RESUMO
Jagaricin is a lipopeptide produced by the bacterial mushroom pathogen Janthinobacterium agaricidamnosum, the causative agent of mushroom soft rot disease. Apart from causing lesions in mushrooms, jagaricin is a potent antifungal active against human-pathogenic fungi. We show that jagaricin acts by impairing membrane integrity, resulting in a rapid flux of ions, including Ca2+, into susceptible target cells. Accordingly, the calcineurin pathway is required for jagaricin tolerance in the fungal pathogen Candida albicans Transcriptional profiling of pathogenic yeasts further revealed that jagaricin triggers cell wall strengthening, general shutdown of membrane potential-driven transport, and the upregulation of lipid transporters, linking cell envelope integrity to jagaricin action and resistance. Whereas jagaricin shows hemolytic effects, it exhibited either no or low plant toxicity at concentrations at which the growth of prevalent phytopathogenic fungi is inhibited. Therefore, jagaricin may have potential for agricultural applications. The action of jagaricin as a membrane-disrupting antifungal is promising but would require modifications for use in humans.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Cálcio/metabolismo , Candida albicans/genética , Candida albicans/isolamento & purificação , Candida glabrata/efeitos dos fármacos , Candida glabrata/genética , Candidíase/microbiologia , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Testes de Sensibilidade Microbiana , MutaçãoRESUMO
The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca(2+). The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca(2+) and regulation of Ca(2+)-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca(2+) release from intracellular stores; (iii) Ca(2+) entry from the extracellular compartment; and (iv) nuclear translocation of the Ca(2+)-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca(2+) signaling.
Assuntos
Sinalização do Cálcio/fisiologia , Fosfolipase C gama/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Substituição de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Galinhas , Humanos , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fatores de Transcrição NFATC/metabolismo , Fosfolipase C gama/química , Fosfolipase C gama/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Human plasmacytoid dendritic cells (pDC) are important modulators of adaptive T cell responses during viral infections. Recently, we found that human pDC produce the serine protease granzyme B (GrB), thereby regulating T cell proliferation in a GrB-dependent manner. In this study, we demonstrate that intrinsic GrB production by pDC is significantly inhibited in vitro and in vivo by clinically used vaccines against viral infections such as tick-borne encephalitis. We show that pDC GrB levels inversely correlate with the proliferative response of coincubated T cells and that GrB suppression by a specific Ab or a GrB substrate inhibitor results in enhanced T cell proliferation, suggesting a predominant role of GrB in pDC-dependent T cell licensing. Functionally, we demonstrate that GrB(high) but not GrB(low) pDC transfer GrB to T cells and may degrade the ζ-chain of the TCR in a GrB-dependent fashion, thereby providing a possible explanation for the observed T cell suppression by GrB-expressing pDC. Modulation of pDC-derived GrB activity represents a previously unknown mechanism by which both antiviral and vaccine-induced T cell responses may be regulated in vivo. Our results provide novel insights into pDC biology during vaccinations and may contribute to an improvement of prophylactic and therapeutic vaccines.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Granzimas/metabolismo , Vacinas Virais/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Encefalite Transmitida por Carrapatos/imunologia , Humanos , Interferon-alfa/biossíntese , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Recently, CD4(+) T helper cells were shown to induce differentiation of human B cells into plasma cells by expressing interleukin (IL-)21 and CD40 ligand (CD40L). In the present study we show, that in the absence of CD40L, CD4(+) T cell-derived IL-21 induces differentiation of B cells into granzyme B (GzmB)-secreting cytotoxic cells. Using fluorescence-activated cell sorting (FACS) analysis, ELISpot and confocal microscopy, we demonstrate that CD4(+) T cells, activated via their T-cell receptor without co-stimulation, can produce IL-21, but do not express CD40L and rapidly induce GzmB in co-cultured B cells in an IL-21 receptor-dependent manner. Of note, we confirmed these results with recombinant reagents, highlighting that CD40L suppresses IL-21-induced GzmB induction in B cells in a dose-dependent manner. Surprisingly, although GzmB-secreting B cells did not express perforin, they were able to transfer active GzmB to tumor cell lines, thereby effectively inducing apoptosis. In contrast, no cytotoxic effects were found when effector B cells were activated with IL-2 instead of IL-21 or when target cells were cultured with IL-21 alone. Our findings suggest GzmB(+) cytotoxic B cells may have a role in early cellular immune responses including tumor immunosurveillance, before fully activated, antigen-specific cytotoxic T cells are on the spot. CD40 ligand determines whether IL-21 induces differentiation of B cells into plasma cells or into granzyme B-secreting cytotoxic cells.
Assuntos
Linfócitos B/citologia , Diferenciação Celular/imunologia , Granzimas/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos B/metabolismo , Ligante de CD40 , Células Cultivadas , Humanos , Imunidade Celular , Interleucina-2/farmacologia , Interleucinas/farmacologiaRESUMO
Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB(+) pDCs potently suppress T-cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly inhibit, GrB secretion by pDCs. GrB-secreting pDCs may play a regulatory role for immune evasion of tumors, antiviral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC-T-cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations.
Assuntos
Artrite Juvenil/imunologia , Células Dendríticas/metabolismo , Granzimas/fisiologia , Ativação Linfocitária/fisiologia , Linfócitos T/imunologia , Artrite Juvenil/patologia , Western Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Criança , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Interleucina-3/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de SinaisRESUMO
BACKGROUND AND AIM: The mechanisms of transmigration of inflammatory cells through the sinusoids are still poorly understood. This study aims to identify in vitro conditions (cytokine treatment) which may allow a better understanding of the changes in PECAM (platelet endothelial cell adhesion molecule)-1-gene-expression observed in vivo. METHODS AND RESULTS: In this study we show by immunohistochemistry, that there is an accumulation of ICAM-1 (intercellular cell adhesion molecule-1) and ED1 positive cells in necrotic areas of livers of CCl4-treated rats, whereas there are few PECAM-1 positive cells observable. After the administration of CCl4, we could detect an early rise of levels of IFN-gamma followed by an enhanced TGF-beta protein level. As shown by Northern blot analysis and surface protein expression analysed by flow cytometry, IFN-gamma-treatment decreased PECAM-1-gene-expression in isolated SECs (sinusoidal endothelial cells) and mononuclear phagocytes (MNPs) in parallel with an increase in ICAM-1-gene-expression in a dose and time dependent manner. In contrast, TGF-beta-treatment increased PECAM-1-expression. Additional administration of IFN-gamma to CCl4-treated rats and observations in IFN-gamma-/- mice confirmed the effect of IFN-gamma on PECAM-1 and ICAM-1-expression observed in vitro and increased the number of ED1-expressing cells 12 h after administration of the toxin. CONCLUSION: The early decrease of PECAM-1-expression and the parallel increase of ICAM-1-expression following CCl4-treatment is induced by elevated levels of IFN-gamma in livers and may facilitate adhesion and transmigration of inflammatory cells. The up-regulation of PECAM-1-expression in SECs and MNPs after TGF-beta-treatment suggests the involvement of PECAM-1 during the recovery after liver damage.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/imunologia , Células Endoteliais/imunologia , Interferon-alfa/imunologia , Interferon gama/imunologia , Fígado/imunologia , Sistema Fagocitário Mononuclear/imunologia , Fator de Crescimento Transformador beta/administração & dosagem , Animais , Tetracloreto de Carbono , Células Cultivadas , Citocinas/imunologia , Regulação para Baixo , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Fatores Imunológicos/imunologia , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Sistema Fagocitário Mononuclear/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
BACKGROUND: Alpha-fetoprotein (AFP) expression can resume in the adult liver under pathophysiological conditions. Orphan nuclear receptors were supposed to regulate AFP gene expression, in vitro. We were interested to study the expression of AFP and orphan nuclear receptors, in vivo. RESULTS: The expression of AFP gene and orphan nuclear receptors in the liver was examined in different rat models: (a) fetal liver (b) liver regeneration [partial hepatectomy (PH) with and without 2-acetyl-aminofluren treatment (2-AAF)], (c) acute liver damage [treatment with CCl4] and (d) acute phase reaction [treatment with turpentine oil]. After PH of 2-AAF treated rats, clusters of AFP positive cells occurred in the periportal region. In the Northern blot analysis, a positive hybridization signal for the full-length AFP-RNA was observed only in liver samples from 2-AAF treated rats after PH. In real-time PCR analysis, the full-length AFP-RNA was highly up regulated in the fetal liver (maximum at day 14: 21,500 fold); after PH of 2-AAF treated rats, the full-length AFP-RNA was also up regulated up to 400 fold (day 7 after PH). The orphan nuclear receptors were down regulated at nearly each time points in all models, also at time point of up regulation of the AFP gene. CONCLUSION: Expression of "fetal" AFP could be demonstrated during liver development and during proliferation of the so-called oval cells. Changes of expression of orphan nuclear receptors, however, did not correlate with AFP expression. Other regulatory pathways were possibly involved in controlling AFP expression, in vivo.
RESUMO
Although systemically applied nanoparticles are quickly taken up by phagocytic cells, mainly macrophages, the interactions between engineered nanoparticles and macrophages are still not well defined. We therefore analyzed the uptake of diagnostically used carboxydextran-coated superparamagnetic iron oxide nanoparticles of 60 nm (SPIO) and 20 nm (USPIO) by human macrophages. By pharmacological and in vitro knockdown approaches, the principal uptake mechanism for both particles was identified as clathrin-mediated, scavenger receptor A-dependent endocytosis. We developed a mathematical model of the uptake process that allows determination of key parameters of endocytosis, including the rate of uptake, the number of nanoparticles per cell in saturation, the mean uptake time, and the correlation between the number of internalized nanoparticles and their extracellular concentration. The calculated parameters correlate well with experimental data obtained by confocal microscopy. Moreover, the model predicts the individual and collective wrapping times of different nanoparticles, describes the relation between cytoskeletal forces, membrane elasticity and the uptake time. We also introduced a new physical parameter 'a' governing the collective uptake process, a reflecting minimal linear spacing between simultaneously acting neighboring endocytotic pits.
Assuntos
Endocitose/fisiologia , Compostos Férricos/química , Macrófagos/metabolismo , Nanopartículas/química , Polímeros/química , Células Cultivadas , Humanos , Macrófagos/ultraestrutura , Imageamento por Ressonância Magnética , Microscopia Confocal , Microscopia Eletrônica de TransmissãoRESUMO
Tumor cell lines are often used as models for the study of nanoparticle-cell interactions. Here we demonstrate that carboxy (PS-COOH) and amino functionalized (PS-NH2) polystyrene nanoparticles of â¼100 nm in diameter are internalized by human macrophages, by undifferentiated and by PMA-differentiated monocytic THP-1 cells via diverse mechanisms. The uptake mechanisms also differed for all cell types and particles when analyzed either in buffer or in medium containing human serum. Macrophages internalized â¼4 times more PS-COOH than THP-1 cells, when analyzed in serum-containing medium. By contrast, in either medium, THP-1 cells internalized PS-NH2 more rapidly than macrophages. Using pharmacological and antisense in vitro knockdown approaches, we showed that, in the presence of serum, the specific interaction between the CD64 receptor and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization in THP-1 cells occurred via dynamin II-dependent endocytosis. PMA-differentiated THP-1 cells differed in their uptake mechanism from macrophages and undifferentiated THP-1 cells by internalizing the particles via macropinocytosis. In line with our in vitro data, more intravenously applied PS-COOH particles accumulated in the liver, where macrophages of the reticuloendothelial system reside. By contrast, PS-NH2 particles were preferentially targeted to tumor xenografts grown on the chorioallantoic membrane of fertilized chicken eggs. Our data show that the amount of internalized nanoparticles, the uptake kinetics, and its mechanism may differ considerably between primary cells and a related tumor cell line, whether differentiated or not, and that particle uptake by these cells is critically dependent on particle opsonization by serum proteins.
Assuntos
Macrófagos/química , Macrófagos/metabolismo , Macrófagos/fisiologia , Monócitos/metabolismo , Nanopartículas/química , Fagocitose/fisiologia , Poliestirenos/farmacocinética , Linhagem Celular , Humanos , Monócitos/química , Poliestirenos/químicaRESUMO
Contrast agents based on dextran-coated superparamagnetic iron oxide nanoparticles (SPIO) are internalized by professional phagocytes such as hepatic Kupffer cells, yet their role in phagocyte biology remains largely unknown. Here we investigated the effects of the SPIO ferucarbotran on murine Kupffer cells and human macrophages. Intravenous injection of ferucarbotran into mice led to rapid accumulation of the particles in phagocytes and to long-lasting increased iron deposition in liver and kidneys. Macrophages incorporate ferucarbotran in lysosomal vesicles containing α-glucosidase, which is capable of degrading the carboxydextran shell of the ferucarbotran particles. Intravenous injection of ferucarbotran into mice followed by incorporation of the nanoparticles into Kupffer cells triggered apoptosis and the subsequent depletion of Kupffer cells. In macrophages, the proinflammatory cytokine TNF-α increased the apoptosis rate, the reactive oxygen species production and the activation of c-Jun N-terminal kinase elicited by ferucarbotran, which might be mediated by the induction of cytoplasmic phospholipase A2 by TNF-α. Notably, the nanoparticle-induced apoptosis of murine Kupffer cells could be prevented by treatment of the mice with the radical scavenger edaravone. Thus, nanosized carboxydextran-coated SPIO-based contrast agents are retained for extended time periods by liver macrophages, where they elicit delayed cell death, which can be antagonized by a therapeutic radical scavenger.
Assuntos
Dextranos/metabolismo , Lisossomos/metabolismo , Nanopartículas/química , Fagócitos/metabolismo , Animais , Antipirina/análogos & derivados , Antipirina/farmacologia , Apoptose/efeitos dos fármacos , Dextranos/farmacocinética , Edaravone , Sequestradores de Radicais Livres/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Cinética , Células de Kupffer/citologia , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/efeitos dos fármacos , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita , Camundongos , Fagócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Superparamagnetic iron oxide nanoparticles are frequently used for cell labeling or as diagnostic contrast media, yet studies analyzing their effects on immune cells remain scarce. Here we investigated how nanosized carboxydextran-coated superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) might affect human macrophages. Within 1 h, both SPIO and USPIO were rapidly taken up by macrophages. Confocal microscopy revealed that after 24 h the particles were almost exclusively localized within the lysosomal compartment. Continued cultivation of the macrophages for several days was associated with apoptosis induction caused by a long-lasting activation of the c-Jun N-terminal kinase (JNK) pathway. JNK activation was due to significantly elevated levels of reactive oxygen species, whereas no TNF-alpha was produced by the macrophages treated with nanoparticles. Compared to SPIO, USPIO induced more pronounced biochemical alterations and cytotoxicity, which could be antagonized by the JNK inhibitor V. Alternatively, treatment of macrophages with Trolox or N-acetyl-L-cysteine, two functionally different scavengers of reactive oxygen species, abolished both the JNK activation and the subsequent cytotoxic effects. These data indicate that nanosized superparamagnetic iron oxide-based contrast media exert cytotoxicity in human macrophages that can be functionally antagonized with radical scavengers.
Assuntos
Apoptose/fisiologia , Materiais Revestidos Biocompatíveis/administração & dosagem , Dextranos/administração & dosagem , Óxido Ferroso-Férrico/administração & dosagem , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/citologia , Macrófagos/enzimologia , Nanopartículas/administração & dosagem , Apoptose/efeitos dos fármacos , Células Cultivadas , Humanos , Macrófagos/efeitos dos fármacos , Nanopartículas de MagnetitaRESUMO
Applications of water-soluble quantum dots (QDs) in the life sciences are limited by their poor colloidal stability in physiological media and nonspecific interaction with biomatter, particularly cell membranes. We have studied colloidal stability and nonspecific interactions with living cells for zwitterionic d-penicillamine-coated QDs (DPA-QDs) and the traditionally used carboxylated 11-mercaptoundecanoic acid-coated QDs (MUA-QDs) and found clear advantages of DPA-QDs. In single molecule fluorescence experiments, DPA-QDs showed no aggregation over the physiologically relevant pH range of 5-9, whereas MUA-QDs showed significant aggregation below pH 9. Upon exposure to living Mono Mac 6 cells, DPA-QDs, which possess overall charge-neutral surfaces, exhibited weak interactions with the cell membrane and were easily removed by flushing with buffer. By contrast, the highly charged MUA-QDs strongly associated with the cells and could not be removed even by extensive rinsing with buffer solution. DPA-QDs exhibit a high chemical stability even in strongly oxidizing conditions, in contrast to cysteine-coated QDs reported earlier. This beneficial property may arise from reduced interactions between DPA ligands due to steric effects of the methyl groups on their beta-carbon atoms.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Monócitos/metabolismo , Pontos Quânticos , Adsorção , Animais , Linhagem Celular Tumoral , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Penicilamina/química , Penicilamina/metabolismo , Solubilidade , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Água/químicaRESUMO
C-reactive protein (CRP) is a prototype acute-phase protein that may be intimately involved in human disease. Its cellular receptors are still under debate; the main candidates are FcR for immunoglobulin G, as CRP was shown to bind specifically to FcgammaRI and FcgammaRIIa. Using ultrasensitive confocal live-cell imaging, we have studied CRP binding to FcgammaR naturally expressed in the plasma membranes of cells from a human leukemia cell line (Mono Mac 6). These macrophage-like cells express high levels of FcgammaRI and FcgammaRII. They were shown to bind fluorescently labeled CRP with micromolar affinity, KD = (6.6 +/- 1.5) microM. CRP binding could be inhibited by pre-incubation with human but not mouse IgG and was thus FcgammaR-specific. Blocking of FcgammaRI by an FcgammaRI-specific antibody abolished CRP binding essentially completely, whereas application of antibodies against FcgammaRII did not have a noticeable effect. In fluorescence images of Mono Mac 6 cells, the intensity patterns of bound CRP were correlated with those of FcgammaRI, but not FcgammaRII. These results provide clear evidence of specific interactions between CRP and FcgammaR (predominantly FcgammaRI) naturally expressed on macrophage-like cells.
Assuntos
Proteína C-Reativa/metabolismo , Macrófagos/metabolismo , Receptores de IgG/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/análise , Antígenos CD/genética , Antígenos CD/metabolismo , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Imunoglobulina G/metabolismo , Macrófagos/química , Ligação Proteica/efeitos dos fármacos , Receptores de IgG/análise , Receptores de IgG/genética , TransfecçãoRESUMO
C-reactive protein (CRP), the prototype human acute phase protein, is widely regarded as a key player in cardiovascular disease, but the identity of its cellular receptor is still under debate. By using ultrasensitive confocal imaging analysis, we have studied CRP binding to transfected COS-7 cells expressing the high-affinity IgG receptor FcgammaRI. Here we show that CRP binds to FcgammaRI on intact cells, with a kd of 10+/-3 micromol/L. Transfection of COS-7 cells with a plasmid coding for both FcgammaRI and its functional counterpart, the gamma-chain, markedly increases CRP affinity to FcgammaRI, resulting in a kd of 0.35+/-0.10 micromol/L. The affinity increase results from an approximately 30-fold enhanced association rate coefficient. The pronounced enhancement of affinity by the gamma-chain suggests its crucial involvement in the CRP receptor interaction, possibly by mediating interactions between the transmembrane moieties of the receptors. Dissociation of CRP from the cell surfaces cannot be detected throughout the time course of several hours and is thus extremely slow. Considering the pentameric structure of CRP, this result indicates that multivalent binding and receptor clustering are crucially involved in the interaction of CRP with nucleated cells.
Assuntos
Proteína C-Reativa/metabolismo , Agregação de Receptores , Receptores de IgG/metabolismo , Animais , Proteína C-Reativa/química , Células COS , Chlorocebus aethiops , Humanos , Ligação Proteica , Estrutura Quaternária de Proteína , Receptores de IgG/química , Receptores de IgG/genéticaRESUMO
The source of serum cytokine-induced neutrophil chemoattractant (CINC-1) and consequences of its presence in the tissue of synthesis have not been clearly elucidated under acute-phase situation. To pursue this question, turpentine oil (TO) was intramuscularly injected into rats, and RNA and local protein levels of acute-phase cytokines and of CINC-1 were studied in the TO injected gluteal muscle, as well as in noninjured muscle, in the liver, kidney, lung and spleen. The serum levels of acute-phase mediators and of CINC-1 were measured together with total leukocyte subpopulations. Recruitment of inflammatory cells in muscle and in the other organs was investigated by quantitative immunohistochemical methods. The effect of acute-phase mediators, including interferon gamma (IFN-gamma) on the synthesis of CINC-1 in cultured hepatocytes was also investigated at the RNA and protein level. We found that the sera of the TO-treated rats contained elevated levels of IL-6, IL-1beta and CINC-1. Increased serum levels of IFN-gamma were also observed not only in the injured muscle but also and to a higher extent in the liver. However, while neutrophils and mononuclear phagocytes were found in the injured muscle, no inflammatory cells were detected at the non-'inflamed' site, namely, the liver or in the other organs. In vitro, treatment of cultured hepatocytes with IL-1beta led to elevated CINC-1 gene expression. This was true to a lesser extent upon IL-6 and tumor necrosis factor (TNF-alpha) exposure. Interestingly, IFN-gamma did not effect CINC-1 gene expression. These results indicate that CINC-1 behaves as an acute-phase protein and its expression is inducible in hepatocytes. However, CINC-1-production in the liver does not lead to recruitment of inflammatory cells into the organ.
Assuntos
Reação de Fase Aguda/metabolismo , Citocinas/fisiologia , Modelos Animais de Doenças , Interleucina-8/metabolismo , Fígado/metabolismo , Animais , Sequência de Bases , Northern Blotting , Creatina Quinase/sangue , Primers do DNA , Imuno-Histoquímica , Fígado/citologia , Masculino , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND/AIMS: Heme oxygenase-1 (HO-1) can be induced by various stimuli, one of which is interleukin-6 (IL-6). Therefore, the aim of this study was to elucidate the molecular mechanisms responsible for IL-6-dependent HO-1 induction in the liver. METHODS: The IL-6-dependent HO-1 regulation in rat primary hepatocytes and HepG2 hepatoma cells was studied by Northern and Western blot analyses, HO-1 promoter reporter gene assays and EMSA. RESULTS: The HO-1 expression was transcriptionally induced by IL-6 in a time- and dose-dependent manner. Activation of signal transducers and activators of transcription (STAT) factors by the IL-6 receptor was crucial for HO-1 induction. By contrast, negative regulation of HO-1 expression appeared to be mediated through the SH2-domain-containing tyrosine phosphatase-2 (SHP2)/ suppressors of cytokine signaling-3 (SOCS3) binding site within the gp130 IL-6 receptor subunit. Among the three putative STAT binding elements (SBE) in the HO-1 promoter, only the distal one was functional and when deleted, the remaining Luc induction was completely obliterated by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. CONCLUSIONS: The HO-1 SBE3 mediates HO-1 gene induction by IL-6 mainly via activation of the Jak/STAT pathway.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase (Desciclizante)/genética , Hepatócitos/enzimologia , Interleucina-6/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Northern Blotting , Técnicas de Cultura de Células , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Cinética , Masculino , Plasmídeos , RNA Mensageiro/genética , Ratos , Ratos Wistar , Transdução de Sinais/imunologia , Ativação TranscricionalRESUMO
In this work, we used two rat models, partial hepatectomy (PH) and CCl(4) administration, to study the changes in iron pathways in response to hepatic damage. Liver injury induced changes in the hepatic gene expression of hepcidin, hemojuvelin (Hjv), several other proteins of iron metabolism, and several cytokines such as IL-1beta, IL-6, TNF-alpha, and IFN-gamma. Hepcidin gene expression was upregulated between 4 and 8 h with a maximum up to 16 h after surgery. However, Hjv gene expression was downregulated at the same time. An early upregulation of hepcidin (3 h) and downregulation of Hjv gene expression was found after CCl(4) administration. Transferrin receptor 1 and ferritin H gene expression was upregulated, whereas ferroportin 1 gene expression was downregulated. Hepatic IL-6 gene expression was upregulated early after PH and reached maximum 8 h after the PH. In CCl(4)-induced liver injury, IL-6, IL-1beta, TNF-alpha, and IFN-gamma upregulation were found at the maximum 12 h after the administration of the toxin. Treatment of isolated rat hepatocytes with IL-6 and, to a lesser extent, with IL-1beta but not with TNF-alpha or IFN-gamma dose dependently upregulated hepcidin and downregulated Hjv gene expression. In hepatic damage, changes of the hepatic gene expression of the main proteins involved in iron metabolism may be induced by locally synthesized mediators.
Assuntos
Hepatócitos/metabolismo , Interleucina-6/metabolismo , Ferro/metabolismo , Cirrose Hepática Experimental/metabolismo , Fígado/lesões , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células Cultivadas , Proteínas Ligadas por GPI , Expressão Gênica , Proteína da Hemocromatose , Hepatectomia , Cirrose Hepática Experimental/induzido quimicamente , Masculino , Ratos , Ratos WistarRESUMO
The aim of this study was to analyse the changes of Prospero-related homeobox 1 (Prox1) gene expression in rat liver under different experimental conditions of liver injury, regeneration and acute phase reaction, and to correlate it with that of markers for hepatoblasts, hepatocytes, cholangiocytes and oval cells. Gene expression was studied at RNA level by RT-PCR, and at protein level by immunohistochemistry. At embryonal stage of rat liver development (embryonal days (ED) 14-16) hepatoblasts were found to be Prox1(+)/Cytokeratin (CK) 19(+) and alpha-fetoprotein (AFP)(+), at this stage Prox1(-)/CK19(+)/AFP(-) small cells (early cholangiocytes?) were identified. In fetal liver (ED 18-22) hepatoblasts were Prox1(+)/CK19(-)/AFP(+). CK7(+) cholangiocytes were detected at this stage, and they were Prox1(-)/AFP(-). In the adult liver hepatocytes were Prox1(+)/CK19(-)/CK7(-)/AFP(-), cholangiocytes were CK19(+) and/or CK7(+) and AFP(-)/Prox1(-). In models of liver damage and regeneration Prox1 remained a stable marker of hepatocytes. After 2-acetyl-aminofluorene treatment with partial hepatectomy (AAF/PH) the amount of Prox1 specific transcripts was low in the liver, when CK19 and AFP gene expression was high, and at no time point AFP(+)/CK19(+ )"oval cells" were found to be Prox1(+). However, a few Prox1(+)/CK19(+) and a few Prox1(+)/CK7(+ )cells were identified in the liver of AAF/PH-animals, which may represent precursors of hepatocytes, or a precancerous state.
Assuntos
Hepatócitos/metabolismo , Proteínas de Homeodomínio/metabolismo , Regeneração Hepática/fisiologia , Fígado/embriologia , Proteínas Supressoras de Tumor/metabolismo , 2-Acetilaminofluoreno , Reação de Fase Aguda/metabolismo , Animais , Ductos Biliares/metabolismo , Biomarcadores/metabolismo , Intoxicação por Tetracloreto de Carbono/patologia , Doença Hepática Induzida por Substâncias e Drogas , Células Epiteliais/metabolismo , Feminino , Imuno-Histoquímica , Queratina-19/biossíntese , Queratina-7/biossíntese , Fígado/patologia , Hepatopatias/metabolismo , Masculino , Gravidez , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , alfa-Fetoproteínas/biossínteseRESUMO
Under certain conditions liver regeneration can be accomplished by hepatic progenitor cells ("oval cells"). So far, only few factors have been identified to be uniquely regulated by the "oval cell" compartment. Using macroarray analysis in a rat model of oval cell proliferation (treatment with 2-acetylaminofluorene and partial hepatectomy, AAF + PH), we identified 12 differentially expressed genes compared to appropriate control models (AAF treatment and sham operation or AAF treatment alone). Further analysis in models of normal liver regeneration (ordinary PH) and acute phase response (turpentine oil-treated rats) revealed that three out of 12 genes (thymidine kinase 1, Jun-D and ADP-ribosylation factor 4) were not affected by the hepatic acute phase reaction but similarly overexpressed in both "oval cell"-dependant and normal liver regeneration. We characterized Jun-D and ADP-ribosylation factors as novel factors upregulated in oval cells and in non-parenchymal liver cells of normally regenerating livers. However, two out of 12 differentially expressed genes were specifically expressed in oval cells: ras-related protein Rab-3b and Ear-2. On protein level, Rab-3b was increased in total liver homogenates and demonstrated only in clusters of oval cells. We postulate that Ear-2 and Rab-3b may represent novel regulatory factors specifically activated in "oval cells".
Assuntos
Hepatócitos/citologia , Regeneração Hepática/fisiologia , Células-Tronco/citologia , 2-Acetilaminofluoreno , Reação de Fase Aguda/induzido quimicamente , Reação de Fase Aguda/metabolismo , Animais , Proliferação de Células , Perfilação da Expressão Gênica , Hepatectomia , Hepatócitos/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Wistar , Receptores de Esteroides/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Terebintina , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
Heme oxygenase-1 (HO-1) is the inducible isoform of an enzyme family responsible for heme degradation and was suggested to be involved in the acute phase response in the liver. However, the mechanisms of the HO-1 regulation under inflammatory conditions are poorly understood. Therefore, the purpose of the current work was to study the expression of HO-1 in the liver and other organs of rats with a localized inflammation after intramuscular injection of turpentine oil (TO). Since interleukin-6 (IL-6) is known to be a principal mediator of inflammation, the levels of this cytokine were also estimated in the animal model used. HO-1 and IL-6 expression was evaluated by Northern blot, in situ hybridization, Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. In the liver and injured muscle, the HO-1 mRNA levels were dramatically increased 4-6 h after TO administration. HO-1 protein levels in the liver were elevated starting from 6-12 h after the treatment. In other internal organs such as the heart, kidney and large intestine, only a slight induction of HO-1 mRNA was observed. IL-6-specific transcripts appeared only in the injured muscle and were in accordance with serum levels of IL-6. In turn, temporal expression of IL-6 in the muscle and circulatory IL-6 levels correlated well with HO-1 expression in the liver and injured muscle. In the liver of control rats HO-1 protein was detected in Kupffer cells, while in TO-injected rats also hepatocytes became strongly HO-1 positive. Conversely, in the injured muscle, HO-1 immunoreactivity was attributed only to macrophages. Our data demonstrate that during localized inflammation HO-1 expression was rapidly and strongly induced in macrophages of injured muscle and in hepatocytes, and IL-6 derived from injured muscle seems to be responsible for the HO-1 induction in the liver.