RESUMO
Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl(-) blocker, whereas CLDN16 functioned as a Na(+) channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl(-) permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC.
Assuntos
Hipercalciúria/genética , Magnésio/metabolismo , Proteínas de Membrana/metabolismo , Nefrocalcinose/genética , Canais de Sódio/metabolismo , Junções Íntimas/metabolismo , Animais , Cátions Monovalentes/metabolismo , Células Cultivadas , Cloro/metabolismo , Claudinas , Imunoprecipitação , Transporte de Íons , Proteínas de Membrana/genética , Mutação , Permeabilidade , Canais de Sódio/genética , SuínosRESUMO
That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-beta-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 h, results in an approximately 80% decrease in cell cholesterol, a fall in transepithelial electrical resistance, and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins-2, -3, and -7. There were negligible changes in levels of actin and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bound particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-beta-cyclodextrin for 30 min. In contrast caveolin-1 is detected only in the apical medium after adding methyl-beta-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-beta-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane-bound particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D did not diminish methyl-beta-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41- and 48-kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Colesterol/metabolismo , Espaço Extracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Metaloproteases/metabolismo , Animais , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/ultraestrutura , Claudinas/metabolismo , Meios de Cultura , Citocalasina D/farmacologia , Dipeptídeos/farmacologia , Cães , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Espaço Extracelular/efeitos dos fármacos , Peso Molecular , Ocludina , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Fatores de Tempo , beta-Ciclodextrinas/farmacologiaAssuntos
Síndrome Hemolítico-Urêmica/patologia , Rim/patologia , Insuficiência Renal/etiologia , Adolescente , Encéfalo/patologia , Diagnóstico Diferencial , Cefaleia/etiologia , Síndrome Hemolítico-Urêmica/complicações , Síndrome Hemolítico-Urêmica/diagnóstico , Síndrome Hemolítico-Urêmica/terapia , Humanos , Hipertensão/etiologia , Rim/diagnóstico por imagem , Masculino , Náusea/etiologia , Nefrite/diagnóstico , Convulsões/etiologia , UltrassonografiaRESUMO
Although the C-terminal cytoplasmic tail of the tight junction protein occludin is heavily phosphorylated, the functional impact of most individual sites is undefined. Here, we show that inhibition of CK2-mediated occludin S408 phosphorylation elevates transepithelial resistance by reducing paracellular cation flux. This regulation requires occludin, claudin-1, claudin-2, and ZO-1. S408 dephosphorylation reduces occludin exchange, but increases exchange of ZO-1, claudin-1, and claudin-2, thereby causing the mobile fractions of these proteins to converge. Claudin-4 exchange is not affected. ZO-1 domains that mediate interactions with occludin and claudins are required for increases in claudin-2 exchange, suggesting assembly of a phosphorylation-sensitive protein complex. Consistent with this, binding of claudin-1 and claudin-2, but not claudin-4, to S408A occludin tail is increased relative to S408D. Finally, CK2 inhibition reversed IL-13-induced, claudin-2-dependent barrier loss. Thus, occludin S408 dephosphorylation regulates paracellular permeability by remodeling tight junction protein dynamic behavior and intermolecular interactions between occludin, ZO-1, and select claudins, and may have therapeutic potential in inflammation-associated barrier dysfunction.
Assuntos
Proteínas de Membrana/química , Junções Íntimas/metabolismo , Células CACO-2 , Claudina-1 , Claudina-4 , Claudinas , Eletrofisiologia , Humanos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Ocludina , Permeabilidade , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
The neurofibromatosis type 2 (NF2) tumor suppressor, Merlin, is a FERM (Four point one, Ezrin, Radixin, Moesin) domain-containing protein whose loss results in defective morphogenesis and tumorigenesis in multiple tissues. Like the closely related ERM proteins (Ezrin, Radixin, and Moesin), Merlin may organize the plasma membrane by assembling membrane protein complexes and linking them to the cortical actin cytoskeleton. We previously found that Merlin is a critical mediator of contact-dependent inhibition of proliferation and is required for the establishment of stable adherens junctions (AJs) in cultured cells. Here, we delineate the molecular function of Merlin in AJ establishment in epidermal keratinocytes in vitro and confirm that a role in AJ establishment is an essential function of Merlin in vivo. Our studies reveal that Merlin can associate directly with α-catenin and link it to Par3, thereby providing an essential link between the AJ and the Par3 polarity complex during junctional maturation.
Assuntos
Junções Aderentes/metabolismo , Polaridade Celular , Epiderme/embriologia , Epiderme/crescimento & desenvolvimento , Neurofibromatose 2/metabolismo , Neurofibromina 2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular , Células Epidérmicas , Epiderme/fisiologia , Queratinócitos/citologia , Queratinócitos/fisiologia , Camundongos , Camundongos Transgênicos , Neurofibromatose 2/genética , Neurofibromina 2/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , alfa Catenina/genética , alfa Catenina/metabolismoRESUMO
Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.
Assuntos
Cátions/metabolismo , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Permeabilidade da Membrana Celular , Células Cultivadas , Claudinas , Simulação por Computador , Cães , Canais Iônicos/química , Canais Iônicos/genética , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Eletricidade EstáticaRESUMO
Claudins are transmembrane proteins of the tight junction that determine and regulate paracellular ion permeability. We previously reported that claudin-8 reduces paracellular cation permeability when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells. Here, we address how the interaction of heterologously expressed claudin-8 with endogenous claudin isoforms impacts epithelial barrier properties. In MDCK II cells, barrier improvement by claudin-8 is accompanied by a reduction of endogenous claudin-2 protein at the tight junction. Here, we show that this is not because of relocalization of claudin-2 into the cytosolic pool but primarily due to a decrease in gene expression. Claudin-8 also affects the trafficking of claudin-2, which was displaced specifically from the junctions at which claudin-8 was inserted. To test whether replacement of cation-permeable claudin-2 mediates the effect of claudin-8 on the electrophysiological phenotype of the host cell line, we expressed claudin-8 in high-resistance MDCK I cells, which lack endogenous claudin-2. Unlike in MDCK II cells, induction of claudin-8 in MDCK I cells (which did not affect levels of endogenous claudins) did not alter paracellular ion permeability. Furthermore, when endogenous claudin-2 in MDCK II cells was downregulated by epidermal growth factor to create a cell model with low transepithelial resistance and low levels of claudin-2, the permeability effects of claudin-8 were also abolished. Our findings demonstrate that claudin overexpression studies measure the combined effect of alterations in both endogenous and exogenous claudins, thus explaining the dependence of the phenotype on the host cell line.
Assuntos
Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Animais , Northern Blotting , Linhagem Celular , Claudinas , Cães , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Immunoblotting , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
Tight junctions are crucial for maintaining the polarity and vectorial transport functions of epithelial cells. We and others have shown that Na-K-ATPase plays a key role in the organization and permeability of tight junctions in mammalian cells and analogous septate junctions in Drosophila. However, the mechanism by which Na-K-ATPase modulates tight junctions is not known. In this study, using a well-differentiated human pancreatic epithelial cell line HPAF-II, we demonstrate that Na-K-ATPase is present at the apical junctions and forms a complex with protein phosphatase-2A, a protein known to be present at tight junctions. Inhibition of Na-K-ATPase ion transport function reduced protein phosphatase-2A activity, hyperphosphorylated occludin, induced rearrangement of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na-K-ATPase is required for controlling the tight junction gate function.
Assuntos
Células Epiteliais/fisiologia , Proteínas de Membrana/metabolismo , Pâncreas/citologia , Pâncreas/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Junções Íntimas/fisiologia , Caderinas/metabolismo , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Técnica de Fratura por Congelamento , Humanos , Microscopia Confocal , Microscopia Imunoeletrônica , Ocludina , Pâncreas/ultraestrutura , Fosfoproteínas/metabolismo , Fosforilação , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH)-depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent-resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [(3)H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH-dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH.
Assuntos
Colesterol/deficiência , Detergentes/farmacologia , Proteínas de Membrana/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Células Cultivadas , Centrifugação , Colesterol/metabolismo , Ácidos Cólicos/farmacologia , Claudina-1 , Cães , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Ocludina , Octoxinol/farmacologia , Ácido Palmítico/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Solubilidade/efeitos dos fármacos , Solubilidade/efeitos da radiação , Junções Íntimas/efeitos da radiação , Raios UltravioletaRESUMO
Epithelial tight junctions form a barrier against passive paracellular flux. This barrier is regulated by complex physiologic and pathophysiologic signals that acutely fine-tune tight junction permeability. Although actomyosin contraction and myosin light chain phosphorylation are clearly involved in some forms of tight junction regulation, the contributions of other signaling events and the role of myosin light chain phosphorylation in this response are poorly understood. Here we ask if activation of myosin light chain kinase alone is sufficient to induce downstream tight junction regulation. We use a confluent polarized intestinal epithelial cell model system in which constitutively active myosin light chain kinase, tMLCK, is expressed using an inducible promoter. tMLCK expression increases myosin light chain phosphorylation, reorganizes perijunctional F-actin, and increases tight junction permeability. TJ proteins ZO-1 and occludin are markedly redistributed, morphologically and biochemically, but effects on claudin-1 and claudin-2 are limited. tMLCK inhibition prevents changes in barrier function and tight junction organization induced by tMLCK expression, suggesting that these events both require myosin light chain phosphorylation. We conclude that myosin light chain phosphorylation alone is sufficient to induce tight junction regulation and provide new insights into the molecular mechanisms that mediate this regulation.
Assuntos
Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Junções Íntimas/metabolismo , Actinas/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular/fisiologia , Claudina-1 , Claudinas , Indução Enzimática , Epitélio/enzimologia , Epitélio/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Quinase de Cadeia Leve de Miosina/biossíntese , Ocludina , Fosfoproteínas/metabolismo , Fosforilação , Junções Íntimas/enzimologia , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
The function of occludin (Occ) in the tight junction is undefined. To gain insight into its role in epithelial cell biology, occludin levels in Madin-Darby canine kidney II cells were suppressed by stably expressing short interfering RNA. Suppression of occludin was associated with a decrease in claudins-1 and -7 and an increase in claudins-3 and -4. Claudin-2 levels were unaffected. The tight junction "fence" function was not impaired in suppressed Occ (Occ-) clones, as determined by BODIPY-sphingomyelin diffusion in the membrane. The most striking changes were those related to control of the cytoskeleton and the "gate" function of tight junctions. A reduced ability of Occ- clones to extrude apoptotic cells from the monolayers suggested that neighbors of apoptotic cells either failed to sense their presence or were unable to coordinate cytoskeletal activity necessary for their extrusion. To further test the extent to which actin cytoskeletal activity depends on the presence of occludin, Occ- and Occ+ monolayers were depleted of cholesterol. Previous studies showed that cholesterol depletion is associated with reorganization of the actin cytoskeleton and a fall in transepithelial electrical resistance. In contrast to control Occ (Occ+) cells, transepithelial electrical resistance did not fall significantly in cholesterol-depleted Occ- monolayers and they failed to generate Rho-GTP, one of the signaling molecules involved in regulating the actin cytoskeleton. While steady-state transepithelial electrical resistance was similar in all clones, tight junction permeability to mono- and divalent inorganic cations was increased in Occ- monolayers. In addition, there was a disproportionately large increase in permeability to monovalent organic cations, up to 6.96 A in diameter. Chloride permeability was unaffected and there was little change in mannitol flux. The data suggest that occludin transduces external (apoptotic cells) and intramembrane (rapid cholesterol depletion) signals via a Rho signaling pathway that, in turn, elicits reorganization of the actin cytoskeleton. Impaired signaling in the absence of occludin may also alter the dynamic behavior of tight junction strands, as reflected by an increase in permeability to large organic cations; the permeability of ion pores formed of claudins, however, is less affected.
Assuntos
Células Epiteliais/fisiologia , Expressão Gênica/fisiologia , Proteínas de Membrana/fisiologia , Junções Íntimas/fisiologia , Animais , Apoptose/fisiologia , Células COS , Adesão Celular/fisiologia , Chlorocebus aethiops , Rim/ultraestrutura , Ocludina , Fenótipo , Fatores de TempoRESUMO
Multicellular organisms are separated from the external environment by a layer of epithelial cells whose integrity is maintained by intercellular junctional complexes composed of tight junctions, adherens junctions, and desmosomes, whereas gap junctions provide for intercellular communication. The aim of this review is to present an updated overview of recent developments in the area of tight junction biology. In a relatively short time, our knowledge of the tight junction has evolved from a relatively simple view of it being a permeability barrier in the paracellular space and a fence in the plane of the plasma membrane to one of it acting as a multicomponent, multifunctional complex that is involved in regulating numerous and diverse cell functions. A group of integral membrane proteins-occludin, claudins, and junction adhesion molecules-interact with an increasingly complex array of tight junction plaque proteins not only to regulate paracellular solute and water flux but also to integrate such diverse processes as gene transcription, tumor suppression, cell proliferation, and cell polarity.
Assuntos
Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Animais , Comunicação Celular/fisiologia , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/fisiologia , Células Epiteliais/ultraestrutura , Homeostase/fisiologia , Humanos , Substâncias Macromoleculares , Junções Íntimas/ultraestruturaRESUMO
To investigate the contribution of dendritic cells (DC) in a pulmonary granulomatous immune response, C57BL/l6 mice, nonimmunized or immunized with purified protein derivative (PPD) of Mycobacterium bovis, were intravenously injected with PPD-coated Sepharose-4B beads. One and three days later lungs were harvested, granuloma size was measured, and immunolabeled cells in granulomas were counted. On Day 1, granulomas in immunized mice were 3-fold larger and contained more major histocompatibility complex class II+, CD11c+ DCs than nonimmunized mice. By Day 3, these differences had diminished. In all granulomas MHC class II+, CD11c+ DCs were in contact with the beads. By in situ hybridization these DCs expressed interleukin (IL)-12 p40 mRNA. MOMA2+ macrophages were present throughout the granulomas, whereas CD4+ and CD8alpha+ T cells were localized at the granuloma periphery. DCs isolated from granulomatous lungs at Day 1, and from thoracic lymph nodes (LNs) at Days 1 and 3, stimulated PPD-specific T cell proliferation without exogenously added antigen, indicating that they had acquired bead-bound antigen. By Day 3, however, granuloma DCs presented little antigen, suggesting that newly immigrated DC lacked access to antigen or that antigen uptake/processing was inhibited. RNase protection assays of whole-lung mRNA showed increased interferon-gamma, IL-1beta, IL-1 receptor antagonist, IL-6, and macrophage inhibitory factor, but no IL-10 mRNA on Days 1 and 3. These observations support the premise that DCs are key in initiating granulomatous cell-mediated immunity. However, factors generated within the granuloma downregulate the antigen presenting function of DC by Day 3 in this experimental model.
Assuntos
Células Dendríticas/imunologia , Granuloma/imunologia , Pulmão/imunologia , Animais , Sequência de Bases , Citocinas/genética , Primers do DNA , Feminino , Hibridização In Situ , Integrina alfaXbeta2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genéticaRESUMO
Dendritic cells (DCs) are the most potent antigen-presenting cells that play a central role in initiating the primary immune response. However, their role in granulomatous inflammation has not been well studied. The aim of the present study was to elucidate the role of DCs in granuloma formation. Using a rat model of bacillus Calmette-Guérin (BCG)-elicited pulmonary granulomas, we investigated the distribution of DCs in the granulomas by immunohistochemistry with a rat-DC-specific monoclonal antibody, OX62. We found numerous large, pleiomorphic OX62(+) cells accumulating at the borders of the pulmonary granulomas. The OX62(+) cells isolated from the granulomatous lung showed intense surface expression of major histocompatibility complex class II, B7-1, and B7-2, and a lack of T cell- and monocyte/macrophage-specific markers. Their ultrastructural morphology was characteristic of DCs. Functionally, they had potent capacity to stimulate allogeneic T cells as well as purified protein derivative-specific syngeneic T cells in the absence of exogenous peptides. Based on these findings, the OX62(+) cells infiltrating the granulomas were considered to be DCs expressing BCG-derived peptides. These results indicate that DCs contribute to pulmonary granuloma formation elicited by BCG by means of their potent antigen-presenting function, providing a novel insight into DC function in T cell-mediated granulomatous immune responses.
Assuntos
Vacina BCG/administração & dosagem , Células Dendríticas/imunologia , Granuloma/imunologia , Pulmão/imunologia , Mycobacterium bovis/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Agregação Celular/imunologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Granuloma/metabolismo , Imuno-Histoquímica , Separação Imunomagnética , Pulmão/citologia , Pulmão/metabolismo , Masculino , Fenótipo , Ratos , Ratos Endogâmicos Lew , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de TempoRESUMO
Intratracheal (IT) administration of heat-killed Listeria monocytogenes (HKL) results in an influx of macrophage and dendritic cell (DC) precursors into the lung interstitium. Low-density, FcR+, interstitial lung cells isolated from rats instilled 24 hr before with HKL or vehicle alone, were > 90% Mar1+. After culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 3 days, up to 24% of the loosely adherent cells were DC that stimulated allogeneic T-cell proliferation in an mixed lymphocyte reaction (MLR) assay. After only an overnight incubation with GM-CSF, however, the capacity of interstitial Mar1+ cells to stimulate HKL immune T-cell proliferation without exogenous antigen was low. By contrast, when DC were isolated as major histocompatibility complex (MHC) class II+ cells from rat lungs at 1, 3, 7 and 14 days after HKL instillation and cultured overnight with GM-CSF, their antigen presentation capacity without added exogenous antigen was robust, but declined over the 2-week period. Interestingly, hilar lymph node DC maintained their HKL antigen-presenting capacity for up to 2 weeks after instillation of HKL. Following IT administration of PKH-26 labelled HKL, fluorescent or immunolabelled organisms were detected in OX62+ DC in airway epithelium, lung interstitium and hilar lymph nodes in situ and in MHC class II+ DC isolated from these sites. We conclude that newly immigrated Mar1+ lung DC precursors, while efficient in endocytosing particulate antigens, are incapable of eliciting a significant proliferative response from HKL-sensitized T cells. By contrast, MHC class II+ DC isolated from lungs and incubated overnight with GM-CSF induce vigorous antigen-specific T-cell proliferation. Antigen-loaded lung DC in hilar lymph nodes maintain their antigen presentation capacity for up to 2 weeks.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos de Bactérias/imunologia , Pulmão/imunologia , Linfonodos/imunologia , Animais , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Listeria monocytogenes/imunologia , Ativação Linfocitária , Ratos , Ratos Endogâmicos LEC , Ratos Endogâmicos Lew , Linfócitos T/imunologia , Fatores de TempoRESUMO
Claudins are a family of integral membrane proteins of the tight junction that are thought to participate in the permeation of solutes across epithelia via the paracellular pathway. Claudin-8 is expressed in the distal renal tubule, which has a characteristically low passive permeability to monovalent cations. To test the hypothesis that claudin-8 plays a role in forming a tight paracellular barrier to cations, stably transfected Madin-Darby canine kidney II cell lines with inducible expression of claudin-8 were generated. Induction of claudin-8 expression was associated with down-regulation of endogenous claudin-2 protein. Other tight junction proteins were expressed and targeted normally, and the number of junctional strands was minimally altered. By Ussing chamber and radiotracer flux studies, claudin-8 expression was found to reduce paracellular permeability to monovalent inorganic and organic cations and to divalent cations but not to anions or neutral solutes. The size selectivity, charge dependence, and activation energy of paracellular cation permeation were all unchanged. These observations are consistent with a model in which claudin-2 encodes a highly cation-permeable channel, whereas claudin-8 acts primarily as a cation barrier. When exogenous claudin-8 is expressed, it replaces endogenous claudin-2, inserting in its place into existing tight junction strands, thereby reducing the apparent number of functional cation pores. Our findings suggest that claudin-8 plays an important role in the paracellular cation barrier of the distal renal tubule.
Assuntos
Junções Comunicantes/genética , Proteínas de Membrana/genética , Animais , Cátions/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular/genética , Claudinas , Cães , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica , Transporte de Íons , Proteínas de Membrana/biossínteseRESUMO
Infection of intestinal epithelial cells with enteropathogenic Escherichia coli (EPEC) disrupts tight junction (TJ) architecture and barrier function. The aim of this study was to determine the impact of EPEC on TJ protein interactions and localization. Human intestinal epithelial cells (T84) were infected for 1, 3 or 6 h with EPEC. To probe the TJ protein-protein interactions, co-immunoprecipitations were performed. The associations between ZO-1, occludin and claudin-1 progressively decreased after infection. Corresponding morphological changes were analysed by immunofluorescence confocal microscopy. Tight junction proteins progressively lost their apically restricted localization. Freeze-fracture electron microscopy revealed the appearance of aberrant strands throughout the lateral membrane that contained claudin-1 and occludin as determined by immunogold labelling. These structural alterations were accompanied by a loss of barrier function. Mutation of the gene encoding EspF, important in the disruption of TJs by EPEC, prevented the disruption of TJs. Tight junction structure normalized following eradication of EPEC with gentamicin and overnight recovery. This is the first demonstration that a microbial pathogen can cause aberrant TJ strands in the lateral membrane of host cells. We speculate that the disruption of integral and cytoplasmic TJ protein interactions following EPEC infection allows TJ strands to form or diffuse into the lateral plasma membrane.
Assuntos
Células Epiteliais/microbiologia , Escherichia coli/patogenicidade , Intestino Delgado/microbiologia , Junções Íntimas/microbiologia , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Membrana Celular/ultraestrutura , Claudina-1 , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Técnica de Fratura por Congelamento , Humanos , Intestino Delgado/metabolismo , Intestino Delgado/ultraestrutura , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Ocludina , Fosfoproteínas/metabolismo , Mapeamento de Interação de Proteínas , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
When sampling inhaled antigens, dendritic cells (DC) must penetrate the tight junction (TJ) barrier while maintaining the TJ seal. In matrix metalloproteinase (MMP)-9-deficient mice, in vivo experiments suggest that migration of DC into air spaces is impaired. To examine the underlying mechanisms, we established a well-defined in vitro model using mouse tracheal epithelial cells and mouse bone marrow DC (BMDC). Transmigration was elicited with either macrophage inflammatory protein (MIP)-1alpha or MIP-3beta in a time-dependent manner. Control MMP-9(+/+) BMDC cultured with granulocyte macrophage-colony-stimulating factor for 7 d showed a 30-fold greater transepithelial migration toward MIP-3beta than MIP-1alpha, indicating a more mature DC phenotype. MMP-9(-/-) BMDC as well as MMP-9(+/+) BMDC in the presence of the MMP inhibitor GM6001, although showing a similar preference for MIP-3beta, were markedly impaired in their ability to traverse the epithelium. Expression levels of CCR5 and CCR7, however, were similar in both MMP-9(-/-) and MMP-9(+/+) BMDC. Expression of the integral TJ proteins, occludin and claudin-1, were examined in BMDC before and after transepithelial migration. Interestingly, occludin but not claudin-1 was degraded following transepithelial migration in both MMP-9(-/-) and control BMDC. In addition, there was a > 2-fold increase in claudin-1 expression in MMP-9(-/-) as compared with control BMDC. These observations indicate that occludin and claudin-1 are differentially regulated and suggest that the lack of MMP-9 may affect claudin-1 turnover.
Assuntos
Movimento Celular/fisiologia , Células Dendríticas/metabolismo , Células Epiteliais/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Mucosa Respiratória/citologia , Junções Íntimas/metabolismo , Traqueia/anatomia & histologia , Animais , Células Cultivadas , Quimiocina CCL19 , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/metabolismo , Claudina-1 , Dipeptídeos/metabolismo , Impedância Elétrica , Células Epiteliais/citologia , Pulmão/anatomia & histologia , Pulmão/imunologia , Pulmão/metabolismo , Proteínas Inflamatórias de Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Ocludina , Inibidores de Proteases/metabolismo , Receptores CCR5/metabolismo , Receptores CCR7 , Receptores de Quimiocinas/metabolismo , Mucosa Respiratória/metabolismoRESUMO
OBJECTIVES: Epithelial cells have distinct apical and basolateral plasma membrane domains separated by tight junctions. This phenotype is essential for the directional transport functions of epithelial cells. Here we characterized a well-differentiated pancreatic epithelial cell line to establish a useful model for understanding the mechanisms involved in the regulation of junctional complexes, polarity, and disease processes in the pancreas. METHODS: Immunofluorescence of cell junction marker proteins and electron microscopy were used to determine the presence of tight junctions, adherens junctions, and desmosomes. The functionality of tight junctions was tested by transepithelial resistance measurements and transepithelial permeability studies of nonionic molecules. Tight junction function in polarity was determined by laser scanning confocal microscopy. RESULTS: Immunofluorescence analysis in HPAF-II cells revealed tight junction localization of ZO-1, occludin, and claudin-4; adherens junction localization of E-cadherin and beta-catenin; and desmosomal localization of desmocollin. Transmission electron microscopy showed the presence of tight junctions, adherens junctions, and des-mosomes, and freeze-fracture electron microscopy revealed the presence of distinct anastomosing tight junction strands. Transepithelial electrical resistance and permeability measurements revealed functional tight junctions. In addition, 3-dimensional images of the monolayer generated by laser scanning confocal microscopy revealed that HPAF-II cells show polarity. Immunoblotting and RT-PCR analyses revealed high expression levels of E-cadherin and Na,K-ATPase beta-subunit but low levels of the transcription factor Snail in HPAF-II cells compared with MiaPaCa-2 cells. CONCLUSION: The HPAF-II cell line is a well-differentiated human pancreatic carcinoma cell line that should be useful as a model for studies aimed at understanding epithelial polarity, regulation of junctional complexes, and disease processes in pancreas.
Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Junções Aderentes/química , Junções Aderentes/ultraestrutura , Biomarcadores , Caderinas/análise , Diferenciação Celular , Linhagem Celular Tumoral/patologia , Linhagem Celular Tumoral/fisiologia , Polaridade Celular , Claudina-4 , Proteínas do Citoesqueleto/análise , Desmossomos/química , Desmossomos/ultraestrutura , Impedância Elétrica , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Humanos , Glicoproteínas de Membrana/análise , Proteínas de Membrana/análise , Ocludina , Fosfoproteínas/análise , Junções Íntimas/química , Junções Íntimas/ultraestrutura , Transativadores/análise , Proteína da Zônula de Oclusão-1 , beta CateninaRESUMO
The pathogenesis of chronic renal allograft rejection (CR) remains obscure. The hypothesis that a subset of CR is mediated by antidonor antibody was tested by determining whether C4d is deposited in peritubular capillaries (PTC) and whether it correlates with circulating antidonor antibodies. All cases (from January 1, 1990, to July 31, 1999) that met histologic criteria for CR and had frozen tissue (28 biopsies, 10 nephrectomies) were included. Controls were renal allograft biopsies with chronic cyclosporine toxicity (n = 21) or nonspecific interstitial fibrosis (n = 10), and native kidneys with end-stage renal disease (n = 10) or chronic interstitial fibrosis (n = 5). Frozen sections were stained by two-color immunofluorescence for C4d, type IV collagen and Ulex europaeus agglutinin I. Antidonor HLA antibody was sought by panel-reactive antibody analysis and/or donor cross matching in sera within 7 wk of biopsy. Overall, 23 of 38 CR cases (61%) had PTC staining for C4d, compared with 1 of 46 (2%) of controls (P < 0.001). C4d in PTC was localized at the interface of endothelium and basement membrane. Most of the C4d-positive CR tested had antidonor HLA antibody (15 of 17; 88%); none of the C4d-negative CR tested (0 of 8) had antidonor antibody (P < 0.0002). The histology of C4d-positive CR was similar to C4d-negative CR, and 1-yr graft survival rates were 62% and 25%, respectively (P = 0.05). Since August 1998, five of six C4d-positive CR cases have been treated with mycophenolate mofetil +/- tacrolimus with a 100% 1-yr graft survival, versus 40% before August 1998 (P < 0.03). These data support the hypothesis that a substantial fraction of CR is mediated by antibody (immunologically active). C4d can be used to separate this group of CR from the nonspecific category of chronic allograft nephropathy and may have the potential to guide successful therapeutic intervention.