RESUMO
The secreted protein developmental endothelial locus 1 (DEL-1) regulates inflammatory cell recruitment and protects against inflammatory pathologies in animal models. Here, we investigated DEL-1 in inflammatory arthritis using collagen-induced arthritis (CIA) and collagen Ab-induced arthritis (CAIA) models. In both models, mice with endothelium-specific overexpression of DEL-1 were protected from arthritis relative to WT controls, whereas arthritis was exacerbated in DEL-1-deficient mice. Compared with WT controls, mice with collagen VI promoter-driven overexpression of DEL-1 in mesenchymal cells were protected against CIA but not CAIA, suggesting a role for DEL-1 in the induction of the arthritogenic Ab response. Indeed, DEL-1 was expressed in perivascular stromal cells of the lymph nodes and inhibited Tfh and germinal center B cell responses. Mechanistically, DEL-1 inhibited DC-dependent induction of Tfh cells by targeting the LFA-1 integrin on T cells. Overall, DEL-1 restrained arthritis through a dual mechanism, one acting locally in the joints and associated with the anti-recruitment function of endothelial cell-derived DEL-1; the other mechanism acting systemically in the lymph nodes and associated with the ability of stromal cell-derived DEL-1 to restrain Tfh responses. DEL-1 may therefore be a promising therapeutic for the treatment of inflammatory arthritis.
Assuntos
Artrite Experimental/prevenção & controle , Proteínas de Ligação ao Cálcio/fisiologia , Moléculas de Adesão Celular/fisiologia , Ativação Linfocitária , Células T Auxiliares Foliculares/imunologia , Animais , Diferenciação Celular , Feminino , Centro Germinativo/imunologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Estromais/química , Células T Auxiliares Foliculares/citologiaRESUMO
Adhesion molecules are known to play major roles in the initiation and stabilization of cell-to-cell contacts during the immunological response. Human immunodeficiency virus type 1 (HIV-1) exploits those interactions to facilitate infection and propagation processes. The primary objective of the present study was to investigate the ability of antagonists specific for lymphocyte function-associated antigen 1 (LFA-1) to diminish HIV-1 infection and transmission. We demonstrate here that LFA-1 antagonists can significantly reduce HIV-1 replication in primary human cells and virus propagation by affecting cell-to-cell interactions. Moreover, the inhibition of LFA-1-mediated adhesion events also potentiates the antiviral efficacy of the peptide fusion inhibitor T-20. Altogether, our data suggest that LFA-1 antagonists represent promising antiviral agents. Antiadhesion therapy could be considered a complementary strategy targeting cellular functions essential for HIV-1 spreading and against which the combined therapy currently used displays a limited efficacy.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/fisiologia , Fragmentos de Peptídeos/farmacologia , Ácidos Ftálicos/farmacologia , beta-Alanina/análogos & derivados , Linhagem Celular , Células Dendríticas/fisiologia , Sinergismo Farmacológico , Enfuvirtida , Humanos , Molécula 1 de Adesão Intercelular/análise , Lovastatina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Vírion/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , beta-Alanina/farmacologiaRESUMO
T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Comunicação Celular/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Ativação Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/fisiologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/metabolismo , Comunicação Celular/genética , Linhagem Celular Tumoral , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/biossíntese , Antígeno-1 Associado à Função Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/citologia , Talina/metabolismo , Regulação para Cima/imunologiaRESUMO
Besides interactions between the viral envelope glycoproteins with cell surface receptors, interactions between cell-derived molecules incorporated onto virions and their ligand could also modulate HIV type-1 (HIV-1) entry inside CD4(+) T lymphocytes. Although incorporation of host ICAM-1 within HIV-1 increases both virus attachment and fusion, the precise mechanism through which this phenomenon is occurring is still unclear. We demonstrate in this study that activation of primary human CD4(+) T lymphocytes increases LFA-1 affinity and avidity states, two events promoting the early events of the HIV-1 replication cycle through interactions between virus-embedded host ICAM-1 and LFA-1 clusters. Confocal analyses suggest that HIV-1 is concentrated in microdomains rich in LFA-1 clusters that also contain CD4 and CXCR4 molecules. Experiments performed with specific inhibitors revealed that entry of HIV-1 in activated CD4(+) T cells is regulated by LFA-1-dependent ZAP70, phospholipase Cgamma1, and calpain enzymatic activities. By using laboratory and clinical strains of HIV-1 produced in primary human cells, we demonstrate the importance of the LFA-1 activation state and cluster formation in the initial step of the virus life cycle. Overall, these data provide new insights into the complex molecular events involved in HIV-1 binding and entry.
Assuntos
Linfócitos T CD4-Positivos/virologia , Citoesqueleto/metabolismo , Citoesqueleto/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Ativação Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Transdução de Sinais/imunologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/fisiologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Calpaína/fisiologia , Adesão Celular/imunologia , Linhagem Celular , Citoesqueleto/imunologia , HIV-1/metabolismo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/metabolismo , Fusão de Membrana/imunologia , Muromonab-CD3/farmacologia , Fosfolipase C gama , Fito-Hemaglutininas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/fisiologia , Proteína-Tirosina Quinase ZAP-70RESUMO
The integrins can activate signaling pathways, but the final downstream outcome of these pathways is often unclear. This study analyzes the consequences of signaling events initiated by the interaction of the leukocyte integrin LFA-1 with its ligand, dimeric ICAM-1. We show that the active form of LFA-1 regulates its own function on primary human T cells by directing the remodeling of the F-actin cytoskeleton to strengthen T cell adhesion to ICAM-1. Confocal microscopy revealed that both F-actin bundling and overall levels of F-actin are increased in the ICAM-1-adhering T cells. This increase in F-actin levels and change in F-actin distribution was quantitated for large numbers of T cells using the technique of laser scanning cytometry and was found to be significant. The study went on to show that clustering of conformationally altered LFA-1 is essential for the changes in F-actin, and a model is proposed in which clustered, high-avidity T cell LFA-1, interacting with multivalent ICAM-1, causes LFA-1 signaling, which results in F-actin polymerization and higher-order F-actin bundling. The findings demonstrate that LFA-1 acts not only as an adhesion receptor but also as a signaling receptor by actively initiating the F-actin reorganization that is essential for many T cell-dependent processes.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Adjuvantes Imunológicos/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Transdução de Sinais/imunologia , Linfócitos T/fisiologia , Cátions Bivalentes/farmacologia , Adesão Celular/imunologia , Citoesqueleto/metabolismo , Dimerização , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Antígeno-1 Associado à Função Linfocitária/metabolismo , Substâncias Macromoleculares , Magnésio/farmacologia , Microscopia Confocal , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/metabolismoRESUMO
Drug antibody conjugates can enhance the activity of monoclonal antibodies (MoAb) and idarubicin-MoAb conjugates have led to tolerance induction with antibodies which are inactive when used alone. It has been reported that, in mice, antibodies to ICAM-1 and LFA-1 have to be used together to induce tolerance to cardiac allografts; here we show that these monoclonal antibodies, conjugated to idarubicin, can lead to tolerance induction to cardiac allografts when used alone.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Coração , Idarubicina/uso terapêutico , Imunoconjugados/uso terapêutico , Imunossupressores/uso terapêutico , Molécula 1 de Adesão Intercelular/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Idarubicina/administração & dosagem , Idarubicina/farmacologia , Tolerância Imunológica , Imunoconjugados/farmacologia , Imunossupressores/farmacologia , Molécula 1 de Adesão Intercelular/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Transplante de Pele/imunologia , Transplante Homólogo/imunologiaRESUMO
Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).
Assuntos
Molécula 1 de Adesão Intercelular/química , Molécula 1 de Adesão Intercelular/ultraestrutura , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Cisteína , DNA Complementar , Dimerização , Humanos , Molécula 1 de Adesão Intercelular/genética , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/fisiologia , Microscopia Eletrônica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , Ressonância de Plasmônio de Superfície , Propriedades de Superfície , TransfecçãoRESUMO
The thiol protease bromelain has been shown to remove T-cell CD44 molecules from lymphocytes and to affect T-cell activation. We investigated the effect of a highly purified bromelain protease F9 (F9) on the adhesion of peripheral blood lymphocytes (PBL) to human umbilical vein endothelial cells (HUVEC). Preincubation of the lymphocytes with F9 reduced the adherence to about 20% of unstimulated and to about 30% of phorbol-dibutyrate (P(Bu)2) stimulated lymphocytes. Using flow cytometry, both crude bromelain and protease F9 reduced the expression of CD44, but not of LFA-1, on PBL. F9 was about 10 times more active than crude bromelain; at 2.5 micrograms/ml of F9 about 97% inhibition of CD44 expression was found. A mAb against CD44 was tested and found to block the F9-induced decrease in PBL-binding to HUVEC. The results indicate that F9 selectively decreases the CD44 mediated binding of PBL to HUVEC.