RESUMO
Tumors characterized by co-expression of S100 and CD34, in the absence of SOX10, remain difficult to classify. Triggered by a few index cases with monomorphic cytomorphology and distinctive stromal and perivascular hyalinization, immunopositivity for S100 and CD34, and RAF1 and NTRK1 fusions, the authors undertook a systematic review of tumors with similar features. Most of the cases selected were previously diagnosed as low-grade malignant peripheral nerve sheath tumors, while others were deemed unclassified. The tumors were studied with targeted RNA sequencing and/or FISH. A total of 25 cases (15 adults and 10 children) with kinase fusions were identified, including 8 cases involving RAF1, 2 BRAF, 14 NTRK1, and 1 NTRK2 gene rearrangements. Most tumors showed a monomorphic spindle cell proliferation with stromal and perivascular keloidal collagen, in a patternless architecture, with only occasional scattered pleomorphic or multinucleated cells. Most cases showed low cellularity, a low mitotic count, and absence of necrosis. Although a subset showed overlap with lipofibromatosis-like neural tumors, the study group showed distinctive hyalinization and overt malignant features, such as highly cellular fascicular growth and primitive appearance. All tumors showed co-expression of S100 and CD34, ranging from focal to diffuse. SOX10 was negative in all cases. NTRK1 immunohistochemistry showed high levels of expression in all tumors with NTRK1 gene rearrangements. H3K27me3 expression performed in a subset of cases was retained. These findings together with the recurrent gene fusions in RAF1, BRAF, and NTRK1/2 kinases suggest a distinct molecular tumor subtype with consistent S100 and CD34 immunoreactivity.
Assuntos
Fusão Gênica , Glicoproteínas de Membrana/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética , Receptor trkA/genética , Receptor trkB/genética , Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Adolescente , Adulto , Criança , Pré-Escolar , Rearranjo Gênico , Genes Neoplásicos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias de Bainha Neural/genética , Receptores de Complemento 3b/biossíntese , Proteínas S100/biossíntese , Fatores de Transcrição SOXE/biossíntese , Sarcoma/classificação , Sarcoma/metabolismo , Sarcoma/patologia , Neoplasias de Tecidos Moles/classificação , Neoplasias de Tecidos Moles/metabolismo , Neoplasias de Tecidos Moles/patologia , Adulto JovemRESUMO
OBJECTIVES: Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. METHODS: Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. RESULTS: The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10(-4), OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10(-7), OR 0.71; case-only pmeta=1.9×10(-4), OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. CONCLUSIONS: These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications.
Assuntos
Anticorpos Antinucleares/sangue , Lúpus Eritematoso Sistêmico/genética , Receptores de Complemento 3d/genética , Adolescente , Adulto , Subpopulações de Linfócitos B/imunologia , Estudos de Casos e Controles , DNA/imunologia , Predisposição Genética para Doença , Variação Genética , Genótipo , Haplótipos , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Receptores de Complemento 3b/biossíntese , Medição de Risco/métodos , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
BACKGROUND: Animal models have shown that CD47-deficient mice develop severe autoimmune hemolytic anemia (AIHA) because the binding of red blood cell (RBC) CD47 to signal-regulatory protein (SIRP-alpha) on macrophages contributes to the inhibition of phagocytosis. In contrast, complement-inhibitory proteins such as CD35, CD55, and CD59 may protect RBCs against the lysis by complement. STUDY DESIGN AND METHODS: With the use of flow cytometric analyses, the expression of CD47, CD35, CD55, and CD59 on RBCs and of SIRP-alpha,beta on peripheral monocytes of 36 patients with warm AIHA (wAIHA; 23 with active wAIHA, 13 with wAIHA in remission) and 20 healthy subjects was evaluated. RESULTS: The mean fluorescence intensities (MFIs) of the expression of CD47, CD35, CD55, and SIRP-alpha,beta of active wAIHA patients, wAIHA in remission, and healthy subjects were not statistically different. Patients with active wAIHA showed significantly lower CD59 expression on RBCs than healthy individuals (MFI, 512.5 +/- 59.6 vs. 553.7 +/- 36.6; p = 0.009), while the CD59 expression in patients with wAIHA in remission was not significantly different from that of healthy controls (MFI, 538.4 +/- 48.3 vs. 553.7 +/- 36.6; p > 0.05). The expression of CD59 on RBCs of 3 patients who died from the wAIHA was lower than that seen on RBCs of healthy controls (MFI, 433.6 +/- 69.6 vs. 553.74 +/- 36.6; p = 0.0001). CONCLUSIONS: Our data show that the expression of CD47 on RBCs and SIRP-alpha,beta on monocytes of patients with wAIHA is not different from that seen in healthy individuals. In addition, we detected that patients with active wAIHA present low expression of CD59 and normal expression of CD35 and CD55 on their RBCs. Complement-regulatory proteins may play an important role in protecting RBC destruction through the activation of complement.
Assuntos
Anemia Hemolítica Autoimune/metabolismo , Antígenos de Diferenciação/biossíntese , Antígeno CD47/biossíntese , Antígenos CD55/biossíntese , Antígenos CD59/biossíntese , Eritrócitos/metabolismo , Monócitos/metabolismo , Receptores de Complemento 3b/biossíntese , Receptores Imunológicos/biossíntese , Adulto , Idoso , Anemia Hemolítica Autoimune/genética , Animais , Antígenos de Diferenciação/genética , Antígeno CD47/genética , Antígenos CD55/genética , Antígenos CD59/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fagocitose/genética , Receptores de Complemento 3b/genética , Receptores Imunológicos/genéticaRESUMO
Cigarette smoke and hemodynamic stress both contribute to vascular inflammation and associated atherosclerosis. We recently demonstrated direct activation of complement components C4 and C3 on human endothelial cells (EC). The present study was designed to explore complement activation on bone marrow microvascular endothelial cells (BMEC) and human umbilical vein endothelial cells (HUVEC) in response to endothelial cell injury by tobacco smoke extract, shear stress, or other known inflammatory and atherogenic mediators, lipopolysaccharide (LPS) and INF-gamma. Following treatment, confluent EC monolayers were exposed to plasma (60 min, 37 degrees C), and cell surface deposition of stable complement derivatives C4d, iC3b and SC5b-9 was measured in situ using an ELISA approach. Consistent with previous results, moderate levels of C4d, iC3b and SC5b-9 deposition were observed on native EC monolayers exposed to human plasma. Tobacco smoke and shear stress enhanced EC C4d deposition. In contrast, LPS and INF-gamma failed to affect EC mediated complement activation, despite evidence of EC activation illustrated by ICAM-1 expression. The combination of tobacco smoke and shear stress nearly doubled EC C4d expression. No increases in iC3b or SC5b-9 were noted, suggesting inhibition of classical and alternative pathway C3 convertase assembly or activity. Indeed, concomitantly increased surface expression of complement regulatory proteins CD35 (CR1) and CD55 was observed following EC exposure to tobacco smoke and shear stress. These results suggest that a balance between complement activation and regulation exists at the EC surface, and may impact vascular injury leading to thrombosis, arteriosclerosis, and atherogenesis.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Células Endoteliais/citologia , Animais , Células da Medula Óssea/citologia , Antígenos CD55/biossíntese , Ativação do Complemento , Células Endoteliais/metabolismo , Hemodinâmica , Humanos , Interferon gama/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Microcirculação/citologia , Receptores de Complemento 3b/biossíntese , Fumar , Estresse Mecânico , Veias Umbilicais/citologiaRESUMO
OBJECTIVE: To research the erythrocyte immunoregulation effects of Patrinia scabra extracts by macroporous adsorptive resins on mice burdened transplanted tumor. METHODS: Extracts of Patrinia scabra Bunge were separated by macroporous adsorptive resins, ingredients were analysised. Mice burdened transplanted tumor were given extracted drugs. Life prolongation rate was observed, erythrocyte immunologic function and the CD35, CD44s contents of red blood cell were evaluated. RESULTS: Polysaccharide and saponin accounted for 8.4% and 48.4%. Extracts could porolong life expectancy of mice, improve erythrocyte immunolgic function and increase the CD35 and CD44s contents of red blood cell. CONCLUSION: Extracts of Patrinia scabra Bunge by macroporous adsorptive resins have erythrocyte immunoregulation effects on mice burdened transplanted tumor.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Eritrócitos/efeitos dos fármacos , Patrinia/química , Sarcoma 180/prevenção & controle , Adsorção , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Eritrócitos/imunologia , Eritrócitos/metabolismo , Feminino , Receptores de Hialuronatos/biossíntese , Masculino , Camundongos , Transplante de Neoplasias , Plantas Medicinais/química , Polissacarídeos/análise , Distribuição Aleatória , Receptores de Complemento 3b/biossíntese , Resinas Sintéticas/química , Saponinas/análise , Sarcoma 180/sangue , Sarcoma 180/patologia , Análise de SobrevidaRESUMO
There is compelling evidence that a unique innate immune response in the CNS plays a critical role in host defense and clearance of toxic cell debris. Although complement has been implicated in neuronal impairment, axonal loss, and demyelination, some preliminary evidence suggests that the initial insult consequently activates surrounding cells to signal neuroprotective activities. Using two different models of experimental autoimmune encephalomyelitis, we herein demonstrate selective C1q complement activation on neuron cell bodies and axons. Interestingly, in brains with chronic but not acute experimental autoimmune encephalomyelitis, C3b opsonization of neuronal cell bodies and axons was consistently associated with robust neuronal expression of one of the most effective complement regulators, decay-accelerating factor (CD55). In contrast, levels of other complement inhibitors, complement receptor 1 (CD35), membrane cofactor protein (CD46), and CD59 were largely unaffected on neurons and reactive glial cells in both conditions. In vitro, we found that proinflammatory stimuli (cytokines and sublytic doses of complement) failed to up-regulate CD55 expression on cultured IMR32 neuronal cells. Interestingly, overexpression of GPI-anchored CD55 on IMR32 was capable of modulating raft-associated protein kinase activities without affecting MAPK activities and neuronal apoptosis. Critically, ectopic expression of decay-accelerating factor conferred strong protection of neurons against complement attack (opsonization and lysis). We conclude that increased CD55 expression by neurons may represent a key protective signaling mechanism mobilized by brain cells to withstand complement activation and to survive within an inflammatory site.
Assuntos
Antígenos CD55/biossíntese , Via Clássica do Complemento/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Neurônios/imunologia , Neurônios/patologia , Doença Aguda , Animais , Antígenos CD/biossíntese , Apoptose/imunologia , Axônios/imunologia , Axônios/metabolismo , Axônios/patologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Antígenos CD55/fisiologia , Antígenos CD59/biossíntese , Callithrix , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/imunologia , Doença Crônica , Ensaio de Atividade Hemolítica de Complemento , Glicosilfosfatidilinositóis/fisiologia , Humanos , Células K562 , Macaca fascicularis , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/biossíntese , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Neurônios/metabolismo , Proteínas Opsonizantes/metabolismo , Receptores de Complemento 3b/biossíntese , Transdução de Sinais/imunologia , Células U937RESUMO
Interleukin-10 (IL-10) is a well known anti-inflammatory cytokine. However, we previously showed that it could present pro-inflammatory properties on human monocytes in the absence of adherence. In the present study, using macroarray technology, we analyzed the effects of IL-10 and adherence on the expression of 1050 genes in human monocytes cultured for 3 hours on plastic or Teflon(R) (to avoid adherence). Adherence alone induced specifically the expression of 12 genes and repressed that of 25 genes. In adherent monocytes, IL-10 induced the expression of 21 genes and repressed that of 50 genes. In non-adherent monocytes, IL-10 induced the expression of 45 genes and repressed that of 67. Only 3 common genes were induced while 35 common genes were repressed by IL-10 in the two culture conditions. Interestingly, we showed that IL-10 modulated conversely on Teflon(R) and plastic the expression of 16 genes, of which SOCS molecules, coproporphyrinogen oxidase, matrix metalloproteinases and complement receptor-1 (CD35). This study demonstrates that adherence has profound modulatory effects on the properties and the signaling induced by IL-10. The discovery that IL-10 can inhibit the production of coproporphyrinogen oxidase (an enzyme involved in the synthesis of heme) may shed some lights on the mechanisms of anaemia induced by IL-10. Furthermore, the inhibition of the expression of SOCS1 by IL-10 in the absence of adherence, may explain its priming effects on a subsequent LPS stimulation that we previously described.
Assuntos
Adesão Celular , Regulação da Expressão Gênica , Interleucina-10/fisiologia , Células Cultivadas , Colagenases/metabolismo , Coproporfirinogênio Oxidase/metabolismo , Citometria de Fluxo , Humanos , Interleucina-10/metabolismo , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Metaloproteinase 13 da Matriz , Monócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Plásticos/farmacologia , Politetrafluoretileno/farmacologia , Receptores de Complemento 3b/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Herein we demonstrate that B cell-activating factor of the TNF family (BAFF), a B cell survival factor, also regulates CD21/35 and CD23 expression. BAFF blockade in wild-type mice down-modulates CD21/35 and CD23 on B cells while survival remains intact, and BAFF exposure causes elevated CD21/35 and CD23 expression. Similar down-modulation is observed in bcl-2-transgenic mice treated with a BAFF inhibitor. This is the first evidence that BAFF has a function independent of B cell survival. Reports using CD21/35 and CD23 expression to assess splenic B cell subsets in BAFF-null mice concluded a lack of B cells beyond the immature stage. Since CD21/35 and CD23 are inadequate for delineating B cell subpopulations in BAFF-null mice, we used expression of BAFF-R and several B cell markers to identify more mature splenic B cells in these mice. These data broaden our understanding of BAFF function and correct the view that BAFF-null mice lack mature B cells.
Assuntos
Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Proteínas de Membrana/fisiologia , Receptores de Complemento 3b/biossíntese , Receptores de Complemento 3d/biossíntese , Receptores de IgE/biossíntese , Fator de Necrose Tumoral alfa/fisiologia , Animais , Fator Ativador de Células B , Receptor do Fator Ativador de Células B , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Feminino , Humanos , Imunoglobulina D/biossíntese , Imunoglobulina M/biossíntese , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Receptores do Fator de Necrose Tumoral/biossíntese , Baço/citologia , Baço/imunologia , Baço/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Primate immune adherence receptors are erythrocyte complement receptors (E-CR) that favorably influence the clearance of circulating immune complexes (IC). The human E-CR is the type one complement receptor (CR1), most commonly expressed as a 220 kDa protein containing 30 short consensus repeats (SCRs). The chimpanzee E-CR is a 75 kDa protein composed of eight SCRs, and is encoded by an ortholog of human CR1-like (CR1L), a genetic element related to CR1. Human CR1L was previously identified from genomic clones that predict exons for seven SCRs, and there have been no reports of CR1L expression. The purpose of this study was to determine if human CR1L is expressed. Amplification of human bone marrow cDNA using primers specific for CR1/CR1L yielded a product similar to chimp CR1L encoding sequence. The first 6.5 SCRs matched 100% with the predicted human CR1L sequence, while the second half of SCR 7 was homologous to the comparable chimp CR1L sequence but with a stop codon. Expression in COS-7 cells yielded a human CR1L protein of approximately 50 kDa that exhibited binding specificity for iC4 but not for iC3. Neither northern nor western blot analysis of human bone marrow revealed the presence of the CR1L transcript or protein. However, northern blot analysis of various other lymphoid tissue identified a candidate CR1L transcript in human fetal liver. PCR amplification of a cDNA panel of human fetal tissue confirmed the presence of the CR1L transcript in fetal liver, and to a lesser extent in fetal spleen and thymus. Thus, expression of the CR1L transcript appears to be limited to hematopoietic and fetal lymphoid tissue.
Assuntos
Feto/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Tecido Linfoide/metabolismo , Receptores de Complemento 3b/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Humanos , Ligantes , Dados de Sequência Molecular , Pan troglodytes/genética , RNA Mensageiro/metabolismo , Receptores de Complemento 3b/biossíntese , Análise de Sequência de DNARESUMO
Hemophagocytic lymphohistiocytosis (HLH) is an extremely rare and highly lethal chronic inflammatory disease, which is mediated by proinflammatory cytokines. In the peripheral blood of a boy suffering from HLH, a chronic expansion of CD14(dim)/CD16(bright) inflammatory monocytes was detected. Compared with CD14(bright) monocytes, their immunophenotype correlated with more mature monocytic cells differentiating to macrophages: they showed lower expression of CD11b, CD64 and CD35. Such CD14(dim)/CD16(bright) monocytes produce the inflammatory cytokines IL-1beta, IL-6 and TNF-alpha. They fit in well with the pathophysiological concept of HLH as an inflammatory state of lymphocytes and of the monocyte/macrophage system. In the presented patient the percentage of these circulating inflammatory monocytes decreased over time during clinical response to immunosuppressive therapy. This finding may indicate that CD14(dim)/CD16(bright) monocytes represented the degree of inflammation in this extremely rare and highly lethal disease.
Assuntos
Histiocitose de Células não Langerhans/imunologia , Receptores de Lipopolissacarídeos/imunologia , Monócitos/imunologia , Receptores de IgG/imunologia , Adolescente , Contagem de Células , Ciclosporina/uso terapêutico , Dexametasona/uso terapêutico , Etoposídeo/uso terapêutico , Antígenos HLA-DR/biossíntese , Histiocitose de Células não Langerhans/tratamento farmacológico , Histiocitose de Células não Langerhans/fisiopatologia , Humanos , Imunossupressores/uso terapêutico , Antígeno de Macrófago 1/biossíntese , Masculino , Monócitos/citologia , Receptores de Complemento 3b/biossíntese , Receptores de IgG/biossínteseRESUMO
Although it is now appreciated that mast cell-mediated release of TNF-alpha is critical for resolution of acute septic peritonitis, questions remain as to how mast cells are activated upon peritoneal bacterial infection. Clues to how this may occur have been derived from earlier studies by Prodeus et al. in which complement proteins C3 and C4 were shown to be required for survival following cecal ligation and puncture (CLP), a model for acute septic peritonitis. To evaluate the mechanism for mast cell activation in the CLP model, complement receptor CD21/CD35-deficient mice (Cr2(null)) were examined in the present study. Along with CD19-deficient (CD19(null)) mice, these animals exhibit decreased survival following CLP compared with wild-type littermates. Injection of IgM before CLP does not change survival rates for Cr2(null) mice and only partially improves survival of CD19(null) mice, implicating CD21/CD35 and CD19 in mast cell activation. Interestingly, early TNF-alpha release is also impaired in Cr2(null) and CD19(null) animals, suggesting that these molecules directly affect mast cell activation. Cr2(null) and CD19(null) mice demonstrate an impairment in neutrophil recruitment and a corresponding increase in bacterial load. Examination of peritoneal mast cells by flow cytometry and confocal microscopy reveals the expression and colocalization of CD21/CD35 and CD19. Taken together, these findings suggest that the engagement of complement receptors CD21/CD35 along with CD19 on the mast cell surface by C3 fragments may be necessary for the full expression of mast cell activation in the CLP model.
Assuntos
Antígenos CD19/fisiologia , Mastócitos/imunologia , Mastócitos/metabolismo , Peritonite/imunologia , Receptores de Complemento 3b/fisiologia , Receptores de Complemento 3d/fisiologia , Sepse/imunologia , Doença Aguda , Animais , Antígenos CD19/biossíntese , Antígenos CD19/genética , Antígenos CD19/metabolismo , Líquido Ascítico/imunologia , Líquido Ascítico/metabolismo , Líquido Ascítico/patologia , Ceco/cirurgia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Citometria de Fluxo , Contagem de Leucócitos , Ligadura , Masculino , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/patologia , Lavagem Peritoneal , Peritonite/genética , Peritonite/mortalidade , Proteínas Proto-Oncogênicas c-kit/biossíntese , Punções , Receptores de Complemento 3b/biossíntese , Receptores de Complemento 3b/metabolismo , Receptores de Complemento 3d/biossíntese , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Análise de Sobrevida , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The respiratory burst of human neutrophils is primed by a number of pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS); however, the mechanism of priming remains unknown. LPS has been shown previously to increase membrane expression of flavocytochrome b(558), a component of the NADPH oxidase. This study shows that TNFalpha also increases membrane expression of flavocytochrome b(558). Mitogen-activated protein kinase (MAPK) modules have been implicated in the action of priming agents. Pharmacologic inhibitors of MAPKs, SB203580 and PD098059, revealed that priming of the respiratory burst and up-regulation of flavocytochrome b(558) are dependent on p38 MAPK but not on extracellular-signal regulated kinase (ERK). TNFalpha and LPS primed respiratory burst activity and increased membrane expression of CD35 and CD66b, specific markers of secretory vesicles and specific granules that contain flavocytochrome b(558), with similar time courses and concentration dependences. These processes also required p38 MAPK but were independent of ERK. TNFalpha failed to prime respiratory burst activity or to increase membrane CD35 expression in enucleated neutrophil cytoplasts. These data suggest that one mechanism by which TNFalpha and LPS prime neutrophil respiratory burst activity is by increasing membrane expression of flavocytochrome b(558) through exocytosis of intracellular granules in a process regulated by p38 MAPK.
Assuntos
Antígenos de Neoplasias , Moléculas de Adesão Celular , Grupo dos Citocromos b/metabolismo , Grânulos Citoplasmáticos/metabolismo , Exocitose , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADPH Oxidases , Neutrófilos/fisiologia , Explosão Respiratória , Antígenos CD , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ativação Enzimática , Proteínas Ligadas por GPI , Humanos , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/biossíntese , Fagocitose/efeitos dos fármacos , Receptores de Complemento 3b/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
In lymph nodes, dendritic cells form a complex meshwork and are linked by intercellular junctions. Intercellular junctions contribute to the integrity of lymphatic follicles and can potentially be affected by malignant processes in neighbouring B cells. We examined whether transmembrane molecules that constitute "adherens junctions" are present in follicular dendritic cells of normal human lymph nodes. We found that follicular dendritic cells but not interdigitating dendritic cells or sinus lining cells expressed cadherin molecules. Follicular dendritic cells also expressed beta-catenin but not vinculin. The cadherin molecules, which were identified in situ with the use of a monoclonal pan-cadherin antibody, were not recognized by antibodies to E-cadherin, N-cadherin or P-cadherin. Intrafollicularly, cadherins were clearly colocalized with beta-catenins, in a dot-like fashion. We also detected intrafollicular expression of desmogleins and desmosomal plaque proteins. These findings indicate the presence of desmosomes within the dendritic meshwork. However, pan-cadherin reactivity was not only colocalized with desmoglein immunoreactivity that was abundantly present. Immunoprecipitation showed that pan-cadherin reactivity was absent in fractions of desmosomal plaque proteins or pan-desmogleins. We speculate that complexes of cadherins of an unknown subclass and beta-catenins form non-desmosomal intercellular junctions in the intrafollicular dendritic meshwork.
Assuntos
Caderinas/biossíntese , Proteínas do Citoesqueleto/biossíntese , Células Dendríticas Foliculares/metabolismo , Linfonodos/metabolismo , Transativadores , Células Dendríticas/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Microscopia de Fluorescência , Fenótipo , Testes de Precipitina , Receptores de Complemento 3b/biossíntese , beta CateninaRESUMO
We have compared the interaction of Mycobacterium tuberculosis with the human macrophage-like cell line THP-1 and with human monocyte-derived macrophages (MDM). The association of M. tuberculosis with THP-1 and MDM was comparable in both the presence and the absence of serum. For both cells, serum-mediated binding was much greater than nonopsonic binding and was mediated by a heat-labile serum component. Nonopsonic binding of M. tuberculosis to both cells could be inhibited by antibodies recognizing CD11b and by mannan and glucan. Intracellular M. tuberculosis grew progressively in infected MDM and THP-1 cells. Treatment of the infected MDM and THP-1 cells with the anti-mycobacterial isoniazid resulted in the rapid killing of the intracellular mycobacteria. Differentiated, adherent THP-1 cells bound IgG and complement-coated particles at levels similar to those of MDM. However, binding of zymosan by THP-1 cells was significantly lower than that seen for MDM.
Assuntos
Macrófagos/microbiologia , Monócitos/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Adulto , Anticorpos/farmacologia , Antituberculosos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/imunologia , Linhagem Celular , Glucanos/imunologia , Glucanos/metabolismo , Humanos , Isoniazida/farmacologia , Antígeno de Macrófago 1/imunologia , Macrófagos/metabolismo , Mananas/imunologia , Testes de Sensibilidade Microbiana , Monócitos/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Polissacarídeos/metabolismo , Receptores de Complemento 3b/biossíntese , Receptores Fc/biossíntese , Receptores Mitogênicos/biossíntese , Zimosan/metabolismoRESUMO
We examined systemic effects of whole-body UVB irradiation on human peripheral blood phagocytes. We found that 24 h after a single erythemal dose of UVB radiation two phagocyte functions, adhesion and phagocytosis, were reduced by 50%. This functional suppression was accompanied by a significant decrease in the expression of complement receptors (CR1 and CR3) and IgG Fc receptors (FcRII and FcRIII). The greatest reduction (47%) was observed in CR3, which is important for both adhesion and phagocytosis. A kinetic analysis showed that both CR1 and CR3 levels started to decrease 15 min after the UVB exposure, reaching the lowest levels at 4.5- and 24-h time points, respectively. The down-modulation of CRs after whole-body UVB exposure was not due to a defective receptor synthesis or translocation from internal stores to plasma membrane because the maximal CR levels in stimulated cells were not affected by UVB. No change in the serum soluble ICAM-1 was detected after UVB, which rules out CD1 1b epitope masking by sICAM-1. UVB did not release low-receptor-density myeloid progenitor cells from storage pools into circulation. Interleukin 10, a mediator of UVB-induced immunosuppression, was unable to modulate CR expression in vitro. When seven suberythemal whole-body UVB exposures were given repeatedly within 2 weeks, a significant decrease in CR expression was seen, which was greatest after three irradiations. Our data suggest that an exposure to UVB has systemic effects in humans which, possibly due to the down-modulation of preexisting cell-surface receptors, suppress some important functions of circulating phagocytic cells.
Assuntos
Terapia de Imunossupressão/efeitos adversos , Neutrófilos/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Irradiação Corporal Total/efeitos adversos , Adulto , Adesão Celular/imunologia , Adesão Celular/efeitos da radiação , Feminino , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Interleucina-10/farmacologia , Antígeno de Macrófago 1/biossíntese , Masculino , Neutrófilos/imunologia , Fagocitose/imunologia , Fagocitose/efeitos da radiação , Receptores de Complemento 3b/biossíntese , Receptores de IgG/biossíntese , Proteínas Recombinantes/farmacologiaRESUMO
Binding to erythrocyte complement receptor type 1 (CR1) clears immune complexes from blood and tissues, preventing complement-mediated pathological inflammation in disease. Previous work has demonstrated that Ara-C, a cytosine analogue, induces an 11-fold increase in CR1 mRNA expression in K-562 erythroleukaemia cells. In this work we therefore investigated whether the Ara-C/K-562 system could be used as a model for studying the pre-erythroid regulation of CR1. We demonstrated that increased CR1 expression could be induced independently of increased haemoglobin expression. Increases in CR1 mRNA levels produced by Ara-C treatment were not a function of increased stability of the message. However, Ara-C induced a protein synthesis-dependent increase in transcription initiation rate as early as 12h after treatment. Further data suggest that the effect of Ara-C on transcription is not a result of its direct DNA-damaging or DNA polymerase-inhibition activities. Induction of receptor transcription was inhibited by tyrosine kinase (TK) and protein kinase C (PKC) inhibitors. These data suggest that TK, PKC and dCTP-adducted phospholipid signalling pathways may all play a role in the mechanism of Ara-C-induced CR1 transcription.
Assuntos
Citarabina/farmacologia , Células Precursoras Eritroides/imunologia , Células K562/imunologia , Receptores de Complemento 3b/genética , Transcrição Gênica/efeitos dos fármacos , Butiratos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/enzimologia , Células Precursoras Eritroides/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Hemoglobinas/biossíntese , Humanos , Células K562/efeitos dos fármacos , Células K562/enzimologia , Células K562/metabolismo , Cinética , Inibidores da Síntese de Ácido Nucleico , Biossíntese de Proteínas , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Receptores de Complemento 3b/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/imunologia , Vidarabina/farmacologiaRESUMO
Complement receptor type 1 (CR1) is expressed principally on erythrocytes, monocytes, neutrophils and B cells, where it acts as a negative regulator of the complement cascade and as a clearance mechanism for immune complexes. As CR1 expression occurs in a number of lineages, its regulation may parallel other steps in haematopoiesis. Several leucocyte-cell lines (including K-562, THP-1, U-937 and HL-60) and B-cell lines, as well as peripheral blood cells (PBCs), were tested for the ability of various compounds to up-regulate their CR1 expression. While most of the compounds tested had minimal effects on CR1 message level, retinoic acid induced increases in mRNA levels in nearly all cell lines studied. Furthermore, in K-562 cells, the cytosine analogue Ara-C, an inducer of erythroid differentiation, caused the highest increases in CR1 mRNA levels as compared to untreated cells. Ara-C also induced significant increases in CR1 message in PBCs. These data suggest that induction of specific CR1 expression could be an integral part of blood cell differentiation and that the identified cell induction systems may be useful as models to study the regulation of CR1 gene expression.
Assuntos
Citarabina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Complemento 3b/biossíntese , Receptores de Complemento 3b/genética , Tretinoína/farmacologia , Relação Dose-Resposta Imunológica , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células HL-60 , Humanos , Interferon gama/farmacologia , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Células K562 , Cinética , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , RNA Mensageiro/efeitos dos fármacos , Receptores de Complemento 3b/sangue , Fator de Necrose Tumoral alfa/farmacologia , Células U937RESUMO
Two mechanisms could account for the impaired humoral immune response found in Cr2-/- mice. The absence of complement receptors 1 and 2 (CR1, CR2) on B cells could affect their activation. Alternatively, impaired Ag trapping by follicular dendritic cells (FDC) could affect B cell maturation into Ig-secreting or memory B cells. To compare the roles of CR1 and CR2 on B cells vs FDC in this abnormal response, bone marrow (BM) chimeric mice were generated and immunized with specific T-dependent Ags. The primary and secondary Ab response was measured. Cr2+/+ animals reconstituted with a Cr2-/- BM generated a diminished but detectable humoral immune response compared with controls. When injected with preformed immune complexes (IC), these mice maintained follicular IC localization. Cr2-/- animals reconstituted with a Cr2+/+ BM had an initial rise in the Ab titer, but were unable to maintain it as shown by a pronounced decrease in the IgG titer. This defect persisted during the secondary immune response. Follicular IC trapping was also impaired. Despite the abnormal Ab response, germinal center formation was retained in all of the chimeric animals. These experiments are the first to demonstrate an absolute requirement for CR1 and CR2 expression on FDC in the generation of a normal humoral immune response.
Assuntos
Linfócitos B/metabolismo , Células Dendríticas/metabolismo , Epitopos de Linfócito B/imunologia , Imunoglobulina G/biossíntese , Receptores de Complemento 3b/biossíntese , Receptores de Complemento 3d/biossíntese , Animais , Complexo Antígeno-Anticorpo/metabolismo , Linfócitos B/imunologia , Transplante de Medula Óssea , Células Dendríticas/imunologia , Centro Germinativo/fisiologia , Deficiência de IgG/genética , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Quimera por Radiação/imunologia , Receptores de Complemento 3b/genética , Receptores de Complemento 3d/genéticaRESUMO
Human complement receptors type 1 (hCR1;CD35) and type 2 (hCR2;CD21) are expressed on B lymphocytes at specific stages during differentiation and activation. These receptors play critical roles in the immune response to T-dependent Ags in addition to germinal center formation. Expression of both hCR2 and hCR1 is decreased on B lymphocytes of patients with systemic lupus erythematosus (SLE). We have studied the expression of mouse CR2 and CR1 on normal populations of mouse B lymphocytes in BALB/c mice. Our results demonstrate that expression of these receptors in the normal state closely parallels that of hCR2. During bone marrow development, expression is first detected on low B220/high IgM cells, demonstrating that complement receptors appear after central tolerance mechanisms are completed. In the splenic microenvironment the highest levels of receptor expression are found on marginal zone B lymphocytes. Mouse CR2 and CR1 are also found on peritoneal B1a and B1b cells in addition to IgA+ Peyer's patch B cells. Activation of splenic B cells under Th2 conditions results in a marked decrease in receptor expression. To determine whether the patterns of receptor expression also parallel those found in human disease, we studied the MRL lpr/lpr (MRL/lpr) model of SLE. Interestingly, we found an early decrease in complement receptor expression that is progressive and first detectable before major clinical manifestations of nephritis. We hypothesize that the early decrease in complement receptor expression such as that demonstrated by MRL/lpr mice plays an important role in the pathogenesis of murine and perhaps human SLE.
Assuntos
Doenças Autoimunes/etiologia , Subpopulações de Linfócitos B/metabolismo , Receptores de Complemento 3b/biossíntese , Receptores de Complemento 3d/biossíntese , Animais , Doenças Autoimunes/metabolismo , Subpopulações de Linfócitos B/imunologia , Western Blotting , Medula Óssea/imunologia , Medula Óssea/metabolismo , Células da Medula Óssea , Região de Troca de Imunoglobulinas , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Cavidade Peritoneal/citologia , Especificidade da Espécie , Baço/citologia , Baço/imunologia , Baço/metabolismo , Subpopulações de Linfócitos T/metabolismo , Timo/citologiaRESUMO
Complement receptor type one (CR1) in primates has several remarkable structural features including a size polymorphism (Mr 190000, 220000, 250000 and 280000) in man, multiple size variants (Mr 55000-220000) among non-human primates, and a partial amino-terminal duplication (CR1-like gene) that appears to encode the short (55000-70000) forms expressed on primate erythrocytes. In general, these short CR1 forms, some of which are GPI anchored, are expressed on erythrocytes and the 220000 molecular weight CR1 form is expressed on PBMC, except in man, where only the 220000 molecular weight form has been detected. In addition, the Mr 220000 human CR1 sequence carries several long internal repeats of up to 99% homology. It has been suggested that the highest homology is maintained by gene conversion and/or unequal crossover. To address further the evolutionary and biologic implications of these multiple forms, a 6 kb cDNA encoding baboon CR1(220) was identified by RTPCR using human CR1 primers. Its sequence contains the expected 30 complement control protein repeats (CCP) and demonstrates an overall homology to human CR1 of 95.4% at the nucleotide level and 93.2% at the amino acid level. As in human CR1, the first 28 CCP maintain the characteristic "seven CCP-long homologous repeats (LHR)" organization. Analysis of baboon CR1(220) indicates that horizontal or concerted evolution has maintained a high degree (> 98%) of identity between corresponding CCP within the LHRs from CCP 4 to CCP 19, while this homology region extends from CCP 3 to CCP 18 in man. In contrast, substitutions occurring in other CCP are not propagated to the corresponding sites of other LHR. Sequence differences in CCP 1, 2 and 3 are likely to be related to the acquisition of enhanced C3b binding capability by this amino-terminal region of the protein. Thus, the sequence data strongly support the hypotheses that gene conversion and or unequal crossover events have driven the evolution of the protein in regions of high homology while selective forces, probably ligand binding requirements, have maintained the regions of divergence.