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1.
Eur J Immunol ; 48(2): 250-257, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28980305

RESUMO

Viruses, particularly the Epstein-Barr virus (EBV) has long been suspected to exacerbate acute arthritic symptoms. However, the cell populations that contribute to import viruses into the inflamed tissues remain to be identified. In the present study, we have investigated the role of monocytes in the transport of Murid herpesvirus 68 (MHV-68), a mouse virus closely related to EBV, using the serum transfer-induced arthritis (STIA) model. We found compelling evidence that MHV-68 infection markedly increased disease severity in NR4A1-/- mice, which are deficient for Ly6Clow monocytes. In contrast, the MHV-68-induced enhancement of joint inflammation was lessened in CCR2-/- mice, suggesting the involvement of inflammatory Ly6Chigh monocytes in viral transport. We also observed that following selective depletion of monocyte subsets by administration of clodronate, MHV-68 transport into the synovium occurs only in the presence of Ly6Chigh monocytes. Tracking of adoptively transferred Ly6Chigh GFP infected monocytes into arthritic CCR2-/- mice by two-photon intravital microscopy showed that this monocyte subset has the capacity to deliver viruses to inflamed AR joints, as confirmed by the detection of viral DNA in inflamed tissues of recipient mice. We thus conclude that Ly6Chigh monocytes import MHV-68 when they are mobilized to the inflamed arthritic joint.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 4/fisiologia , Monócitos/imunologia , Rhadinovirus/fisiologia , Infecções Tumorais por Vírus/imunologia , Transferência Adotiva , Animais , Antígenos Ly/metabolismo , Artrite Experimental/virologia , Artrite Reumatoide/virologia , Células Cultivadas , DNA Viral/análise , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/virologia , Feminino , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/transplante , Muridae , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Receptores CCR2/genética , Rhadinovirus/patogenicidade , Infecções Tumorais por Vírus/virologia
2.
Eur J Immunol ; 46(12): 2789-2800, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27600773

RESUMO

Monocytes are central to the physiopathology of arthritis, but their roles in progression and resolution of the disease remain to be clarified. Using NR4A1-/- mice, which lack patrolling lymphocyte antigen 6C (Ly6Clow ) monocytes, we found that inflammatory Ly6Chigh monocytes contribute to rapid development of arthritis in a serum transfer-induced arthritis (STIA) model. Our experiments suggest that patrolling monocytes do not promote the initiation and progression of arthritis in mice, as severity of symptoms was amplified in NR4A1-/- mice. Moreover, we show that treatment of arthritic wild type (WT) mice with cytosporone B (Csn-B), a NR4A1-specific agonist, significantly reduces severity of disease. Effects of Csn-B were absent in monocyte-depleted mice treated with clodronate until Ly6Clow monocytes were restored. Adoptive transfer of Ly6Clow monocytes in arthritic NR4A1-/- mice treated with Csn-B reduces joint inflammation, supporting the regulatory role of Ly6Clow subset on disease development. Our results also reveal that administration of Csn-B to arthritic mice enhances levels of circulating CD4+ CD25+ FoxP3+ Treg cells, a process requiring the presence of Ly6Clow monocytes. Together, these data indicate that Ly6Chigh monocytes are involved in the initiation and progression of arthritis and Ly6Clow monocytes contribute to reduce joint inflammation through the mobilization of Treg cells.


Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite/imunologia , Inflamação/imunologia , Articulações/imunologia , Monócitos/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Antígenos Ly/metabolismo , Artrite/tratamento farmacológico , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Fenilacetatos/uso terapêutico
3.
Sensors (Basel) ; 17(10)2017 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-28976942

RESUMO

We introduce here a microfluidic cell culture platform or spheroid culture chamber array (SCCA) that can synthesize, culture, and enable fluorescence imaging of 3D cell aggregates (typically spheroids) directly on-chip while specifying the flow of reagents in each chamber via the use of an array of passive magnetic valves. The SCCA valves demonstrated sufficient resistance to burst (above 100 mBar), including after receiving radiotherapy (RT) doses of up to 8 Gy combined with standard 37 °C incubation for up to 7 days, enabling the simultaneous synthesis of multiple spheroids from different cell lines on the same array. Our results suggest that SCCA would be an asset in drug discovery processes, seeking to identify combinatorial treatments.


Assuntos
Microfluídica , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Neoplasias , Esferoides Celulares
4.
J Immunol ; 187(4): 1547-51, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21768396

RESUMO

In multivesicular bodies, HLA-DM (DM) assists the loading of antigenic peptides on classical MHC class II molecules such as HLA-DR. In cells expressing HLA-DO (DO), DM is redistributed from the internal vesicles to the limiting membrane of these organelles. This suggests that DO might reduce DM incorporation into exosomes, which are shed upon fusion of multivesicular bodies with the plasma membrane. To test this hypothesis, we used the 721.45 B lymphoblastoid cell line and different HeLa cell transfectants. We demonstrate that the poor recovery of DM in exosomes as compared with HLA-DR is not the mere reflection of differences in protein expression. Indeed, we found that DO contributes to the inefficient transfer of DM to exosomes. This negative regulation requires an intact di-leucine endosomal sorting motif in the cytoplasmic tail of HLA-DOß. These results demonstrate that canonical sorting signals and protein-protein interactions modulate the selection of MHC protein cargos.


Assuntos
Exossomos/imunologia , Antígenos HLA-D/imunologia , Motivos de Aminoácidos , Exossomos/genética , Exossomos/metabolismo , Antígenos HLA-D/genética , Antígenos HLA-D/metabolismo , Células HeLa , Humanos , Transporte Proteico/genética , Transporte Proteico/imunologia
5.
Elife ; 112022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35192455

RESUMO

Virtually all fishes rely on flows of water to transport food to the back of their pharynx. While external flows that draw food into the mouth are well described, how intraoral waterflows manage to deposit food at the esophagus entrance remains unknown. In theory, the posteriorly moving water must, at some point, curve laterally and/or ventrally to exit through the gill slits. Such flows would eventually carry food away from the esophagus instead of toward it. This apparent paradox calls for a filtration mechanism to deviate food from the suction-feeding streamlines. To study this gap in our fundamental understanding of how fishes feed, we developed and applied a new technique to quantify three-dimensional (3D) patterns of intraoral waterflows in vivo. We combined stereoscopic high-speed X-ray videos to quantify skeletal motion (XROMM) with 3D X-ray particle tracking (XPT) of neutrally buoyant spheres of 1.4 mm in diameter. We show, for carp (Cyprinus carpio) and tilapia (Oreochromis niloticus), that water tracers displayed higher curvatures than food tracers, indicating an inertia-driven filtration. In addition, tilapia also exhibited a 'central jet' flow pattern, which aids in quickly carrying food to the pharyngeal jaw region. When the food was trapped at the branchial basket, it was resuspended and carried more centrally by periodical bidirectional waterflows, synchronized with head-bone motions. By providing a complete picture of the suction-feeding process and revealing fundamental differences in food transport mechanisms among species, this novel technique opens a new area of investigation to fully understand how most aquatic vertebrates feed.


Assuntos
Carpas , Tilápia , Animais , Fenômenos Biomecânicos , Comportamento Alimentar , Boca , Sucção , Água
6.
Traffic ; 10(10): 1518-27, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19566897

RESUMO

Major histocompatibility complex class II (MHC-II) molecules accumulate in exocytic vesicles, called exosomes, which are secreted by antigen presenting cells. These vesicles are released following the fusion of multivesicular bodies (MVBs) with the plasma membrane. The molecular mechanisms regulating cargo selection remain to be fully characterized. As ubiquitination of the MHC-II beta-chain cytoplasmic tail has recently been demonstrated in various cell types, we sought to determine if this post-translational modification is required for the incorporation of MHC-II molecules into exosomes. First, we stably transfected HeLa cells with a chimeric HLA-DR molecule in which the beta-chain cytoplasmic tail is replaced by ubiquitin. Western blot analysis did not indicate preferential shedding of these chimeric molecules into exosomes. Next, we forced the ubiquitination of MHC-II in class II transactivator (CIITA)-expressing HeLa and HEK293 cells by transfecting the MARCH8 E3 ubiquitin ligase. Despite the almost complete downregulation of MHC-II from the plasma membrane, these molecules were not enriched in exosomes. Finally, site-directed mutagenesis of all cytoplasmic lysine residues on HLA-DR did not prevent inclusion into these vesicles. Taken together, these results demonstrate that ubiquitination of MHC-II is not a prerequisite for incorporation into exosomes.


Assuntos
Exossomos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Western Blotting , Citoplasma/metabolismo , Regulação para Baixo , Citometria de Fluxo , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Células HeLa , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
7.
Immunology ; 127(3): 408-17, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19019088

RESUMO

Human leucocyte antigen (HLA)-DO is a non-classical major histocompatibility complex class II molecule which modulates the function of HLA-DM and the loading of antigenic peptides on molecules such as HLA-DR. The bulk of HLA-DO associates with HLA-DM and this interaction is critical for HLA-DO egress from the endoplasmic reticulum. HLA-DM assists the early steps of HLA-DO maturation presumably through the stabilization of the interactions between the N-terminal regions of the alpha and beta chains. To evaluate a possible role for HLA-DM in influencing the conformation of HLA-DO, we made use of a monoclonal antibody, Mags.DO5, that was raised against HLA-DO/DM complexes. Using transfected cells expressing mismatched heterodimers between HLA-DR and -DO chains, we found that the epitope for Mags.DO5 is located on the DObeta chain and that Mags.DO5 reactivity was increased upon cotransfection with HLA-DM. Our results suggest that HLA-DM influences the folding of HLA-DO in the endoplasmic reticulum. A mutant HLA-DO showing reduced capacity for endoplasmic reticulum egress was better recognized by Mags.DO5 in the presence of HLA-DM. On the other hand, an HLA-DO mutant capable of endoplasmic reticulum egress on its own was efficiently recognized by Mags.DO5, irrespective of the presence of HLA-DM. Taken together, our results suggest that HLA-DM acts as a private chaperone, directly assisting the folding of HLA-DO to promote egress from the endoplasmic reticulum.


Assuntos
Antígenos HLA-D/imunologia , Dobramento de Proteína , Anticorpos Monoclonais/imunologia , Apresentação de Antígeno/imunologia , Linfócitos B/imunologia , Retículo Endoplasmático/imunologia , Epitopos de Linfócito B/análise , Antígenos HLA-D/química , Antígenos HLA-D/genética , Células HeLa , Humanos , Transfecção
8.
Cell Rep ; 20(8): 1830-1843, 2017 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-28834747

RESUMO

The signals that regulate the fate of circulating monocytes remain unknown. In the present study, we demonstrate that triggering of the NOD2 receptor by muramyl dipeptide (MDP) converts inflammatory Ly6Chigh monocytes into patrolling Ly6Clow monocytes. Administration of MDP to Nr4a1-/- mice, which lack Ly6Clow monocytes, or to Ly6Clow-depleted mice led to the emergence of blood-patrolling monocytes with a profile similar to that of Ly6Clow monocytes, including high expression of CX3CR1 and LFA1. Using intravital microscopy in animal models of inflammatory diseases, we also found that converted Ly6Chigh monocytes patrol the endothelium of blood vessels and that their presence contributes to a reduction in the inflammatory response following MDP injection. Our results demonstrate that NOD2 contributes to the regulation of blood monocytes and suggest that it could be therapeutically targeted to treat inflammatory diseases.


Assuntos
Antígenos Ly/imunologia , Monócitos/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Adaptadora de Sinalização NOD2/genética
9.
Arthritis Res Ther ; 18: 10, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26759164

RESUMO

BACKGROUND: Synovial infiltration of monocytes is commonly associated with inflammation in rheumatoid arthritis (RA). Toll-like receptors (TLRs) are innate sensors that recognize cell debris and microbial components in host, a process contributing to maintain chronic inflammation in RA. We assessed the expression levels of TLR2 and TLR9 in monocyte subsets of active RA patients and characterized their cytokine profiles in response to synthetic and viral TLR2 and TLR9 agonists, including Epstein-Barr virus (EBV) which is suspected to contribute to RA symptoms. METHODS: Prevalence of monocyte subsets CD14(++) CD16(-), CD14(+) CD16(+) and CD14(low) CD16(++) was evaluated in blood and synovial fluids of active RA patients and levels of TLR2 and TLR9 in monocyte subsets were measured by flow cytometry. Enriched monocytes derived from RA patients and healthy donors were stimulated in vitro with synthetic TLR2 and TLR9 agonists and with EBV particles or viral DNA. Intracellular cytokine profiles were determined in respective monocyte subsets. Finally, the presence of EBV genome was evaluated by real-time PCR in blood and synovial monocytes of RA patients. RESULTS: Numbers of CD14(+) CD16(+) and CD14(low) CD16(++) were found to increase in blood of RA patients compared to healthy controls, while all three subsets were detected in synovial fluids. TLR2 is abundantly expressed on blood and synovial CD14(++) CD16(-) and CD14(+) CD16(+) monocytes from RA patients. Levels of TLR9 were increased on all three subsets of blood monocytes but markedly enhanced in monocytes isolated from synovial fluids. Compared to healthy controls, CD14(++) CD16(-) monocytes of RA patients displayed an enlarged capacity to produce proinflammatory cytokines after stimulation with synthetic TLR2 and TLR9 agonists while both CD14(++) CD16(-) and CD14(+) CD16(+) monocytes showed increased response to EBV stimulation. The presence of EBV genome was also detected in monocytes and neutrophils of a significant proportion of patients. CONCLUSION: Patients with active RA show an increased expression of TLR2 and TLR9 on monocyte subsets and display higher production of inflammatory cytokines in response to TLR agonists. The presence of EBV genome in monocytes and neutrophils reinforces the suspected role of the virus in the exacerbation of RA symptoms.


Assuntos
Artrite Reumatoide/metabolismo , Monócitos/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/biossíntese , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/biossíntese , Idoso , Feminino , Citometria de Fluxo/métodos , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Ácidos Teicoicos/farmacologia , Resultado do Tratamento
10.
Anal Chim Acta ; 906: 98-109, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26772129

RESUMO

The soil redox potential (Eh) can provide essential information to characterise soil conditions. In practice, however, numerous problems may arise regarding: (i) Eh determination in soils, especially aerobic soils, e.g. variations in the instrumentation and methodology for Eh measurement, high spatial and temporal Eh variability in soils, irreversibility of the redox reaction at the surface electrode, chemical disequilibrium; and (ii) measurement interpretation. This study aimed at developing a standardised method for redox potential measurement in soils, in order to use Eh as a soil quality indicator. This paper presents practical improvements in soil Eh measurement, especially regarding the control of electromagnetic perturbations, electrode choice and preparation, soil sample preparation (drying procedure) and soil:water extraction rate. The repeatability and reproducibility of the measurement method developed are highlighted. The use of Eh corrected at pH7, pe+pH or rH2, which are equivalent notions, is proposed to facilitate interpretation of the results. The application of this Eh measurement method allows characterisation of soil conditions with sufficient repeatability, reproducibility and accuracy to demonstrate that conservation agriculture systems positively alter the protonic and electronic balance of soil as compared to conventional systems.


Assuntos
Produtos Agrícolas , Solo/química , Concentração de Íons de Hidrogênio , Oxirredução
11.
J Innate Immun ; 6(2): 159-68, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-23988515

RESUMO

Leukotriene B4 (LTB4) is an endogenous lipid mediator of inflammation derived from arachidonic acid by the sequential action of cytosolic phospholipase A2 and 5-lipoxygenase. This mediator was initially recognized for its involvement in the recruitment of neutrophils. However, in the last decade, LTB4 has been clearly demonstrated to play a significant role in the control of microbial infections through its ability to activate host innate defenses. In this review, we will focus on the modulator effects of LTB4 on the innate defenses and discuss its therapeutic potential against viral pathogens.


Assuntos
Imunidade Inata/imunologia , Mediadores da Inflamação/imunologia , Leucotrieno B4/imunologia , Neutrófilos/imunologia , Animais , Infecções Bacterianas/imunologia , Humanos , Modelos Imunológicos , Receptores Toll-Like/imunologia , Viroses/imunologia
12.
Eur J Immunol ; 38(5): 1225-30, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18389477

RESUMO

IL-10 is a potent anti-inflammatory cytokine interfering with antigen presentation by inducing the intracellular sequestration of MHC class II (MHC-II) molecules. Here we studied the contribution of membrane-associated RING-CH (MARCH) ubiquitin ligase family members to the IL-10-induced down-regulation of MHC-II molecules. We found that MARCH1 and MARCH8 proteins are the most potent family members for the down-regulation of MHC-II surface expression in transfected cells, but only MARCH1 mRNA expression is strongly induced by IL-10 in human primary monocytes. We detected mono- and poly-ubiquitinated forms of MHC-II molecules both in IL-10-treated monocytes and in cells transfected with MARCH1. We also show direct interaction between MHC-II and MARCH1 molecules in co-immunoprecipitation assays. Finally, we found that siRNA-mediated knockdown of MARCH1 reverses IL-10-induced MHC-II down-regulation in primary monocytes. Thus, the immunosuppressive effect of IL-10 on antigen presentation is mediated through induced expression of MARCH1.


Assuntos
Antígenos HLA-D/metabolismo , Interleucina-10/fisiologia , Monócitos/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Antígeno B7-2/metabolismo , Regulação para Baixo , Expressão Gênica/efeitos dos fármacos , Antígenos HLA-DR/metabolismo , Células HeLa , Humanos , Interferon gama/farmacologia , Interleucina-10/farmacologia , Proteínas de Membrana/metabolismo , Monócitos/efeitos dos fármacos , Proteínas Nucleares/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Transativadores/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/imunologia
13.
J Virol ; 80(19): 9789-97, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16973583

RESUMO

Human immunodeficiency virus type 1 (HIV-1) assembly, budding, and release occur mostly at the plasma membrane in T lymphocytes as well as in established nonlymphoid cell lines, while in macrophages these processes occur primarily in intracellular compartments that harbor late endosomal/multivesicular body (LE/MVB) markers, including human leukocyte antigen DR (HLA-DR). Major histocompatibility complex class II molecules (MHC-II), which are expressed in macrophages and activated T cells, have been previously reported to induce the formation of multilaminar and multivesicular endocytic MHC-II-like structures analogous to MVB upon their expression in HEK 293 cells. Here, we have examined the role of MHC-II in HIV-1 Gag targeting as well as in virus assembly and release. Expression of HLA-DR in nonlymphoid cell lines induced a relocation of Gag to intracellular compartments that harbored LE/MVB markers and increased the accumulation of viral particles assembling intracellularly. Consequently, viral production and release from the cell surface was found to be substantially decreased in HLA-DR-expressing cells. This process was specific, since it was not observed with HLA-DR molecules lacking their cytoplasmic tails, nor with structurally related but functionally distinct MHC-II molecules such as HLA-DM or HLA-DO. Importantly, virus released intracellularly in HLA-DR-expressing cells retained infectivity. Overall, these results suggest a role of MHC-II molecules in promoting HIV-1 assembly and budding to LE/MVB and raise the possibility that this activity might be part of a normal pathway of virus production in cell types physiologically expressing MHC-II molecules, such as macrophages.


Assuntos
Endossomos/imunologia , HIV-1/fisiologia , Antígenos de Histocompatibilidade Classe II/imunologia , Montagem de Vírus , Linhagem Celular , Produtos do Gene gag/imunologia , HIV-1/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Fatores de Tempo , Vírion/metabolismo
14.
J Cell Sci ; 118(Pt 20): 4679-87, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16188937

RESUMO

The p35 isoform of the human invariant chain (Iip35) contains an N-terminal RXR endoplasmic-reticulum (ER) retention signal that becomes nonfunctional only after assembly with MHC-class-II molecules. We have previously shown that the MHC-class-II beta-chain cytoplasmic tail is crucial for the maturation of class-II/Iip35 complexes. In order to shed some light on the molecular determinants involved in shielding the RXR motif, we performed site-directed mutagenesis of the DRbeta chain and Ii cytoplasmic domains. Chimeric beta chains with irrelevant cytoplasmic tails allowed the efficient transport of Iip35 out of the ER in transiently transfected HEK 293T cells. An alanine scan of the cytoplasmic tail of HLA-DRbeta confirmed that no specific motif is required to overcome ER retention. Surprisingly, a beta chain with a three-amino-acid-long cytoplasmic tail (Tyr-Phe-Arg) was sufficient to overcome the Iip35 RXR motif. Moreover, replacement of residues F231 and R232 with alanines created a cytoplasmic tail (Tyr-Ala-Ala) that allowed ER egress. Given the limited length of this tail, steric hindrance would only be possible if the Ii ER retention motif was close to the membrane in the first place. However, this is not likely because an Ii molecule with an internal cytoplasmic deletion bringing the RXR motif closer to the membrane is not retained in the ER, even in the absence of class-II molecules. These results suggest that MHC-class-II molecules overcome ER retention and prevent COPI binding to the Iip35 RXR motif through a mechanism distinct from steric hindrance by its beta chain.


Assuntos
Aminoácidos/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Linhagem Celular , Membrana Celular , Citometria de Fluxo , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Transporte Proteico , Alinhamento de Sequência , Transfecção
15.
Proc Natl Acad Sci U S A ; 102(18): 6443-8, 2005 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-15849268

RESUMO

B lymphocytes express the nonclassical class II molecule HLA-DO, which modulates the peptide loading activity of HLA-DM in the endocytic pathway. Binding to HLA-DM is required for HLA-DO to egress from the endoplasmic reticulum (ER). To gain insights into the mode of action of DO and on the role of DM in ER release, we sought to identify DM-binding residues on DO. Our results show that DOalpha encompasses the binding site for HLA-DM. More specifically, mutation of residue DOalpha41 on an exposed lateral loop of the alpha1 domain affects the binding to DM, ER egress, and activity of DO. Using a series of chimeric DR/DO molecules, we confirmed the role of the alpha chain and established that a second DM-binding region is located C-terminal to the DOalpha80 residue, most probably in the alpha2 domain. Interestingly, after mutation of a buried proline (alpha11) on the floor of the putative peptide-binding groove, HLA-DO remained functional but became independent of HLA-DM for ER egress and intracellular trafficking. Collectively, these results suggest that the binding of HLA-DM to DOalpha allows the complex to egress from the ER by stabilizing intramolecular contacts between the N-terminal antiparallel beta-strands of the DOalphabeta heterodimer.


Assuntos
Linfócitos B/metabolismo , Retículo Endoplasmático/metabolismo , Antígenos HLA-D/genética , Antígenos HLA-D/metabolismo , Mutação Puntual/genética , Anticorpos Monoclonais/metabolismo , Western Blotting , Citometria de Fluxo , Células HeLa , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Mutagênese , Plasmídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/metabolismo
16.
Int Immunol ; 15(10): 1249-63, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-13679394

RESUMO

The human-specific p35 isoform of the invariant chain (Ii) includes an R-X-R endoplasmic reticulum (ER) retention motif that is inactivated upon HLA-DR binding. Although the masking is assumed to involve the cytoplasmic tails of class II molecules, the mechanism underlying this function remains to be investigated. Moreover, in light of the polymorphic nature of the class II cytosolic tails, little is known about the capacity of various isotypes or alleles to overcome the retention signal of Iip35. To gain further insights into these issues, we first addressed the proposed role of the HLA-DR cytoplasmic tails. As shown by flow cytometry, the presence of Iip35 in transfected HeLa cells prevented surface expression of HLA-DR molecules lacking their cytoplasmic tails (DRalphaTM/betaTM). These truncated class II molecules and Iip35 accumulated in the ER, and co-localized with calnexin, as determined by confocal microscopy. Sensitivity of DRalphaTM/betaTM to endoglycosidase H treatment confirmed that these molecules do not reach the trans-Golgi network when associated with Iip35. Further characterization revealed that the beta chain cytosolic tail is critical for efficient ER egress of class II/Iip35 complexes. Interestingly, our results clearly demonstrate for the first time that DP and DQ isotypes can also overcome the retention motif of Iip35 through a mechanism involving their very distinctive polymorphic beta chain cytoplasmic tails. Altogether, these results further dissect the masking of di-basic retention signals, and emphasize the interplay between class II molecules and Ii for the transport of the complex to the endocytic pathway.


Assuntos
Antígenos de Diferenciação de Linfócitos B/química , Retículo Endoplasmático/metabolismo , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Sequência de Aminoácidos , Citoplasma/metabolismo , Endocitose/fisiologia , Feminino , Antígenos HLA-DR/análise , Células HeLa , Humanos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Ácidos Siálicos/metabolismo , Transdução de Sinais
17.
J Biol Chem ; 279(18): 18472-80, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-14976194

RESUMO

Whereas the association of major histocompatibility complex (MHC) class II molecules with the cytoskeleton and their recruitment into lipid rafts play a critical role during cognate T/antigen-presenting cell interactions, MHC class II-induced signals, regions, and residues involved in their association and recruitment have not yet been fully deciphered. In this study, we show that oligomerization of HLA-DR molecules induces their association with the cytoskeleton and their recruitment into lipid rafts. The association of oligomerized HLA-DR molecules with the cytoskeleton and their recruitment into lipid rafts occur independently. Furthermore, the association with the cytoskeleton is HLA-DR-specific, since oligomerization of HLA-DP triggers its recruitment only into lipid rafts. HLA-DR molecules devoid of both alpha and beta cytoplasmic tails did not associate with the cytoskeleton, but their recruitment into lipid rafts was unimpeded. Deletion of either the alpha or beta cytoplasmic tail did not affect the association of HLA-DR with the cytoskeleton and/or recruitment into lipid rafts. HLA-DR molecules that were devoid of the alpha cytoplasmic chain and that had their beta cytoplasmic chain replaced with the HLA-DP beta chain or with a beta chain in which the residues at positions Gly(226)-His(227)-Ser(228) were substituted by alanine no longer associated with the cytoskeleton. They were, however, still recruited into lipid rafts. Together, these results support the involvement of different regions of the cytoplasmic tails in the association and the recruitment of HLA-DR into different compartments. The differential behavior of HLA-DP and -DR with respect to their association with the cytoskeleton may explain the previously described difference in their transduced signals.


Assuntos
Citoesqueleto/metabolismo , Antígenos HLA-DR/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Complexo Antígeno-Anticorpo , Sítios de Ligação , Linhagem Celular Tumoral , Citoesqueleto/fisiologia , Antígenos HLA-DP/metabolismo , Antígenos HLA-DP/fisiologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Humanos , Microdomínios da Membrana/metabolismo , Ligação Proteica , Transporte Proteico , Transdução de Sinais , Transfecção
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