development, immunologic disease, and current immune reconstitution therapies.


Assuntos
Células da Medula Óssea/imunologia , Medula Óssea/imunologia , Animais , Células da Medula Óssea/citologia , Comunicação Celular , Células-Tronco Hematopoéticas/imunologia , Humanos , Doenças do Sistema Imunitário/metabolismo , Células-Tronco Mesenquimais/imunologia
2.
Stem Cell Res Ther ; 13(1): 37, 2022 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-35093170

RESUMO

BACKGROUND: The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling. METHODS: Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures. RESULTS: GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of ß-arrestin-2 in sphingosine-1-phosphate receptor internalization. CONCLUSIONS: Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting ß-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Animais , Diferenciação Celular , Proliferação de Células , Células-Tronco Mesenquimais/metabolismo , Camundongos , Receptores de Esfingosina-1-Fosfato , Microtomografia por Raio-X
3.
Mol Immunol ; 106: 12-21, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30576947

RESUMO

Chemerin receptor (CMKLR1) is a G protein-coupled receptor (GPCR) implicated in macrophage-mediated inflammation and in several forms of human arthritis. Analogous to other GPCR, CMKLR1 is likely regulated by G protein-coupled receptor kinase (GRK) phosphorylation of intracellular domains in an activation-dependent manner, which leads to recruitment and termination of intracellular signaling via desensitization and internalization of the receptor. The ubiquitously expressed GRK family members include GRK2, GRK3, GRK5, and GRK6, but it is unknown which GRK regulates CMKLR1 cellular and signaling functions. Our data show that activation of CMKLR1 by chemerin in primary macrophages leads to signaling and functional outcomes that are regulated by GRK6 and ß-arrestin 2. We show that arrestin recruitment to CMKLR1 following chemerin stimulation is enhanced with co-expression of GRK6. Further, internalization of endogenous CMKLR1, following the addition of chemerin, is decreased in inflammatory macrophages from GRK6- and ß-arrestin 2-deficient mice. These GRK6- and ß-arrestin 2-deficient macrophages display increased migration toward chemerin and altered AKT and Extracellular-signal Related Kinase (ERK) signaling. Our findings show that chemerin-activated CMKLR1 regulation in inflammatory macrophages is largely GRK6 and ß-arrestin mediated, which may impact innate immunity and have therapeutic implications in rheumatic disease.


Assuntos
Quimiocinas/imunologia , Quinases de Receptores Acoplados a Proteína G/imunologia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Macrófagos/imunologia , Receptores Acoplados a Proteínas G/imunologia , beta-Arrestina 2/imunologia , Animais , Linhagem Celular , Quimiocinas/genética , Quinases de Receptores Acoplados a Proteína G/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Macrófagos/patologia , Camundongos , Camundongos Knockout , Receptores de Quimiocinas , Receptores Acoplados a Proteínas G/genética , Doenças Reumáticas/genética , Doenças Reumáticas/imunologia , Doenças Reumáticas/patologia , beta-Arrestina 2/genética
4.
PLoS One ; 11(4): e0152856, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27049755

RESUMO

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.


Assuntos
Neoplasias da Mama/patologia , Quinase 3 de Receptor Acoplado a Proteína G/fisiologia , Animais , Feminino , Quinase 3 de Receptor Acoplado a Proteína G/genética , Inativação Gênica , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica
5.
Immunol Res ; 27(1): 71-84, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12637769

RESUMO

T lymphocytes are the primary cells responsible for maintaining the immune system. There are many intricate mechanisms involved in the regulation of T cells and the integrin family of adhesive surface proteins plays a pivotal role in the control of T lymphocyte activation and functions. Integrins are heterodimeric transmembrane proteins that are not merely adhesion molecules but also function in T cell coactivation by providing a scaffold for signaling and cytoskeletal proteins that are adept at transmitting signals from the inside of the cell to the outside ("inside-out signaling") or from the outside of the cell to the inside ("outside-in signaling"). The signaling property of integrins allows for rapid responses to changes in the microenviroment of the lymphocyte. Therefore, whether the T cell needs to adhere or detach, integrins can quickly accommodate either state of the cell. Once cells are guided to sites of infection, inflammation, or antigen presentation, integrins can also participate in the initiation, maintenance, or termination of the response. This review will focus on the aspects of integrin-mediated T cell coactivation, affinity and avidity control of integrins, signaling molecules involved with integrins, association of integrins in lipid microdomains, and negative regulation of integrins.


Assuntos
Integrinas/imunologia , Linfócitos T/imunologia , Proteínas de Bactérias/imunologia , Adesão Celular/imunologia , Humanos , Ativação Linfocitária/imunologia , Microdomínios da Membrana/imunologia , Transdução de Sinais/imunologia
6.
Am J Clin Exp Immunol ; 3(2): 97-106, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25143870

RESUMO

G protein signaling modulator-3 (GPSM3), also known as G18 or AGS4, is a member of a family of proteins containing one or more copies of a small regulatory motif known as the GoLoco (or GPR) motif. GPSM3 interacts directly with Gα and Gß subunits of heterotrimeric G proteins to regulate downstream intracellular signals initiated by G protein coupled receptors (GPCRs) that are activated via binding to their cognate ligands. GPSM3 has a selective tissue distribution and is highly expressed in immune system cells; genome-wide association studies (GWAS) have recently revealed that single nucleotide polymorphisms (SNPs) in GPSM3 are associated with chronic inflammatory diseases. This review highlights the current knowledge of GPSM3 function in normal and pathologic immune-mediated conditions.

7.
Mol Immunol ; 54(2): 193-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23280397

RESUMO

Polymorphism at the GPSM3 gene locus is inversely associated with four systemic autoimmune diseases, including rheumatoid arthritis and ankylosing spondylitis. G-protein signaling modulator-3 (GPSM3) expression is most pronounced in myeloid cells, in which it targets heterotrimeric G-protein Gαi subunits of chemokine receptors, critical to immune function. To begin to explore the regulatory role of GPSM3 in monocytes, human THP-1 and primary mouse myeloid cells were cultured under stimulus conditions; GPSM3 was found by immunoblotting to be expressed at highest levels in the mature monocyte. To evaluate the effects of GPSM3 deficiency on a myeloid-dependent autoimmune disease, collagen antibody-induced arthritis (CAIA) was induced in Gpsm3-/- and control mice, which were then analyzed for clinical score, paw swelling, intra-articular proinflammatory markers, and histopathology. Mice lacking GPSM3 were protected from CAIA, and expression of monocyte-representative pro-inflammatory chemokine receptors and cytokines in paws of Gpsm3-/- mice were decreased. Flow cytometry, apoptosis, and transwell chemotaxis experiments were conducted to further characterize the effect of GPSM3 deficiency on survival and chemokine responsiveness of monocytes. GPSM3-deficient myeloid cells had reduced migration ex vivo to CCL2, CX3CL1, and chemerin and enhanced apoptosis in vitro. Our results suggest that GPSM3 is an important regulator of monocyte function involving mechanisms of differentiation, survival, and chemotaxis, and deficiency in GPSM3 expression is protective in acute inflammatory arthritis.


Assuntos
Artrite Experimental/genética , Artrite Experimental/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Monócitos/imunologia , Animais , Sobrevivência Celular/genética , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Inibidores de Dissociação do Nucleotídeo Guanina/imunologia , Mediadores da Inflamação/imunologia , Camundongos , Camundongos Knockout , Monócitos/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia
8.
J Leukoc Biol ; 94(6): 1243-51, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23935208

RESUMO

Chemokine receptor interactions coordinate leukocyte migration in inflammation. Chemokine receptors are GPCRs that when activated, are phosphorylated by GRKs to turn off G protein-mediated signaling yet recruit additional signaling machinery. Recently, GRK3 was identified as a negative regulator of CXCL12/CXCR4 signaling that is defective in human WHIM syndrome. Here, we report that GRK3-/- mice exhibit numerous features of human WHIM, such as impaired CXCL12-mediated desensitization, enhanced CXCR4 signaling to ERK activation, altered granulocyte migration, and a mild myelokathexis. Moreover, GRK3-/- protects mice from two acute models of inflammatory arthritis (K/BxN serum transfer and CAIA). In these granulocyte-dependent disease models, protection of GRK3-/- mice is mediated by retention of cells in the marrow, fewer circulating granulocytes in the peripheral blood, and reduced granulocytes in the joints during active inflammation. In contrast to WHIM, GRK3-/- mice have minimal hypogammaglobulinemia and a peripheral leukocytosis with increased lymphocytes and absent neutropenia. Thus, we conclude that the loss of GRK3-mediated regulation of CXCL12/CXCR4 signaling contributes to some, but not all, of the complete WHIM phenotype and that GRK3 inhibition may be beneficial in the treatment of inflammatory arthritis.


Assuntos
Quinase 3 de Receptor Acoplado a Proteína G/imunologia , Síndromes de Imunodeficiência/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Verrugas/imunologia , Animais , Linhagem Celular Transformada , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , Quinase 3 de Receptor Acoplado a Proteína G/genética , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Granulócitos/enzimologia , Granulócitos/imunologia , Granulócitos/patologia , Humanos , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Inflamação/enzimologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Doenças da Imunodeficiência Primária , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Receptores CXCR4/metabolismo , Verrugas/enzimologia , Verrugas/genética , Verrugas/patologia
9.
PLoS One ; 6(3): e17940, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21437240

RESUMO

BACKGROUND: Productive thymopoiesis is essential for a robust and healthy immune system. Thymus unfortunately is acutely sensitive to stress resulting in involution and decreased T cell production. Thymic involution is a complication of many clinical settings, including infection, malnutrition, starvation, and irradiation or immunosuppressive therapies. Systemic rises in glucocorticoids and inflammatory cytokines are known to contribute to thymic atrophy. Little is known, however, about intrathymic mechanisms that may actively contribute to thymus atrophy or initiate thymic recovery following stress events. METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic, histologic and transcriptome/pathway analysis of murine thymic tissue during the early stages of endotoxemia-induced thymic involution was performed to identify putative mechanisms that drive thymic involution during stress. Thymus atrophy in this murine model was confirmed by down-regulation of genes involved in T cell development, cell activation, and cell cycle progression, correlating with observed phenotypic and histologic thymus involution. Significant gene changes support the hypothesis that multiple key intrathymic pathways are differentially activated during stress-induced thymic involution. These included direct activation of thymus tissue by LPS through TLR signaling, local expression of inflammatory cytokines, inhibition of T cell signaling, and induction of wound healing/tissue remodeling. CONCLUSIONS/SIGNIFICANCE: Taken together, these observations demonstrated that in addition to the classic systemic response, a direct intrathymic response to endotoxin challenge concurrently contributes to thymic involution during endotoxemia. These findings are a substantial advancement over current understanding of thymus response to stress and may lead to the development of novel therapeutic approaches to ameliorate immune deficiency associated with stress events.


Assuntos
Endotoxinas/toxicidade , Inflamação/patologia , Timo/efeitos dos fármacos , Timo/patologia , Cicatrização/efeitos dos fármacos , Doença Aguda , Animais , Atrofia , Citocinas/sangue , Citocinas/genética , Endotoxemia/sangue , Endotoxemia/complicações , Endotoxemia/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/genética , Inflamação/sangue , Inflamação/complicações , Camundongos , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estresse Fisiológico/efeitos dos fármacos , Timo/metabolismo , Cicatrização/genética
10.
Hum Immunol ; 71(1): 23-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19815047

RESUMO

The alpha(4)beta(1) integrin VLA-4 (very-late activation antigen-4) and the lineage-specific CD4 and CD8 receptors have been proposed as putative co-stimulatory receptors on T cells. To assess the relative contribution of signaling through the TCR, CD28 and these accessory molecules, we activated human T cells using soluble antibodies recognizing all four of these T-cell receptor classes (CD3, CD28, CD4/CD8, and VLA-4), and we assessed the degree of activation using higher-order flow cytometry detecting intracellular Erk1/2 phosphorylation and production of IL-2 and IFN-gamma. We found that: (1) co-stimulation via CD4/CD8, in addition to CD28, is required for optimal T-cell activation; (2) VLA-4 binding consistently potentiates CD4(+) and CD8(+) T-cell activation; (3) augmentation of T-cell activation through VLA-4 binding is most pronounced following engagement of CD4/CD8. These results confirm that multiple signals, including VLA-4 engagement, are necessary for maximal T-cell activation beyond that induced via the TCR and CD28.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Integrina alfa4beta1/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Citocinas/biossíntese , Ativação Enzimática , Humanos , Ativação Linfocitária , Sistema de Sinalização das MAP Quinases , Fosforilação , Receptores de Antígenos de Linfócitos T/imunologia
11.
Immunol Cell Biol ; 86(4): 381-4, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18195724

RESUMO

CD45RA T cells are fully co-activated by natural beta1 integrin ligands fibronectin (FN) and VCAM-1, as well as monoclonal antibody (mAb) 19H8, which binds a combinatorial epitope of the alpha4beta1 heterodimer. These integrin ligands stimulate CD3-dependent proliferation and the upregulation of early activation markers CD25 and CD69. However, beta1-specific antibody 33B6, which binds to a similar range of the predominant T-cell integrins as natural ligands FN (alpha4beta1 and alpha5beta1) and VCAM-1 (alpha4beta1), failed to costimulate proliferation in the CD45RA subset, while retaining the ability to costimulate early activation markers CD25 and CD69. After addition of exogenous human interleukin-2 to the culture media, 33B6 costimulation of proliferation is restored. These data provide evidence that a branch of the alpha4beta1 integrin-signaling pathway in CD45RA T cells can be independently regulated and exploited through the use of partial agonist ligands, including mAbs to the integrin heterodimer.


Assuntos
Anticorpos Monoclonais/imunologia , Integrina beta1/imunologia , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Proliferação de Células , Humanos , Interleucina-2/imunologia , Linfócitos T/citologia
12.
J Immunol ; 176(8): 5041-9, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16585601

RESUMO

Cell adhesion mediated by the interaction between integrin alpha4beta1 and VCAM-1 is important in normal physiologic processes and in inflammatory and autoimmune disease. Numerous studies have mapped the alpha4beta1 binding sites in VCAM-1 that mediate cell adhesion; however, little is known about the regions in VCAM-1 important for regulating soluble binding. In the present study, we demonstrate that 6D VCAM-1 (an alternatively spliced isoform of VCAM-1 lacking Ig-like domain 4) binds alpha4beta1 with a higher relative affinity than does the full-length form of VCAM-1 containing 7 Ig-like extracellular domains (7D VCAM-1). In indirect binding assays, the EC50 of soluble 6D VCAM-1 binding to alpha4beta1 on Jurkat cells (in 1 mM MnCl2) was 2 x 10(-9) M, compared with 7D VCAM-1 at 11 x 10(-9) M. When used in solution to inhibit alpha4beta1 mediated cell adhesion, the IC50 of 6D VCAM-1 was 13 x 10(-9) M, compared with 7D VCAM-1 measured at 150 x 10(-9) M. Removal of Ig-like domains 4, 5, or 6, or simply substituting Asp328 in domain 4 of 7D VCAM-1 with alanine, caused increased binding of soluble 7D VCAM-1 to alpha4beta1. In contrast, cells adhered more avidly to 7D VCAM-1 under shear force, as it induced cell spreading at lower concentrations than did 6D VCAM-1. Finally, soluble 6D VCAM-1 acts as an agonist through alpha4beta1 by augmenting cell migration and inducing cell aggregation. These results indicate that the domain 4 of VCAM-1 plays a contrasting role when VCAM-1 is presented in solution or as a cell surface-expressed adhesive substrate.


Assuntos
Adesão Celular/fisiologia , Integrina alfa4beta1/metabolismo , Molécula 1 de Adesão de Célula Vascular/química , Molécula 1 de Adesão de Célula Vascular/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Agregação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , DNA/genética , Humanos , Técnicas In Vitro , Molécula 1 de Adesão Intercelular/fisiologia , Células Jurkat , Cinética , Mutagênese , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Molécula 1 de Adesão de Célula Vascular/genética
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