RESUMO
Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.
Assuntos
COVID-19/complicações , COVID-19/imunologia , Síndrome da Liberação de Citocina/complicações , Monócitos/patologia , Ativação de Neutrófilo , Idoso , Células Apresentadoras de Antígenos/imunologia , COVID-19/sangue , COVID-19/virologia , Estudos de Casos e Controles , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/patologia , Síndrome da Liberação de Citocina/virologia , Citocinas/sangue , Armadilhas Extracelulares/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/fisiologia , Índice de Gravidade de DoençaRESUMO
Periodontal diseases originate from a dysbiosis within the oral microbiota, which is associated with a deregulation of the host immune response. Although little is known about the initiation of dysbiosis, it has been shown that H2O2 production is one of the main mechanisms by which some commensal bacteria suppress the outgrowth of pathobionts. Current models emphasize the critical nature of complex microbial biofilms that form unique microbial ecologies and of their change during transition from health (homeostatic) to disease (dysbiotic). However, very little is known on how this alters their virulence and host responses. The objective of this study was to determine differences in virulence gene expression by pathobionts and the inflammatory host response in homeostatic and dysbiotic biofilms originating from the same ecology. Quantitative polymerase chain reaction was performed to quantify the pathobiont outgrowth. Expression analysis of bacterial virulence and cellular inflammatory genes together with cytokine enzyme-linked immunosorbent assays were used to detect differences in bacterial virulence and to analyze potential differences in inflammatory response. An increase in pathobionts in induced dysbiotic biofilms was observed compared to homeostatic biofilms. The main virulence genes of all pathobionts were upregulated in dysbiotic biofilms. Exposure of these dysbiotic biofilms to epithelial and fibroblast cultures increased the expression of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, and matrix metalloprotease 8, but especially the chemokine CXCL8 (IL-8). Conversely, homeostatic and beneficial biofilms had a minor immune response at the messenger RNA and protein level. Overall, induced dysbiotic biofilms enriched in pathobionts and virulence factors significantly increased the inflammatory response compared to homeostatic and commensal biofilms.
Assuntos
Biofilmes , Disbiose/fisiopatologia , Periodontite/microbiologia , Disbiose/imunologia , Disbiose/microbiologia , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Metaloproteinase 8 da Matriz/metabolismo , Periodontite/fisiopatologia , Reação em Cadeia da Polimerase , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Lymphocytic airway inflammation is a major risk factor for chronic lung allograft dysfunction, for which there is no established treatment. We investigated whether azithromycin could control lymphocytic airway inflammation and improve allograft function. Fifteen lung transplant recipients demonstrating acute allograft dysfunction due to isolated lymphocytic airway inflammation were prospectively treated with azithromycin for at least 6 months (NCT01109160). Spirometry (FVC, FEV1 , FEF25-75 , Tiffeneau index) and FeNO were assessed before and up to 12 months after initiation of azithromycin. Radiologic features, local inflammation assessed on airway biopsy (rejection score, IL-17(+) cells/mm(2) lamina propria) and broncho-alveolar lavage fluid (total and differential cell counts, chemokine and cytokine levels); as well as systemic C-reactive protein levels were compared between baseline and after 3 months of treatment. Airflow improved and FeNO decreased to baseline levels after 1 month of azithromycin and were sustained thereafter. After 3 months of treatment, radiologic abnormalities, submucosal cellular inflammation, lavage protein levels of IL-1ß, IL-8/CXCL-8, IP-10/CXCL-10, RANTES/CCL5, MIP1-α/CCL3, MIP-1ß/CCL4, Eotaxin, PDGF-BB, total cell count, neutrophils and eosinophils, as well as plasma C-reactive protein levels all significantly decreased compared to baseline (p < 0.05). Administration of azithromycin was associated with suppression of posttransplant lymphocytic airway inflammation and clinical improvement in lung allograft function.
Assuntos
Azitromicina/uso terapêutico , Bronquite/tratamento farmacológico , Rejeição de Enxerto/tratamento farmacológico , Transplante de Pulmão/efeitos adversos , Linfócitos/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Complicações Pós-Operatórias , Adolescente , Adulto , Antibacterianos/uso terapêutico , Bronquite/etiologia , Lavagem Broncoalveolar , Proteína C-Reativa , Citocinas/metabolismo , Feminino , Seguimentos , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Humanos , Pneumopatias/complicações , Pneumopatias/cirurgia , Linfócitos/patologia , Masculino , Pessoa de Meia-Idade , Pneumonia/etiologia , Prognóstico , Estudos Prospectivos , Testes de Função Respiratória , Estudos Retrospectivos , Espirometria , Transplante Homólogo , Adulto JovemRESUMO
Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced ß-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to ß-cell protection against cytokine-induced ß-cell dysfunction and death. Human and mouse islets were exposed to IL-1ß and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and ß-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in ß-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic ß-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced ß-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.
Assuntos
Calcitriol/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica , Inflamação , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Idoso , Animais , Morte Celular , Linhagem Celular , Células Cultivadas , Quimiotaxia , Ensaio de Imunoadsorção Enzimática , Glucose/metabolismo , Humanos , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Ratos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Variovorax sp. strain WDL1, which mineralizes the phenylurea herbicide linuron, expresses a novel linuron-hydrolyzing enzyme, HylA, that converts linuron to 3,4-dichloroaniline (DCA). The enzyme is distinct from the linuron hydrolase LibA enzyme recently identified in other linuron-mineralizing Variovorax strains and from phenylurea-hydrolyzing enzymes (PuhA, PuhB) found in Gram-positive bacteria. The dimeric enzyme belongs to a separate family of hydrolases and differs in Km, temperature optimum, and phenylurea herbicide substrate range. Within the metal-dependent amidohydrolase superfamily, HylA and PuhA/PuhB belong to two distinct protein families, while LibA is a member of the unrelated amidase signature family. The hylA gene was identified in a draft genome sequence of strain WDL1. The involvement of hylA in linuron degradation by strain WDL1 is inferred from its absence in spontaneous WDL1 mutants defective in linuron hydrolysis and its presence in linuron-degrading Variovorax strains that lack libA. In strain WDL1, the hylA gene is combined with catabolic gene modules encoding the downstream pathways for DCA degradation, which are very similar to those present in Variovorax sp. SRS16, which contains libA. Our results show that the expansion of a DCA catabolic pathway toward linuron degradation in Variovorax can involve different but isofunctional linuron hydrolysis genes encoding proteins that belong to evolutionary unrelated hydrolase families. This may be explained by divergent evolution and the independent acquisition of the corresponding genetic modules.
Assuntos
Comamonadaceae/enzimologia , Comamonadaceae/metabolismo , Herbicidas/metabolismo , Hidrolases/metabolismo , Linurona/metabolismo , Compostos de Anilina/metabolismo , Biotransformação , Comamonadaceae/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrolases/química , Hidrolases/genética , Hidrolases/isolamento & purificação , Dados de Sequência Molecular , Multimerização Proteica , Análise de Sequência de DNA , TemperaturaRESUMO
Chemokines, adhesion molecules, cytokines and proteases regulate the extravasation of leucocytes during acute and chronic inflammation and leucocyte homing. Chemokines are produced after transcriptional activation by inflammatory mediators such as cytokines or microbial Toll-like receptor ligands and their effect depends on the expression of chemokine receptors on specific cell types. More and more evidence points towards a role for post-translational modifications in the fine-tuning of chemokine activity. Although both glycosylation and proteolytic processing of the C- and/or N-terminus of chemokines has been reported, mainly proteolytic processing of the N-terminus appears to affect the receptor specificity, chemotactic property and signalling potency of these low-molecular-mass proteins. N-terminal processing of chemokines by aminopeptidases or endoproteases may alter the receptor specificity and may result in up- or down-regulation of their chemotactic, antiviral or angiogenic activity.
Assuntos
Quimiocinas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Humanos , Peptídeo Hidrolases/metabolismo , Receptores de Quimiocinas/metabolismoRESUMO
Gelatinase B/matrix metalloproteinase-9 (MMP-9) is an inflammatory mediator and effector. Considerable amounts of gelatinase B are released by neutrophils in the synovial cavity of patients with rheumatoid arthritis, and gelatinase B-deficient mice are resistant against antibody-induced arthritis. Native human collagen type II is susceptible to cleavage by various collagenases (MMP-1, MMP-8, and MMP-13), which cleave at a single position in the triple helix. Although the triple-helical structure may persist after this single cleavage, we show that gelatinase B degrades the resulting fragments into small remnant peptides. These were identified by mass spectrometry and Edman degradation. Localization of 31 cleavage sites shows that the immunodominant epitopes remain intact after cleavage and may become available, processed as antigens and presented in MHC-II molecules. Furthermore, most post-translational modifications were identified on the fragments, including nine glycosylation sites. In particular, it is shown for the first time by structural analysis that in natural human collagen II, lysines in the main immunodominant epitope are modified by partial hydroxylation and partial glycosylation. Determination of T-cell reactivity against such fragments indicates that, besides the two known main immunodominant epitopes, other glyco-epitopes may be present in collagen II. This reinforces the role of glycopeptide antigens in autoimmunity.
Assuntos
Colágeno Tipo II/metabolismo , Epitopos Imunodominantes/análise , Metaloproteinase 9 da Matriz/metabolismo , Fragmentos de Peptídeos/imunologia , Apresentação de Antígeno , Colágeno Tipo II/imunologia , Glicopeptídeos/análise , Glicopeptídeos/imunologia , Humanos , Fragmentos de Peptídeos/análise , Processamento de Proteína Pós-Traducional , Linfócitos T/imunologiaRESUMO
AIMS/HYPOTHESIS: Cytokines and chemokines are important mediators of immune responses due to their ability to recruit and activate leukocytes. Using microarray analysis we observed that rat beta cells exposed to IL-1beta and IFN-gamma have increased mRNA levels of chemokines and IL-15. The aim of this study was to characterize the expression of IP-10, MIP-3alpha, fractalkine and IL-15 in rat beta cells, human pancreatic islets, and in islets isolated from NOD mice, both during the pre-diabetic period and following islet transplantation. METHODS: FACS-purified rat beta cells and human islets were cultured with IL-1beta, IFN-gamma and/or TNF-alpha. Islets were isolated from NOD or BALB/c mice at different ages. For syngeneic islet transplantation, 2- or 3-week-old NOD islets were grafted under the kidney capsule of spontaneously diabetic NOD recipients. Chemokine and IL-15 mRNA expression and protein release were evaluated, respectively, by RT-PCR and ELISA. RESULTS: Human islets and rat beta cells express IP-10, MIP-3alpha, fractalkine and IL-15 mRNAs upon exposure to cytokines. The expression of IL-15, IP-10 and fractalkine is regulated by IFN-gamma, while the expression of MIP-3alpha is IL-1beta-dependent. Moreover, cytokines induced IL-15, IP-10, Mig, I-TAC and MIP-3alpha protein accumulation in culture medium from human islets. In vivo, there was an age-related increase in IL-15, IP-10 and MIP-3alpha expression in islets isolated from NOD mice. Following syngeneic islet transplantation, increased expression of IL-1beta, IFN-gamma, fractalkine, IP-10, MCP-1 and MIP-3alpha mRNAs were observed in the grafts. CONCLUSION/INTERPRETATION: Cytokine-exposed islets or beta cells express chemokines and IL-15. This could contribute to the recruitment and activation of mononuclear cells and development of insulitis in early Type 1 diabetes and during graft destruction.
Assuntos
Quimiocinas/metabolismo , Interferon gama/farmacologia , Interleucina-15/metabolismo , Interleucina-1/farmacologia , Ilhotas Pancreáticas/metabolismo , Camundongos Endogâmicos NOD/metabolismo , Adulto , Animais , Quimiocina CCL20 , Quimiocina CX3CL1 , Quimiocina CXCL10 , Quimiocinas CC/genética , Quimiocinas CX3C/genética , Quimiocinas CXC/genética , Humanos , Interleucina-15/genética , Proteínas Inflamatórias de Macrófagos/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
The N-terminal fragment of mouse pro-opiomelanocortin (N-POMC) was isolated from AtT-20 cell-conditioned medium on the basis of immunoreactivity to an anti-POMC1-50 monoclonal antibody by a concentration step, a cation exchange step, reversed phase high-performance liquid chromatography (HPLC) and size exclusion HPLC. Two groups of N-POMC isoforms with a molecular weight (MW) of approximately 11 kDa and 13 kDa, respectively, were identified by mass spectrometry and N-terminal amino acid sequencing. C-terminal sequencing indicated that 11 kDa isoforms correspond to POMC1-74 and 13 kDa isoforms to POMC1-95. Isoforms from both groups enhanced the prolactin mRNA content (measured by means of TaqMan real-time reverse transcription-polymerase chain reaction) in cultured rat pituitary cell aggregates in a dose-dependent manner, but not all of them showed this activity. POMC1-74 compounds were significantly more potent than POMC1-95 isoforms. The observed effects were abolished by coincubation with the monoclonal anti-POMC1-50 antibody, showing the specificity of this biological action. Incorporation of bromodeoxyuridine into DNA of immunostained lactotrophs was enhanced by only a minor part of the isoforms. Some of these had no effect on prolactin mRNA expression. The N-POMC isoforms appeared to be N- and at least in part O-glycosylated. After enzymatic N-deglycosylation of selected N-POMC isoforms, the stimulatory effect on the prolactin mRNA level was depressed (in case of the POMC1-95 isoforms) or totally abolished (in case of the POMC1-74 isoforms). The present findings show that N-POMC is a mixture of differentially glycosylated isoforms, that the isoforms of POMC1-74 are the biologically more effective forms and that different isoforms induce different biological responses in the same cell population. The data also show the essential role of N-glycosylation in the biological response.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Pró-Opiomelanocortina/química , Pró-Opiomelanocortina/metabolismo , Animais , Anticorpos/farmacologia , Bromodesoxiuridina/metabolismo , Células Cultivadas , Feminino , Glicosilação , Camundongos , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologia , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Pró-Opiomelanocortina/isolamento & purificação , Pró-Opiomelanocortina/farmacologia , Prolactina/genética , Prolactina/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Human FPRL1 and its mouse homologue FPR2 are functional receptors for several exogenous and host-derived chemotactic peptides, including amyloid beta(42) (A beta(42)), a critical pathogenic factor in Alzheimer's disease. We investigated the effect of TNF alpha on the expression and function of FPR2 in mouse microglial cells, a crucial inflammatory cell type in the CNS. Primary murine microglia and a cell line N9 in resting state expressed low levels of FPR2 gene and lacked the response to chemotactic agonists for this receptor. Incubation with TNF alpha, however, increased microglial expression of FPR2 gene, in association with potent chemotactic responses to FPR2-specific agonists including A beta(42). The effect of TNF alpha was dependent on the p55 TNF alpha receptor and activation of MAP kinase p38. TNF alpha concomitantly down-regulated microglial response to the chemokine SDF-1 alpha. Thus, by selectively up-regulating FPR2 in microglia, TNF alpha has the capacity to amplify host response in inflammatory diseases in the CNS.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Microglia/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Receptores Imunológicos/biossíntese , Receptores de Lipoxinas , Receptores de Peptídeos/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/fisiologia , Animais , Animais Recém-Nascidos , Linhagem Celular , Relação Dose-Resposta Imunológica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microglia/metabolismo , Receptores de Formil Peptídeo , Fator de Necrose Tumoral alfa/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
In order to infect a target cell, the HIV envelope glycoprotein gp 120 has to interact with both the cellular receptor CD4 and an HIV-coreceptor, i.e. the CC or CXC chemokine receptor CCR5 or CXCR4. Both coreceptors were immediately recognized as novel targets for anti-HIV-therapy. Blocking these coreceptors would protect the cell from viral entry and would reduce the viral transmission and pathogenesis. Here we describe the purification and characterization of natural chemokine variants and compare their antiviral activity. In addition, the role of proteases for the processing of the CC chemokines RANTES, eotaxin, MDC and MIP-1 alpha and of the CXC chemokine SDF-1 are studied. The MIP-1 alpha-isoform LD78 beta, that was purified form natural sources, inhibited HIV-infection completely in CCR5-transfected cells, mononuclear leukocytes and purified monocytes at low (ng/ml) concentrations. This research will make it feasible to develop specific chemokine-analogs that block HIV-entry. Deciphering the processes that play a role during the complicated interactions between HIV-gp120 and the cellular membrane may lead to a more efficient treatment of HIV-infections.
Assuntos
Quimiocinas/fisiologia , Infecções por HIV/imunologia , HIV/imunologia , Receptores de HIV/fisiologia , Replicação Viral , HIV/fisiologia , Infecções por HIV/virologia , Humanos , Receptores CCR5/metabolismo , Receptores CCR5/fisiologia , Receptores CXCR4/metabolismo , Receptores CXCR4/fisiologiaRESUMO
The interferon (IFN)-inducible chemokines, specifically, IFN-gamma-inducible protein-10 (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), share a unique CXC chemokine receptor (CXCR3). Recently, the highly specific membrane-bound protease and lymphocyte surface marker CD26/dipeptidyl peptidase IV (DPP IV) was found to be responsible for posttranslational processing of chemokines. Removal of NH(2)-terminal dipeptides by CD26/DPP IV alters chemokine receptor binding and signaling, and hence inflammatory and anti-human immunodeficiency virus (HIV) activities. CD26/DPP IV and CXCR3 are both markers for Th1 lymphocytes and, moreover, CD26/DPP IV is present in a soluble, active form in human plasma. This study reports that at physiologic enzyme concentrations CD26/DPP IV cleaved 50% of I-TAC within 2 minutes, whereas for IP-10 and Mig the kinetics were 3- and 10-fold slower, respectively. Processing of IP-10 and I-TAC by CD26/DPP IV resulted in reduced CXCR3-binding properties, loss of calcium-signaling capacity through CXCR3, and more than 10-fold reduced chemotactic potency. Moreover, IP-10 and I-TAC cleaved by CD26/DPP IV acted as chemotaxis antagonists and CD26/DPP IV-truncated IP-10 and Mig retained their ability to inhibit the angiogenic activity of interleukin-8 in the rabbit cornea micropocket model. These data demonstrate a negative feedback regulation by CD26/DPP IV in CXCR3-mediated chemotaxis without affecting the angiostatic potential of the CXCR3 ligands IP-10 and Mig.
Assuntos
Inibidores da Angiogênese , Quimiocinas CXC/química , Quimiotaxia de Leucócito/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Linfócitos/fisiologia , Receptores de Quimiocinas/agonistas , Transdução de Sinais/efeitos dos fármacos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Cálcio/metabolismo , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacologia , Dipeptidil Peptidase 4/metabolismo , Receptores CXCR3 , Receptores de Quimiocinas/fisiologia , Relação Estrutura-AtividadeRESUMO
Dipeptidyl-peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady-state kinetics of DPPIV/CD26-mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating peptide (PACAP27 and PACAP38), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C-terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Neuropeptídeos/metabolismo , Peptídeo Liberador de Gastrina/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Cinética , Espectrometria de Massas , Neuropeptídeo Y/metabolismo , Pâncreas/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Especificidade por Substrato , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Chemokines are important mediators of cell migration during inflammation and normal leukocyte trafficking. Inflammatory chemokines are induced in multiple cell types at sites of infection. Here, we describe a novel bovine CC chemokine, designated regakine-1, that is constitutively present at high concentrations in plasma. Cloning of its gene revealed an expected two intron/three exon organization, with a rather long first intron. In addition to a 21-residue signal peptide, the coding sequence corresponded to a 71-residue secreted protein. However, the natural regakine-1 protein missed the COOH-terminal lysine residue. Regakine-1 has only weak sequence similarity (<50% identical residues) with other animal or human chemokines. Northern blot analysis demonstrated regakine-1 RNA expression in spleen and lung. At physiological concentrations (30-100 ng/mL), natural 7.5 kDa regakine-1 stimulated gelatinase B release from neutrophils and chemoattracted immature myeloid HL-60 cells, as well as mature granulocytes. Regakine-1 was more potent on human myeloid cells than the human plasma CC chemokine hemofiltrate CC chemokine-1 (HCC-1). Moreover, regakine-1 synergized with the bacterial peptide N-formylmethionylleucylphenylalanine (fMLP), yielding a 10-fold increase in neutrophil chemotactic response above their additive effect. Regakine-1 did not compete with interleukin-8 (IL-8) for binding to neutrophils, nor did it affect fMLP-induced calcium signaling, suggesting that regakine-1 recognizes a different receptor. In view of its high constitutive plasma concentration, regakine-1 is believed to recruit myeloid cells into the circulation, whereas its synergy with other neutrophil chemoattractants suggests that it also enhances the inflammatory response to infection.
Assuntos
Quimiocinas CC/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Bovinos , Linhagem Celular , Quimiocinas CC/sangue , Quimiocinas CC/química , Quimiotaxia de Leucócito , Clonagem Molecular , DNA Complementar , Humanos , Dados de Sequência Molecular , N-Formilmetionina Leucil-Fenilalanina/metabolismoRESUMO
Recently, the LD78beta isoform of the CC chemokine macrophage inflammatory protein (MIP)-1alpha was shown to efficiently chemoattract lymphocytes and monocytes and to inhibit infection of mononuclear cells by R5 HIV-1 strains. We have now demonstrated that after cleavage of the NH2-terminal Ala-Pro dipeptide by CD26, LD78beta(3 - 70) became the most potent chemokine blocking HIV-1. LD78beta(3 - 70) competed tenfold more efficiently than LD78beta(1 - 70) with [125I] RANTES for binding to the CC chemokine receptors CCR5 and CCR1. Contrary to LD78alpha, LD78beta(1 - 70) at 30 ng/ml efficiently competed with [125I] RANTES for binding to CCR3 and mobilized calcium in CCR3 transfectants, whereas LD78beta(3 - 70) showed a 30-fold decrease in CCR3 affinity compared to LD78beta(1 - 70). This demonstrates the importance of the penultimate proline in LD78beta(1 - 70) for CCR3 recognition. Both LD78beta isoforms efficiently chemoattracted eosinophils from responsive donors. In contrast, only the CCR3 agonist LD78beta(1 - 70) and not LD78beta(3 - 70), induced calcium increases in eosinophils with low levels of CCR1. In responder neutrophils, LD78beta(3 - 70) elicited calcium fluxes at a 30-fold lower dose (10 ng/ml) compared to intact LD78beta and LD78alpha, whereas the three MIP-1alpha isoforms were equipotent neutrophil chemoattractants. Taken together, both LD78beta isoforms are potent HIV-1 inhibitors (CCR5) and activators for neutrophils (CCR1) and eosinophils (CCR1, CCR3), affecting infection and inflammation.
Assuntos
Quimiotaxia de Leucócito , HIV-1/efeitos dos fármacos , Proteínas Inflamatórias de Macrófagos/metabolismo , Proteínas Inflamatórias de Macrófagos/farmacologia , Receptores de Quimiocinas/metabolismo , Animais , Células CHO , Sinalização do Cálcio , Linhagem Celular , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , Cricetinae , Eosinófilos/imunologia , Humanos , Células K562 , Neutrófilos/imunologia , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Processamento de Proteína Pós-Traducional , Receptores CCR1 , Receptores CCR3 , Receptores CCR5/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Matrix metalloproteinases (MMPs) form a family of enzymes with major actions in the remodeling of extracellular matrix (ECM) components. Gelatinase B (MMP-9) is the most complex family member in terms of domain structure and regulation of its activity. Gelatinase B activity is under strict control at various levels: transcription of the gene by cytokines and cellular interactions; activation of the pro-enzyme by a cascade of enzymes comprising serine proteases and other MMPs; and regulation by specific tissue inhibitors of MMPs (TIMPs) or by unspecific inhibitors, such as alpha2-macroglobulin. Thus, remodeling ECM is the result of the local protease load, i.e., the net balance between enzymes and inhibitors. Glycosylation has a limited effect on the net activity of gelatinase B, and in contrast to the all-or-none effect of enzyme activation or inhibition, it results in a higher-level, fine-tuning effect on the ECM catalysis by proteases in mammalian species. Fast degranulation of considerable amounts of intracellularly stored gelatinase B from neutrophils, induced by various types of chemotactic factors, is another level of control of activity. Neutrophils are first-line defense leukocytes and do not produce gelatinase A or TIMP. Thus, neutrophils contrast sharply with mononuclear leukocytes, which produce gelatinase A constitutively, synthesize gelatinase B de novo after adequate triggering, and overproduce TIMP-1. Gelatinase B is also endowed with functions other than cleaving the ECM. It has been shown to generate autoimmune neo-epitopes and to activate pro-IL-1beta into active IL-1beta. Gelatinase B ablation in the mouse leads to altered bone remodeling and subfertility, results in resistance to several induced inflammatory or autoimmune pathologies, and indicates that the enzyme plays a crucial role in development and angiogenesis. The major human neutrophil chemoattractant, IL-8, stimulates fast degranulation of gelatinase B from neutrophils. Gelatinase B is also found to function as a regulator of neutrophil biology and to truncate IL-8 at the amino terminus into a tenfold more potent chemokine, resulting in an important positive feedback loop for neutrophil activation and chemotaxis. The CXC chemokines GRO-alpha, CTAP-III, and PF-4 are degraded by gelatinase B, whereas the CC chemokines MCP-2 and RANTES are not cleaved.
Assuntos
Leucócitos/enzimologia , Metaloproteinase 9 da Matriz/fisiologia , Doenças Autoimunes/enzimologia , Quimiocinas/fisiologia , Cromossomos Humanos Par 20/genética , Citocinas/fisiologia , Ativação Enzimática , Indução Enzimática , Matriz Extracelular/enzimologia , Humanos , Leucócitos/citologia , Leucócitos Mononucleares/enzimologia , Substâncias Macromoleculares , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/deficiência , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Neoplasias/enzimologia , Neutrófilos/citologia , Neutrófilos/enzimologia , Especificidade de Órgãos , Fenótipo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Chemokines coordinate many aspects of leukocyte migration. As chemoattractants they play an important role in the innate and acquired immune response. There is good experimental evidence that N-terminal truncation by secreted or cell surface proteases is a way of modulating chemokine action. The localization of CD26/dipeptidyl peptidase IV on cell surfaces and in biological fluids, its primary specificity, and the type of naturally occurring truncated chemokines are consistent with such a function. We determined the steady-state catalytic parameters for a relevant selection of chemokines (CCL3b, CCL5, CCL11, CCL22, CXCL9, CXCL10, CXCL11, and CXCL12) previously reported to alter their chemotactic behavior due to CD26/dipeptidyl peptidase IV-catalyzed truncation. The results reveal a striking selectivity for stromal cell-derived factor-1alpha (CXCL12) and macrophage-derived chemokine (CCL22). The kinetic parameters support the hypothesis that CD26/dipeptidyl peptidase IV contributes to the degradation of certain chemokines in vivo. The data not only provide insight into the selectivity of the enzyme for specific chemokines, but they also contribute to the general understanding of CD26/dipeptidyl peptidase IV secondary substrate specificity.
Assuntos
Quimiocinas/metabolismo , Dipeptidil Peptidase 4/biossíntese , Sequência de Aminoácidos , Catálise , Quimiocina CCL8 , Quimiocina CXCL11 , Quimiocina CXCL12 , Quimiocinas/química , Quimiocinas CXC/biossíntese , Quimiocinas CXC/metabolismo , Dipeptidil Peptidase 4/química , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Quimioatraentes de Monócitos/metabolismo , Ligação Proteica , Receptores CCR4 , Receptores CXCR3 , Receptores CXCR4/metabolismo , Receptores de Quimiocinas/metabolismo , Especificidade por Substrato , Fatores de TempoRESUMO
The CC-chemokines RANTES, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta are natural ligands for the CC-chemokine receptor CCR5. MIP-1alpha, also known as LD78alpha, has an isoform, LD78beta, which was identified as the product of a nonallelic gene. The two isoforms differ in only 3 amino acids. LD78beta was recently reported to be a much more potent CCR5 agonist than LD78alpha and RANTES in inducing intracellular Ca2+ signaling and chemotaxis. CCR5 is expressed by human monocytes/macrophages (M/M) and represents an important coreceptor for macrophage-tropic, CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains to infect the cells. We compared the antiviral activities of LD78beta and the other CC-chemokines in M/M. LD78beta at 100 ng/ml almost completely blocked HIV-1 replication, while at the same concentration LD78alpha had only weak antiviral activity. Moreover, when HIV-1 infection in M/M was monitored by a flow cytometric analysis using p24 antigen intracellular staining, LD78beta proved to be the most antivirally active of the chemokines. RANTES, once described as the most potent chemokine in inhibiting R5 HIV-1 infection, was found to be considerably less active than LD78beta. LD78beta strongly downregulated CCR5 expression in M/M, thereby explaining its potent antiviral activity.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Proteínas Inflamatórias de Macrófagos/farmacologia , Macrófagos/virologia , Receptores CCR5/metabolismo , Replicação Viral/efeitos dos fármacos , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/imunologia , HIV-1/metabolismo , HIV-1/fisiologia , Humanos , Isoformas de Proteínas/farmacologiaRESUMO
Chemokines constitute a large family of chemotactic cytokines that selectively attract different blood cell types. Although most inflammatory chemoattractants are only induced and released in the circulation during acute infection, a restricted number of CXC and CC chemokines are constitutively present in normal plasma at high concentrations. Here, such a chemotactic protein was purified to homogeneity from serum and fully identified as a novel CC chemokine by mass spectrometry and amino acid sequence analysis. The protein, tentatively designated Regakine-1, shows less than 50% sequence identity with any known chemokine. This novel CC chemokine chemoattracts both neutrophils and lymphocytes but not monocytes or eosinophils. Its modest chemotactic potency but high blood concentration is similar to that of other chemokines present in the circulation, such as hemofiltrate CC chemokine-1, platelet factor-4, and beta-thromboglobulin. Regakine-1 did not induce neutrophil chemokinesis. However, it synergized with the CXC chemokines interleukin-8 and granulocyte chemotactic protein-2, and the CC chemokine monocyte chemotactic protein-3, resulting in an at least a 2-fold increase of the neutrophil and lymphocyte chemotactic response, respectively. The biologic effects of homogeneous natural Regakine-1 were confirmed with chemically synthesized chemokine. Like other plasma chemokines, it is expected that Regakine-1 plays a unique role in the circulation during normal or pathologic conditions.
Assuntos
Quimiocinas CC/isolamento & purificação , Quimiotaxia/efeitos dos fármacos , Citocinas , Linfócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Bovinos , Quimiocina CCL7 , Quimiocina CXCL6 , Quimiocinas/química , Quimiocinas CC/sangue , Quimiocinas CC/farmacologia , Quimiocinas CXC/farmacologia , Cromatografia Líquida de Alta Pressão , Sinergismo Farmacológico , Eletroforese em Gel de Poliacrilamida , Eosinófilos/efeitos dos fármacos , Sangue Fetal/química , Humanos , Interleucina-8/farmacologia , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Quimioatraentes de Monócitos/farmacologia , Concentração Osmolar , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
AIMS/HYPOTHESIS: Monocyte chemoattractant protein-1 (MCP-1) attracts monocytes and T lymphocytes, and could thus contribute to mononuclear cell infiltration in Type I (insulin-dependent) diabetes mellitus. Cytokines induce MCP-1 mRNA expression in pancreatic rat beta cells. To investigate this issue, we analysed the signal transduction for IL-1 beta-induced MCP-1 expression in rat beta cells and in vitro MCP-1 mRNA expression and protein release by human islets as well as in vivo islet MCP-1 mRNA expression in prediabetic non-obese diabetic mice. METHODS: Fluorescence-activated cell sorting-purified rat beta cells were cultured for 6 h with IL-1 beta (30 U/ml) or MAPK inhibitors or both. Human islets were cultured for 6-72 h with the cytokines IL-1 beta, IFN-gamma or the inducible nitric oxide synthase (iNOS) inhibitor NG-methyl-L-arginine or both. We measured MCP-1 mRNA by RT-PCR and protein by ELISA. The MCP-1 mRNA expression in islets from male and female non-obese diabetic mice (2-12 weeks of age) was measured by real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Interleukin-1 beta induced MCP-1 mRNA expression in rat beta cells, with a maximum induction after 6 h. A combination of p38 and ERK1/2 inhibitors decreased MCP-1 expression by 70%. IL-1 beta induced both MCP-1 mRNA expression and a threefold increase in medium MCP-1 protein accumulation in human islet cells. This effect was not prevented by iNOS blockers. In vivo there was an age-related increase in MCP-1 mRNA expression in islets from male and female non-obese diabetic mice, reaching a peak at 8 weeks. CONCLUSIONS/INTERPRETATION: In rat and human islet cells MCP-1 mRNA is induced by IL-1 beta. Both ERK1/2 and p38 MAPK, but not nitric oxide, contribute to MCP-1 expression. In non-obese diabetic mice MCP-1 mRNA expression increases with age, peaking at the early phases of insulitis. The production of MCP-1 by pancreatic beta cells could contribute to the recruitment of mononuclear cells into pancreatic islets in early Type I diabetes.