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1.
Immunity ; 51(3): 561-572.e5, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31402260

RESUMO

Lymphatic vessels form a critical component in the regulation of human health and disease. While their functional significance is increasingly being recognized, the comprehensive heterogeneity of lymphatics remains uncharacterized. Here, we report the profiling of 33,000 lymphatic endothelial cells (LECs) in human lymph nodes (LNs) by single-cell RNA sequencing. Unbiased clustering revealed six major types of human LECs. LECs lining the subcapsular sinus (SCS) of LNs abundantly expressed neutrophil chemoattractants, whereas LECs lining the medullary sinus (MS) expressed a C-type lectin CD209. Binding of a carbohydrate Lewis X (CD15) to CD209 mediated neutrophil binding to the MS. The neutrophil-selective homing by MS LECs may retain neutrophils in the LN medulla and allow lymph-borne pathogens to clear, preventing their spread through LNs in humans. Our study provides a comprehensive characterization of LEC heterogeneity and unveils a previously undefined role for medullary LECs in human immunity.


Assuntos
Células Endoteliais/imunologia , Neutrófilos/imunologia , Animais , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Humanos , Lectinas Tipo C/imunologia , Antígenos CD15/imunologia , Linfonodos/imunologia , Vasos Linfáticos/imunologia , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/imunologia , Inquéritos e Questionários
2.
Nat Immunol ; 16(4): 386-96, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25665101

RESUMO

In the lymphatic sinuses of draining lymph nodes, soluble lymph-borne antigens enter the reticular conduits in a size-selective manner and lymphocytes transmigrate to the parenchyma. The molecular mechanisms that control these processes are unknown. Here we unexpectedly found that PLVAP, a prototypic endothelial protein of blood vessels, was synthesized in the sinus-lining lymphatic endothelial cells covering the distal conduits. In PLVAP-deficient mice, both small antigens and large antigens entered the conduit system, and the transmigration of lymphocytes through the sinus floor was augmented. Mechanistically, the filtering function of the lymphatic sinus endothelium was dependent on diaphragms formed by PLVAP fibrils in transendothelial channels. Thus, in the lymphatic sinus, PLVAP forms a physical sieve that regulates the parenchymal entry of lymphocytes and soluble antigens.


Assuntos
Proteínas de Transporte/imunologia , Células Endoteliais/imunologia , Linfonodos/imunologia , Linfócitos/imunologia , Proteínas de Membrana/imunologia , Animais , Antígenos/imunologia , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas de Transporte/genética , Caveolina 1/deficiência , Caveolina 1/genética , Caveolina 1/imunologia , Células Endoteliais/citologia , Endotélio Linfático/citologia , Endotélio Linfático/imunologia , Feminino , Regulação da Expressão Gênica , Linfonodos/citologia , Vasos Linfáticos/citologia , Vasos Linfáticos/imunologia , Linfócitos/citologia , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Migração Transendotelial e Transepitelial/imunologia
3.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34949714

RESUMO

The interaction of signal regulatory protein α (SIRPα) on macrophages with CD47 on cancer cells is thought to prevent antibody (Ab)-dependent cellular phagocytosis (ADCP) of the latter cells by the former. Blockade of the CD47-SIRPα interaction by Abs to CD47 or to SIRPα, in combination with tumor-targeting Abs such as rituximab, thus inhibits tumor formation by promoting macrophage-mediated ADCP of cancer cells. Here we show that monotherapy with a monoclonal Ab (mAb) to SIRPα that also recognizes SIRPß1 inhibited tumor formation by bladder and mammary cancer cells in mice, with this inhibitory effect being largely dependent on macrophages. The mAb to SIRPα promoted polarization of tumor-infiltrating macrophages toward an antitumorigenic phenotype, resulting in the killing and phagocytosis of cancer cells by the macrophages. Ablation of SIRPα in mice did not prevent the inhibitory effect of the anti-SIRPα mAb on tumor formation or its promotion of the cancer cell-killing activity of macrophages, however. Moreover, knockdown of SIRPß1 in macrophages attenuated the stimulatory effect of the anti-SIRPα mAb on the killing of cancer cells, whereas an mAb specific for SIRPß1 mimicked the effect of the anti-SIRPα mAb. Our results thus suggest that monotherapy with Abs to SIRPα/SIRPß1 induces antitumorigenic macrophages and thereby inhibits tumor growth and that SIRPß1 is a potential target for cancer immunotherapy.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Antineoplásicos/farmacologia , Imunoterapia/métodos , Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Camundongos , Receptores de Superfície Celular/genética , Receptores Imunológicos/genética , Rituximab , Resultado do Tratamento , Bexiga Urinária
4.
Cancer Sci ; 113(4): 1338-1351, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35133060

RESUMO

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor-mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7-dependent signaling in MDA-MB-231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7-dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA-MB-231-derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE-1+ intratumoral and peritumoral lymphatic vessels. In the three-dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21-reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand-expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis.


Assuntos
Neoplasias da Mama , Movimento Celular , Quimiocina CXCL12 , Vasos Linfáticos , Receptores CCR7 , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quimiocina CCL21/metabolismo , Quimiocina CXCL12/metabolismo , Feminino , Humanos , Ligantes , Metástase Linfática , Vasos Linfáticos/patologia , Invasividade Neoplásica , Receptores CCR7/metabolismo , Receptores CXCR4
6.
Int Immunol ; 33(10): 507-513, 2021 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-33569606

RESUMO

Understanding the precise nature and durability of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential in order to gain insight into the pathophysiology of coronavirus disease 2019 (COVID-19) and to develop novel treatment strategies to this disease. Here, I succinctly summarize what is currently known and unknown about the immune response during COVID-19 and discuss whether natural infections can lead to herd immunity.


Assuntos
COVID-19/imunologia , Imunidade Coletiva/imunologia , Imunidade Inata/imunologia , Animais , Humanos , SARS-CoV-2/imunologia
7.
Nat Immunol ; 9(7): 769-76, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18516037

RESUMO

The intestinal cell types responsible for defense against pathogenic organisms remain incompletely characterized. Here we identify a subset of CD11c(hi)CD11b(hi) lamina propria dendritic cells (LPDCs) that expressed Toll-like receptor 5 (TLR5) in the small intestine. When stimulated by the TLR5 ligand flagellin, TLR5(+) LPDCs induced the differentiation of naive B cells into immunoglobulin A-producing plasma cells by a mechanism independent of gut-associated lymphoid tissue. In addition, by a mechanism dependent on TLR5 stimulation, these LPDCs promoted the differentiation of antigen-specific interleukin 17-producing T helper cells and type 1 T helper cells. Unlike spleen DCs, the LPDCs specifically produced retinoic acid, which, in a dose-dependent way, supported the generation and retention of immunoglobulin A-producing cells in the lamina propria and positively regulated the differentiation interleukin 17-producing T helper cells. Our findings demonstrate unique properties of LPDCs and the importance of TLR5 for adaptive immunity in the intestine.


Assuntos
Formação de Anticorpos , Células Dendríticas/imunologia , Imunidade Celular , Imunidade nas Mucosas , Receptor 5 Toll-Like/biossíntese , Animais , Linfócitos B/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Flagelina/imunologia , Citometria de Fluxo , Imunoglobulina A/biossíntese , Imunoglobulina A/imunologia , Imuno-Histoquímica , Ativação Linfocitária/imunologia , Camundongos , Mucosa/citologia , Mucosa/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Tretinoína/imunologia , Tretinoína/metabolismo
8.
J Immunol ; 198(2): 895-907, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27903737

RESUMO

Allergic asthma develops in the mucosal tissue of small bronchi. At these sites, local cytokine production by Th2/Th17 cells is believed to be critical for the development of tissue eosinophilia/neutrophilia. Using the mouse trachea as a relevant model of human small airways, we performed advanced in vivo dynamic and in situ static imaging to visualize individual cytokine-producing T cells in the airway mucosa and to define their immediate cellular environment. Upon allergen sensitization, newly recruited CD4+ T cells formed discrete Ag-driven clusters with dendritic cells (DCs). Within T cell-DC clusters, a small fraction of CD4+ T cells produced IL-13 or IL-17 following prolonged Ag-specific interactions with DCs. As a result of local Th2 cytokine signaling, eosinophils were recruited into these clusters. Neutrophils also infiltrated these clusters in a T cell-dependent manner, but their mucosal distribution was more diffuse. Our findings reveal the focal nature of allergen-driven responses in the airways and define multiple steps with potential for interference with the progression of asthmatic pathology.


Assuntos
Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Citocinas/biossíntese , Células Dendríticas/imunologia , Transferência Adotiva , Animais , Asma/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Hipersensibilidade/imunologia , Imunidade nas Mucosas/imunologia , Imuno-Histoquímica , Masculino , Camundongos , Microscopia Confocal , Mucosa Respiratória/imunologia
9.
Proc Natl Acad Sci U S A ; 113(40): E5916-E5924, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27647916

RESUMO

Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules.


Assuntos
Microscopia/métodos , Microvilosidades/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos CD/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Humanos , Imageamento Tridimensional , Selectina L/metabolismo , Linfonodos/citologia , Linfonodos/metabolismo , Proteínas de Membrana/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/ultraestrutura , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/ultraestrutura , Tiazolidinas/farmacologia
10.
Proc Natl Acad Sci U S A ; 113(38): 10643-8, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27601677

RESUMO

Afferent lymphatic vessels bring antigens and diverse populations of leukocytes to draining lymph nodes, whereas efferent lymphatics allow only lymphocytes and antigens to leave the nodes. Despite the fundamental importance of afferent vs. efferent lymphatics in immune response and cancer spread, the molecular characteristics of these different arms of the lymphatic vasculature are largely unknown. The objective of this work was to explore molecular differences behind the distinct functions of afferent and efferent lymphatic vessels, and find possible molecules mediating lymphocyte traffic. We used laser-capture microdissection and cell sorting to isolate lymphatic endothelial cells (LECs) from the subcapsular sinus (SS, afferent) and lymphatic sinus (LS, efferent) for transcriptional analyses. The results reveal marked differences between afferent and efferent LECs and identify molecules on lymphatic vessels. Further characterizations of Siglec-1 (CD169) and macrophage scavenger receptor 1 (MSR1/CD204), show that they are discriminatively expressed on lymphatic endothelium of the SS but not on lymphatic vasculature of the LS. In contrast, endomucin (EMCN) is present on the LS endothelium and not on lymphatic endothelium of the SS. Moreover, both murine and human MSR1 on lymphatic endothelium of the SS bind lymphocytes and in in vivo studies MSR1 regulates entrance of lymphocytes from the SS to the lymph node parenchyma. In conclusion, this paper reports surprisingly distinct molecular profiles for afferent and efferent lymphatics and a function for MSR1. These results may open avenues to explore some of the now-identified molecules as targets to manipulate the function of lymphatic vessels.


Assuntos
Neoplasias/genética , Neovascularização Patológica/genética , Receptores Depuradores Classe A/genética , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Sialoglicoproteínas/genética , Animais , Movimento Celular/genética , Endotélio Linfático/metabolismo , Endotélio Linfático/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imunidade Celular/genética , Linfonodos/metabolismo , Linfonodos/patologia , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Linfócitos/patologia , Camundongos , Análise em Microsséries/métodos , Neoplasias/imunologia
11.
FASEB J ; 31(6): 2477-2491, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28251930

RESUMO

In pulmonary fibrosis, an inflammatory reaction and differentiation of myofibroblasts culminate in pathologic deposition of collagen. Amine oxidase copper containing-3 (AOC3) is a cell-surface-expressed oxidase that regulates leukocyte extravasation. Here we analyzed the potential role of AOC3 using gene-modified and inhibitor-treated mice in a bleomycin-induced pulmonary fibrosis model. Inflammation and fibrosis of lungs were assessed by histologic, flow cytometric, and quantitative PCR analysis. AOC3-deficient mice showed a 30-50% reduction in fibrosis, collagen synthesis, numbers of myofibroblasts, and accumulation of CD4+ lymphocytes, NK T cells, macrophages, and type 2 innate lymphoid cells compared with wild-type control mice. AOC3-knock-in mice, which express a catalytically inactive form of AOC3, were also protected from lung fibrosis. In wild-type mice, a small-molecule AOC3 inhibitor treatment reduced leukocyte infiltration, myofibroblast differentiation, and fibrotic injury both in prophylactic and early therapeutic settings by about 50% but was unable to reverse the established fibrosis. AOC3 was also induced in myofibroblasts in human idiopathic pulmonary fibrosis. Thus, the oxidase activity of AOC3 contributes to the development of lung fibrosis mainly by regulating the accumulation of pathogenic leukocyte subtypes, which drive the fibrotic response.-Marttila-Ichihara, F., Elima, K., Auvinen, K., Veres, T. Z., Rantakari, P., Weston, C., Miyasaka, M., Adams, D., Jalkanen, S., Salmi, M. Amine oxidase activity regulates the development of pulmonary fibrosis.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Moléculas de Adesão Celular/metabolismo , Fibrose Pulmonar/enzimologia , Amina Oxidase (contendo Cobre)/genética , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Ácidos Carboxílicos , Moléculas de Adesão Celular/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Pulmão/enzimologia , Pulmão/patologia , Linfócitos/fisiologia , Camundongos , Camundongos Knockout , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Pirrolidinas
12.
Int Immunol ; 28(6): 283-92, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26714589

RESUMO

Naive lymphocytes continuously migrate from the blood into lymph nodes (LNs) via high endothelial venules (HEVs). To extravasate from the HEVs, lymphocytes undergo multiple adhesion steps, including tethering, rolling, firm adhesion and transmigration. We previously showed that autotaxin (ATX), an enzyme that generates lysophosphatidic acid (LPA), is highly expressed in HEVs, and that the ATX/LPA axis plays an important role in the lymphocyte transmigration across HEVs. However, the detailed mechanism underlying this axis's involvement in lymphocyte transmigration has remained ill-defined. Here, we show that two LPA receptors, LPA4 and LPA6, are selectively expressed on HEV endothelial cells (ECs) and that LPA4 plays a major role in the lymphocyte transmigration across HEVs in mice. In the absence of LPA4 expression, lymphocytes accumulated heavily within the HEV EC layer, compared to wild-type (WT) mice. This accumulation was also observed in the absence of LPA6 expression, but it was less pronounced. Adoptive transfer experiments using WT lymphocytes revealed that the LPA4 deficiency in ECs specifically compromised the lymphocyte transmigration process, whereas the effect of LPA6 deficiency was not significant. These results indicate that the signals evoked in HEV ECs via the LPA4 and LPA6 differentially regulate lymphocyte extravasation from HEVs in the peripheral LNs.


Assuntos
Células Endoteliais/imunologia , Linfonodos/imunologia , Linfócitos/fisiologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores Purinérgicos/metabolismo , Transferência Adotiva , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Diester Fosfórico Hidrolases/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Receptores Purinérgicos/genética , Transdução de Sinais , Migração Transendotelial e Transepitelial
13.
Proc Jpn Acad Ser B Phys Biol Sci ; 93(4): 183-195, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28413196

RESUMO

Lymphocyte recirculation between the blood and the lymphoid/non-lymphoid tissues is an essential homeostatic mechanism that regulates humoral and cellular immune responses in vivo. This system promotes the encounter of naïve T and B cells with their specific cognate antigen presented by dendritic cells, and with the regulatory cells with which they need to interact to initiate, maintain, and terminate immune responses. The constitutive lymphocyte trafficking is mediated by particular types of blood vessels, including the high endothelial venules (HEVs) in lymph nodes and Peyer's patches, and the flat-walled venules in non-lymphoid tissues including the skin. The lymphocyte migration across HEVs involves tethering/rolling, arrest/firm adhesion/intraluminal crawling, and transendothelial migration. On the other hand, relatively little is known about how lymphocytes and other types of cells migrate across the venules of non-lymphoid tissues. Here we summarize recent findings about the molecular mechanisms that govern immune cell trafficking, including the roles of chemokines and lysophospholipids in regulating immune cell motility and endothelial permeability.


Assuntos
Movimento Celular , Sistema Imunitário/citologia , Animais , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Sistema Imunitário/imunologia , Linfócitos/citologia , Linfócitos/imunologia
14.
Proc Natl Acad Sci U S A ; 110(46): 18620-5, 2013 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-24167252

RESUMO

Developmental processes of hematopoietic cells are orchestrated by transcriptional networks. GATA-1, the founding member of the GATA family of transcription factors, has been demonstrated to play crucial roles in the differentiation of erythroid cells, magakaryocytes, eosinophils, and mast cells. However, the role of GATA-1 in basophils remains elusive. Here we show that basophils abundantly express Gata1 mRNAs, and that siRNA-mediated knockdown of Gata1 resulted in impaired production of IL-4 by basophils in response to the stimulation with IgE plus antigens. ΔdblGATA mice that carry the mutated Gata1 promoter and are widely used for functional analysis of eosinophils owing to their selective loss of eosinophils showed a decreased number of basophils with reduced expression of Gata1 mRNAs. The number of basophil progenitors in bone marrow was reduced in these mice, and the generation of basophils from their bone marrow cells in culture with IL-3 or thymic stromal lymphopoietin was impaired. ΔdblGATA basophils responded poorly ex vivo to stimulation with IgE plus antigens compared with wild-type basophils as assessed by degranulation and production of IL-4 and IL-6. Moreover, ΔdblGATA mice showed impaired responses in basophil-mediated protective immunity against intestinal helminth infection. Thus, ΔdblGATA mice showed numerical and functional aberrancy in basophils in addition to the known deficiency of eosinophils. Our findings demonstrate that GATA-1 plays a key role in the generation and function of basophils and underscore the need for careful distinction of the cell lineage responsible for each phenotype observed in ΔdblGATA mice.


Assuntos
Basófilos/imunologia , Diferenciação Celular/imunologia , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/imunologia , Animais , Basófilos/metabolismo , Primers do DNA/genética , Citometria de Fluxo , Fator de Transcrição GATA1/genética , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real
15.
J Immunol ; 190(5): 2036-48, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23365076

RESUMO

Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC's effect was abrogated by ATX inhibition, whereas LPA's effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte-HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state.


Assuntos
Membrana Basal/efeitos dos fármacos , Endotélio/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Lisofosfolipídeos/farmacologia , Diester Fosfórico Hidrolases/genética , Vênulas/efeitos dos fármacos , Animais , Membrana Basal/metabolismo , Endotélio/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Lisofosfatidilcolinas/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Vênulas/metabolismo
16.
Arthritis Rheum ; 65(8): 2037-47, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23666827

RESUMO

OBJECTIVE: Lysophosphatidic acid (LPA) is a bioactive lipid that binds to a group of cell surface G protein-coupled receptors (LPA receptors 1-6 [LPA1-6 ]) and has been implicated as an important mediator of angiogenesis, inflammation, and cancer growth. This study was undertaken to analyze the effects of LPA1 on the development of arthritis. METHODS: Expression of LPA receptors on synovial tissue was analyzed by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. The effects of abrogation of LPA1 on collagen-induced arthritis (CIA) were evaluated using LPA1 -deficient mice or LPA1 antagonist. Migrating fluorescence-labeled CD11b+ splenocytes, which were transferred into the synovium of mice with CIA, were counted. CD4+ naive T cells were incubated under Th1-, Th2-, or Th17-polarizing conditions, and T helper cell differentiation was assessed. Osteoclast formation from bone marrow cells was examined. RESULTS: LPA1 was highly expressed in the synovium of patients with rheumatoid arthritis (RA) compared with that of patients with osteoarthritis. LPA1 -deficient mice did not develop arthritis following immunization with type II collagen (CII). LPA1 antagonist also ameliorated murine CIA. Abrogation of LPA1 was associated with reductions in cell infiltration, bone destruction in the joints, and interleukin-17 production from CII-stimulated splenocytes. Infiltration of transferred CD11b+ macrophages from LPA1 -deficient mice into the synovium was suppressed compared with infiltration of macrophages from wild-type mice. LPA1 antagonist inhibited the infiltration of macrophages from wild-type mice. Differentiation into Th17, but not Th1 or Th2, and osteoclast formation were also suppressed under conditions of LPA1 deficiency or LPA1 inhibition in vitro. CONCLUSION: Collectively, these results indicate that LPA/LPA1 signaling contributes to the development of arthritis via cellular infiltration, Th17 differentiation, and osteoclastogenesis. Thus, LPA1 may be a promising target molecule for RA therapy.


Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Membrana Sinovial/metabolismo , Idoso , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Antígeno CD11b , Diferenciação Celular , Transplante de Células , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/deficiência , Transdução de Sinais , Baço/metabolismo , Baço/patologia , Membrana Sinovial/patologia , Células Th17
17.
J Immunol ; 189(1): 191-9, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22634622

RESUMO

Although the spleen plays an important role in host defense against infection, the mechanism underlying the migration of the innate immune cells, plasmacytoid dendritic cells (pDCs), into the spleen remains ill defined. In this article, we report that pDCs constitutively migrate into the splenic white pulp (WP) in a manner dependent on the chemokine receptors CCR7 and CXCR4. In CCR7-deficient mice and CCR7 ligand-deficient mice, compared with wild-type (WT) mice, substantially fewer pDCs were found in the periarteriolar lymphoid sheath of the splenic WP under steady-state conditions. In addition, the migration of adoptively transferred CCR7-deficient pDCs into the WP was significantly worse than that of WT pDCs, supporting the idea that pDC trafficking to the splenic WP requires CCR7 signaling. WT pDCs responded to a CCR7 ligand with modest chemotaxis and ICAM-1 binding in vitro, and priming with the CCR7 ligand enabled the pDCs to migrate efficiently toward low concentrations of CXCL12 in a CXCR4-dependent manner, raising the possibility that CCR7 signaling enhances CXCR4-mediated pDC migration. In agreement with this hypothesis, CCL21 and CXCL12 were colocalized on fibroblastic reticular cells in the T cell zone and in the marginal zone bridging channels, through which pDCs appeared to enter the WP. Furthermore, functional blockage of CCR7 and CXCR4 abrogated pDC trafficking into the WP. Collectively, these results strongly suggest that pDCs employ both CCR7 and CXCR4 as critical chemokine receptors to migrate into the WP under steady-state conditions.


Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Receptores CCR7/fisiologia , Receptores CXCR4/fisiologia , Transdução de Sinais/imunologia , Baço/imunologia , Animais , Movimento Celular/genética , Células Dendríticas/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais/genética , Baço/citologia , Baço/metabolismo
18.
Biophys J ; 105(3): 555-64, 2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23931303

RESUMO

Single particle tracking is widely used to study protein movement with high spatiotemporal resolution both in vitro and in cells. Quantum dots, which are semiconductor nanoparticles, have recently been employed in single particle tracking because of their intense and stable fluorescence. Although single particles inside cells have been tracked in three spatial dimensions (X, Y, Z), measurement of the angular orientation of a molecule being tracked would significantly enhance our understanding of the molecule's function. In this study, we synthesized highly polarized, rod-shaped quantum dots (Qrods) and developed a coating method that optimizes the Qrods for biological imaging. We describe a Qrod-based single particle tracking technique that blends optical nanometry with nanomaterial science to simultaneously measure the three-dimensional and angular movements of molecules. Using Qrods, we spatially tracked a membrane receptor in living cells in four dimensions with precision close to the single-digit range in nanometers and degrees.


Assuntos
Imagem Óptica/métodos , Pontos Quânticos/metabolismo , Animais , Imageamento Tridimensional/métodos , Macrófagos/metabolismo , Camundongos , Imagem Óptica/instrumentação , Pontos Quânticos/química
19.
Int Immunol ; 24(11): 705-17, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22875842

RESUMO

Moesin is a member of the ezrin-radixin-moesin (ERM) family of cytoskeletal proteins. These proteins organize membrane domains by interacting with plasma membrane proteins and the actin cytoskeleton. Because of their high sequence similarity, ERM proteins are usually thought to be functionally redundant. Lymphocytes express two ERM proteins, ezrin and moesin. Whether each ERM plays a specialized role in lymphocytes, particularly in vivo, remains unknown. Here, we show that moesin has a crucial, non-redundant role in lymphocyte homeostasis. Moesin-deficient mice exhibited decreases in both T and B cells in the peripheral blood and lymph nodes, but not in the spleen. This phenotype was recapitulated in bone marrow (BM) chimeras with a hematopoietic moesin deficiency. Although the T and B cells apparently developed without major defects in the moesin-deficient mice, T cell egress from the thymus and immature B cell egress from the BM were impaired. In the periphery, both T and B cells showed delayed egress from lymphoid organs. We showed that moesin is the primary phosphorylated ERM subject to dynamic regulation during cell shape changes and migration. Our findings identify a previously unknown, non-redundant function of moesin in lymphocyte homeostasis in regulating lymphocyte egress from lymphoid organs.


Assuntos
Linfócitos B/imunologia , Homeostase/imunologia , Proteínas dos Microfilamentos/imunologia , Linfócitos T/imunologia , Animais , Animais Recém-Nascidos , Linfócitos B/metabolismo , Western Blotting , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/ultraestrutura , Movimento Celular/imunologia , Forma Celular/imunologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Feminino , Expressão Gênica , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica de Varredura , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/imunologia , Baço/metabolismo , Linfócitos T/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismo
20.
J Immunol ; 187(5): 2268-77, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21775684

RESUMO

Eosinophils are abundant in the lamina propria of the small intestine, but they rarely show degranulation in situ under steady-state conditions. In this study, using two novel mAbs, we found that intestinal eosinophils constitutively expressed a high level of an inhibitory receptor signal regulatory protein α (SIRPα)/CD172a and a low, but significant, level of a tetraspanin CD63, whose upregulation is closely associated with degranulation. Cross-linking SIRPα/CD172a on the surface of wild-type eosinophils significantly inhibited the release of eosinophil peroxidase induced by the calcium ionophore A23187, whereas this cross-linking effect was not observed in eosinophils isolated from mice expressing a mutated SIRPα/CD172a that lacks most of its cytoplasmic domain (SIRPα Cyto(-/-)). The SIRPα Cyto(-/-) eosinophils showed reduced viability, increased CD63 expression, and increased eosinophil peroxidase release with or without A23187 stimulation in vitro. In addition, SIRPα Cyto(-/-) mice showed increased frequencies of Annexin V-binding eosinophils and free MBP(+)CD63(+) extracellular granules, as well as increased tissue remodeling in the small intestine under steady-state conditions. Mice deficient in CD47, which is a ligand for SIRPα/CD172a, recapitulated these phenomena. Moreover, during Th2-biased inflammation, increased eosinophil cell death and degranulation were obvious in a number of tissues, including the small intestine, in the SIRPα Cyto(-/-) mice compared with wild-type mice. Collectively, our results indicated that SIRPα/CD172a regulates eosinophil homeostasis, probably by interacting with CD47, with substantial effects on eosinophil survival. Thus, SIRPα/CD172a is a potential therapeutic target for eosinophil-associated diseases.


Assuntos
Eosinófilos/metabolismo , Homeostase/imunologia , Receptores Imunológicos/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Western Blotting , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Degranulação Celular/imunologia , Separação Celular , Cromatografia Líquida , Eosinófilos/imunologia , Feminino , Citometria de Fluxo , Imunofluorescência , Imunidade nas Mucosas/imunologia , Imunoprecipitação , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Glicoproteínas da Membrana de Plaquetas/imunologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Imunológicos/metabolismo , Tetraspanina 30
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