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1.
Innate Immun ; 20(3): 239-48, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23839527

RESUMO

Microparticles (MPs) are small, membrane-bound vesicles that arise from dead and dying cells, and display pro-inflammatory and pro-thrombotic activity. As shown previously, the RAW 264.7 murine macrophage cell line can release MPs following stimulation with LPS or polyinosinic:polycytidylic acid [poly (I:C)], ligands of TLR4 and TLR3 respectively. To determine the relationship of these MPs to those released during apoptosis, the nucleic acid content of MPs from cultures stimulated with LPS or poly (I:C) was compared with the nucleic acid content of MPs from untreated cells or cells induced to undergo apoptosis by treatment with etoposide or staurosporine (STS). As results of these studies showed, MPs from activated, apoptotic and untreated cells had features in common, as demonstrated by binding of the nucleic acid dyes SYTO 13 and propidium iodide; molecular mass of DNA; and binding of monoclonal anti-DNA and anti-nucleosome Abs. While MPs from the different culture conditions all contained ribosomal RNA, ribosomal RNA from MPs from STS-treated cells showed cleavage and degradation. Taken together, these studies indicate that the nucleic acid content of MPs from activated and apoptotic cells have important similarities, suggesting that events during TLR activation may lead to apoptosis and subsequent MP release.


Assuntos
Apoptose/fisiologia , Micropartículas Derivadas de Células/química , Ativação de Macrófagos/fisiologia , Macrófagos/fisiologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinógenos/farmacologia , Linhagem Celular , DNA/análise , DNA/imunologia , DNA/metabolismo , Etoposídeo/farmacologia , Citometria de Fluxo , Histonas/imunologia , L-Lactato Desidrogenase/análise , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/química , Macrófagos/efeitos dos fármacos , Camundongos , Poli I-C/farmacologia , RNA/análise , RNA/metabolismo , Estaurosporina/farmacologia
2.
Rheumatology (Oxford) ; 51(10): 1737-46, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22403183

RESUMO

Microparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells and enter the blood to display pro-inflammatory and pro-thrombotic activities. MPs are 0.1-1.0 µm in size and incorporate nuclear, cytoplasmic and membrane molecules as they detach from cells. This process can occur with cell activation as well as cell death, with particles likely corresponding to blebs that form on the cell surface during apoptosis. To measure particle expression, flow cytometry allows determination of particle numbers based on size as well as surface markers that denote the cell of origin; platelet MPs are usually the most abundant type in blood. As shown in in vitro and in vivo systems, MPs can promote inflammation and thrombosis resulting from their content of cytokines like IL-1 and pro-coagulant molecules like tissue factor. Certain particle types can be anti-inflammatory, however, suggesting a range of immunomodulatory activities depending on the cell of origin. Studies on patients with a wide range of rheumatic disease show increased MP numbers in blood, with platelet and endothelial particles associated with vascular manifestations; increased numbers of particles also occur in the joint fluid where they may drive cytokine production and activate synoviocytes. In autoimmune diseases such as SLE and RA, MPs may also contribute to disease pathogenesis by the formation of immune complexes. MPs thus represent novel subcellular structures that can impact on the pathogenesis of rheumatic disease and serve as biomarkers of underlying cellular disturbances.


Assuntos
Micropartículas Derivadas de Células/imunologia , Doenças Reumáticas/imunologia , Biomarcadores/metabolismo , Micropartículas Derivadas de Células/metabolismo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Doenças Reumáticas/metabolismo
3.
Antioxid Redox Signal ; 15(8): 2209-19, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21194388

RESUMO

In a wide variety of diseases, cell death represents both an outcome and an important step in pathogenesis. This duality occurs because cell death leads to the extracellular release of molecules and structures that can potently induce the innate immune system. These mediators include the alarmins which are endogenous cellular constituents that exit activated or dying cells to stimulate toll-like receptors (TLRs) as well as non-TLR receptors. Of alarmins, the nonhistone protein HMGB1 is the prototype. Like DNA and RNA, HMGB1 can translocate from cells as they die. The activity of HMGB1 may reflect its interaction with other molecules such as LPS, DNA, and cytokines. In addition to alarmins, dead and dying cells can release subcellular organelles called microparticles that contain cytoplasmic and nuclear constituents, including DNA and RNA. These particles can impact on many cell types to induce inflammation. The release of HMGB1 and microparticles shows important similarities, occurring with cell death as well as stimulation of certain but not all TLRs. Furthermore, nitric oxide can induce the release of both. These observations suggest that the products of dead cells can serve as important mediators to drive immune responses and promote inflammation and autoreactivity.


Assuntos
Morte Celular/fisiologia , Proteína HMGB1/metabolismo , Animais , DNA/metabolismo , Humanos , Imunidade Inata/fisiologia , RNA/metabolismo , Receptores Toll-Like/metabolismo
4.
Immunol Res ; 49(1-3): 227-34, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21132466

RESUMO

Microparticles are small membrane-bound vesicles that display pro-inflammatory and pro-thrombotic activities important in the pathogenesis of a wide variety of diseases. These particles are released from activated and dying cells and incorporate nuclear and cytoplasmic molecules for extracellular export. Of these molecules, DNA is a central autoantigen in systemic lupus erythematosus (SLE). As studies in our laboratory show, DNA occurs prominently in microparticles, translocating into these structures during apoptotic cell death. This DNA is antigenically active and can bind to lupus anti-DNA autoantibodies. These findings suggest that microparticles are an important source of extracellular DNA to serve as an autoantigen and autoadjuvant in SLE.


Assuntos
Micropartículas Derivadas de Células/genética , Micropartículas Derivadas de Células/imunologia , DNA/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Anticorpos Antinucleares/imunologia , Apoptose/genética , Autoanticorpos/imunologia , Autoantígenos/imunologia , Linhagem Celular , Citocinas/imunologia , Espaço Extracelular/imunologia , Humanos , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/genética , Receptores Toll-Like/imunologia
5.
J Leukoc Biol ; 87(6): 1115-23, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20335312

RESUMO

MPs are small membrane-bound particles that originate from activated and dying cells and mediate intercellular communication. Once released from cells, MPs can serve as novel signaling elements in innate immunity, with levels elevated in immune-mediated diseases. This study tested the hypothesis that TLR stimulation can induce MP release by macrophages. In these experiments, using the RAW 264.7 murine macrophage cell line as a model, LPS, a TLR4 ligand, and poly(I:C), a TLR3 ligand, induced MP release effectively, as measured by flow cytometry; in contrast, a CpG oligonucleotide, which can stimulate TLR9, induced much lower levels of particle release. To determine the role of other mediators in this response, the effects of NO were tested. Thus, MP release from RAW 264.7 cells stimulated by LPS or poly(I:C) correlated with NO production, and treatment with the iNOS inhibitor 1400W decreased particle release and NO production. Furthermore, treatment of RAW 264.7 cells with NO donors induced MP production. As TLR ligands can induce apoptosis, the effect of caspase inhibition on MP release by stimulated cells was assessed. These experiments showed that the pan-caspase inhibitor, ZVAD, although decreasing NO production, increased MP release by stimulated cells. Together, these experiments demonstrate that TLR stimulation of macrophages can lead to MP release, and NO plays a key role in this response.


Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Poli I-C/farmacologia , Receptores Toll-Like/metabolismo , Animais , Inibidores de Caspase , Caspases/metabolismo , Células Cultivadas , Citometria de Fluxo , Ligantes , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais
6.
Autoimmunity ; 42(4): 299-301, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19811282

RESUMO

High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein with alarmin activity. When present in an extracellular location, HMGB1 can activate the innate immune system and promote inflammation in conditions such as sepsis. To exert these activities, HMGB1 must transit from the nucleus, through the cytoplasm, to the outside of the cell. This process can occur during cell activation as well as cell death. In murine macrophages (MPhi), stimulation of TLR3 and TLR4, but not TLR9, can cause HMGB1 translocation. With cell death, necrosis can lead to extracellular HMGB1 by a passive mechanism. With apoptosis, HMGB1 is only released during secondary necrosis, when cell permeability barriers break down. Since agents that stimulate MPhi can also induce apoptosis, HMGB1 release following TLR stimulation may also reflect a contribution from dead cells, suggesting a common mechanism for protein release in activation and death.


Assuntos
Apoptose/imunologia , Proteína HMGB1/imunologia , Ativação de Macrófagos/imunologia , Transporte Proteico/imunologia , Animais , Humanos
7.
Artigo em Inglês | MEDLINE | ID: mdl-19358893

RESUMO

In this study, we examined the effect of concurrent low concentrations of sodium arsenite and mild heat shock temperatures on hsp30 and hsp70 gene expression in Xenopus A6 kidney epithelial cells. RNA blot hybridization and immunoblot analysis revealed that exposure of A6 cells to 1-10 microM sodium arsenite at a mild heat shock temperature of 30 degrees C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 microM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26 degrees C with larger responses at 28 and 30 degrees C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 microM) and heat shock (30 degrees C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge (37 degrees C). Acquired thermotolerance was not observed with cells treated with the two mild stresses individually.


Assuntos
Adaptação Fisiológica , Arsenitos/farmacologia , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP70/genética , Temperatura Alta , Rim/efeitos dos fármacos , Compostos de Sódio/farmacologia , Animais , Northern Blotting , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imuno-Histoquímica , Rim/citologia , Rim/metabolismo , Rim/fisiologia , Microscopia Confocal , Xenopus
8.
Artigo em Inglês | MEDLINE | ID: mdl-17267255

RESUMO

In previous studies, the only small HSPs that have been studied in Xenopus laevis are members of the HSP30 family. We now report the analysis of Xenopus HSP27, a homolog of the human small HSP, HSP27. To date the presence of both hsp30 and hsp27 genes has been demonstrated only in minnow and chicken. Xenopus HSP27 cDNA encodes a 213 aa protein that contains an alpha-crystallin domain as well as a polar C-terminal extension. Xenopus HSP27 shares 71% identity with chicken HSP24 but only 19% identity with Xenopus HSP30C. Northern blot analysis revealed that Xenopus HSP27 gene expression was developmentally regulated. Constitutive and heat shock-induced hsp27 mRNA accumulation was first detectable at the early tailbud stage while HSP27 protein was detected at the tadpole stage. Furthermore, hsp27 mRNA was enriched in selected tissues under both control and heat shock conditions. Whole mount in situ hybridization analysis detected the presence of this message in the lens vesicle, heart, head, somites, and tail region. Purified recombinant HSP27 protein displayed molecular chaperone properties since it had the ability to inhibit heat-induced aggregation of target proteins including citrate synthase, malate dehydrogenase and luciferase. Thus, Xenopus HSP27, like HSP30, is a developmentally-regulated heat-inducible molecular chaperone.


Assuntos
Embrião não Mamífero/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Citrato (si)-Sintase/química , DNA Complementar/genética , Regulação da Expressão Gênica , Resposta ao Choque Térmico , Luciferases/química , Malato Desidrogenase/química , Dados de Sequência Molecular , Filogenia , Estrutura Quaternária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Xenopus laevis/genética
9.
Artigo em Inglês | MEDLINE | ID: mdl-16861019

RESUMO

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP110/genética , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Arsenitos/farmacologia , Cloreto de Cádmio/farmacologia , Células Cultivadas , Cricetinae , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Choque Térmico HSP110/biossíntese , Temperatura Alta , Rim/embriologia , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Compostos de Sódio/farmacologia , Proteínas de Xenopus/biossíntese , Xenopus laevis/embriologia
10.
Immunogenetics ; 55(4): 253-61, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12774179

RESUMO

A full-length cDNA encoding CCAAT/enhancer-binding protein beta (C/EBPbeta) was cloned from rainbow trout by anchored PCR. The putative 291 amino acid protein has 53% and 32% identity to the zebrafish and Japanese flounder sequences, respectively, and 30-34% identity to tetrapod homologues. This clone contains most conserved C/EBPbeta domains except the second transactivation domain just like the zebrafish homologue. Also similar to zebrafish, rainbow trout produces only shorter C/EBPbeta isoforms (LAP and LIP) but not the longer isoform (LAP*). However, unlike the zebrafish and Japanese flounder homologues, trout C/EBPbeta has the short open reading frame (uORF) upstream of the start codon for LAP but in an alternate reading frame, a feature of tetrapod C/EBPbeta genes. In normal rainbow trout, C/EBPbeta mRNA was detected in peripheral blood leukocytes, head kidney, posterior kidney, liver, spleen, gills, intestine and muscle. RT-PCR revealed that transcript levels of trout C/EBPbeta are clearly higher in sodium alginate-induced peritoneal cells than in head kidney and peritoneal cells of saline-injected fish or head kidney cells of alginate-injected fish. Together with expression in immunologically important tissues, this indicates that C/EBPbeta is likely to be involved in the immune response just as in mammals. Southern hybridization suggested that C/EBPbeta is a single copy gene. There are no introns in this C/EBPbeta gene, just like the mammalian homologues. These data suggest that we have obtained the trout ortholog of C/EBPbeta.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Oncorhynchus mykiss/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Clonagem Molecular , DNA Complementar/genética , Evolução Molecular , Dosagem de Genes , Dados de Sequência Molecular , Oncorhynchus mykiss/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
11.
Immunogenetics ; 55(2): 126-31, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12695860

RESUMO

A cDNA clone, designated IL-8nL, was obtained by suppression subtractive hybridisation between lipopolysaccharide-stimulated and non-stimulated populations of the rainbow trout macrophage-like cell line, RTS11. IL-8nL was similar but not identical to a recently published sequence of the gene encoding rainbow trout interleukin-8 (IL-8). Amplification of genomic DNA by the polymerase chain reaction (genomic PCR) using a single outbred trout with common primers in the 5' and 3' untranslated regions gave six distinct genomic sequences, including one ( IL-8A) almost identical to that of the published IL-8 gene and another identical to IL-8nL. The other four clones were termed IL-8B, IL-8C, IL-8D and IL-8E. The deduced amino acid sequences of IL-8A through IL-8E are all identical to the published IL-8, while the IL-8nL protein has a substitution of Arg87 to Lys. Analysis of ten outbred trout by genomic PCR of a repeat region in exon 4, which has three different sizes in the above alleles, revealed a shorter, fourth fragment termed IL-8X and another of the same size as IL-8nL, but with a different single nucleotide replacement, called IL-8nL2. These results, together with a Southern blot of the same ten individuals showing up to five bands, indicate that rainbow trout has at least four copies of the IL-8 gene. Like IL-8nL, IL-8X lacks the repeat sequence in exon 4 and encodes a protein identical to IL-8nL protein. Polymerase chain reaction of the repeat region was useful for typing rainbow trout into four categories, and the type III and IV fish have a new allele, IL-8F, which lacks one repeat unit compared with IL-8A.


Assuntos
Interleucina-8/genética , Oncorhynchus mykiss/genética , Animais , Sequência de Bases , Southern Blotting , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Alinhamento de Sequência
12.
Immunogenetics ; 54(8): 604-9, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12439625

RESUMO

In comparison to mammals, relatively few of the molecules involved in teleost immune responses have been isolated and characterized. A rapid method of isolating molecules important for immune function is subtractive hybridization. One such experiment using infectious hematopoietic necrosis virus-infected Atlantic salmon produced several cDNA clones with similarity to mammalian immune-specific genes, including granzyme M (accession no. AF434669) and CD9. After cloning the rainbow trout version of CD9, sequence analysis showed that both salmonid sequences contained many features of the tetraspanin receptor family to which CD9 belongs. Phylogenetic analysis revealed a close association of the trout and salmon sequences to known CD9 and CD81 receptors. Southern blotting demonstrated that the rainbow trout gene is single copy. Reverse transcriptase PCR showed strong expression of this clone in many tissues, but liver expression was very low - an observation consistent with the clone being a CD9, not a CD81, equivalent. The evidence suggests that the sequences reported here are bona fide teleost CD9 homologues and we are currently producing recombinant proteins and polyclonal antisera for use in functional studies.


Assuntos
Antígenos CD/genética , Glicoproteínas de Membrana/genética , Oncorhynchus mykiss/genética , Salmo salar/genética , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Sequência de Bases , Clonagem Molecular , DNA Complementar , Glicoproteínas de Membrana/biossíntese , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Tetraspanina 29 , Distribuição Tecidual
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