RESUMO
The endothelium is critically involved in the pathogenesis of atherosclerosis by producing pro-inflammatory mediators, including IL-1ß. Coronary arteries from patients with ischemic heart disease express large amounts of IL-1ß in the endothelium. However, the mechanism by which endothelial cells (ECs) release IL-1ß remains to be elucidated. We investigated neutrophil elastase (NE), a potent serine protease detected in vulnerable areas of human carotid plaques, as a potential "trigger" for IL-1ß processing and release. This study tested the hypothesis that NE potentiates the processing and release of IL-1ß from human coronary endothelium. We found that NE cleaves the pro-isoform of IL-1ß in ECs and causes significant secretion of bioactive IL-1ß via extracellular vesicles. This release was attenuated significantly by inhibition of neutrophil elastase but not caspase-1. Transient increases in intracellular Ca(2+) levels were observed prior to secretion. Inside ECs, and after NE treatment only, IL-1ß was detected within LAMP-1-positive multivesicular bodies. The released vesicles contained bioactive IL-1ß. In vivo, in experimental atherosclerosis, NE was detected in mature atherosclerotic plaques, predominantly in the endothelium, alongside IL-1ß. This study reveals a novel mechanistic link between NE expression in atherosclerotic plaques and concomitant pro-inflammatory bioactive IL-1ß secretion from ECs. This could reveal additional potential anti-IL-1ß therapeutic targets and provide further insights into the inflammatory process by which vascular disease develops.
Assuntos
Vasos Coronários/metabolismo , Endotélio Vascular/metabolismo , Regulação Enzimológica da Expressão Gênica , Interleucina-1beta/metabolismo , Elastase de Leucócito/metabolismo , Animais , Apolipoproteínas E/genética , Apoptose , Cálcio/metabolismo , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/enzimologia , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microcirculação , Fosforilação , Placa Aterosclerótica/metabolismoRESUMO
Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+) T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+) T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+) T-cells in PBMC cultures required 'classical' CD14(+) monocytes, which enhanced T-cell activation. CD3(+) T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+) T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.
Assuntos
Apoptose , Proteína Ligante Fas/metabolismo , Monócitos/imunologia , Infecções Pneumocócicas/imunologia , Linfócitos T/fisiologia , Animais , Bacteriemia , Proteínas de Bactérias , Complexo CD3/biossíntese , Células Cultivadas , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Receptores de Lipopolissacarídeos/biossíntese , Pulmão/microbiologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/microbiologia , Necrose , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/patogenicidade , Estreptolisinas , Linfócitos T/imunologia , Linfócitos T/microbiologiaRESUMO
BACKGROUND: Therapeutic strategies to modulate the host response to bacterial pneumonia are needed to improve outcomes during community-acquired pneumonia. This study used mice with impaired Fas signalling to examine susceptibility to pneumococcal pneumonia and decoy receptor 3 analogue (DcR3-a) to correct factors associated with increased susceptibility. METHODS: Wild-type mice and those with varying degrees of impairment of Fas (lpr) or Fas ligand signalling (gld) were challenged with Streptococcus pneumoniae and microbiological and immunological outcomes measured in the presence or absence of DcR3-a. RESULTS: During established pneumonia, neutrophils became the predominant cell in the airway and gld mice were less able to clear bacteria from the lungs, demonstrating localised impairment of pulmonary neutrophil function in comparison to lpr or wild-type mice. T-cells from gld mice had enhanced activation and reduced apoptosis in comparison to wild-type and lpr mice during established pneumonia. Treatment with DcR3-a reduced T-cell activation and corrected the defect in pulmonary bacterial clearance in gld mice. CONCLUSIONS: The results suggest that imbalance in tumour necrosis factor superfamily signalling and excessive T-cell activation can impair bacterial clearance in the lung but that DcR3-a treatment can reduce T-cell activation, restore optimal pulmonary neutrophil function and enhance bacterial clearance during S pneumoniae infection.
Assuntos
Proteína Ligante Fas/metabolismo , Neutrófilos/imunologia , Fagócitos/imunologia , Pneumonia Pneumocócica/imunologia , Membro 6b de Receptores do Fator de Necrose Tumoral/farmacologia , Animais , Modelos Animais de Doenças , Proteína Ligante Fas/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neutrófilos/efeitos dos fármacos , Fagócitos/efeitos dos fármacos , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/terapia , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Streptococcus pneumoniae/imunologiaRESUMO
Peripheral blood monocytes represent the rapid response component of mononuclear phagocyte host defense, generating vigorous but finite antibacterial responses. We investigated the fate of highly purified primary human monocytes following phagocytosis of different bacteria. Exposure to high bacterial loads resulted in rapid loss of cell viability and decreased functional competence. Cell death typically involved classical apoptosis. Exposure to high numbers of Escherichia coli and Klebsiella pneumoniae induced nonapoptotic death with loss of cell membrane integrity, marked disruption of phagolysosomes, and caspase-1 activation, while a subset of cells also released caspase-1-regulated extracellular traps. Classical apoptosis increased if extracellular bacterial replication was reduced and decreased if intracellular ATP levels were reduced during these infections. Both classical apoptosis and the alternative forms of cell death allowed monocytes, whose functional competence was exhausted, to downregulate reactive oxygen species and proinflammatory cytokine responses. In contrast, sustained stimulation of glycolytic metabolism and mitochondrial oxidative phosphorylation, with associated hypoxia inducible factor-1alpha upregulation, maintained intracellular ATP levels and prolonged monocyte functional longevity, as assessed by maintenance of phagocytosis, reactive oxygen species production, and proinflammatory cytokine generation. Monocyte innate responses to bacteria are short-lived and are limited by an intrinsic program of apoptosis, a response that is subverted by overwhelming infection with E. coli and K. pneumoniae or bacterial stimulation of cell metabolism. In this regard, the fate of monocytes following bacterial challenge more closely resembles neutrophils than macrophages.
Assuntos
Infecções por Escherichia coli/imunologia , Imunidade Inata , Infecções por Klebsiella/imunologia , Infecções Meningocócicas/imunologia , Monócitos/imunologia , Monócitos/patologia , Infecções por Neisseriaceae/imunologia , Morte Celular/imunologia , Permeabilidade da Membrana Celular/genética , Permeabilidade da Membrana Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , DNA/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/patologia , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/patologia , Monócitos/metabolismo , Monócitos/microbiologia , Infecções por Neisseriaceae/microbiologia , Infecções por Neisseriaceae/patologiaRESUMO
Loss of RARß2 expression by promoter methylation is an early event in oral carcinogenesis. Understanding the mechanisms and consequences of RARß loss may aid in understanding the disappointing results of retinoid chemoprevention trials. This study aimed to describe the effects of all-trans retinoic acid (ATRA) and the de-methylating agent 5-Aza-2' deoxycytidine (5-AZA-CdR) on a panel of immortal potentially malignant oral lesion (PMOL) cell cultures. RARß expression was assessed in PMOL tissues by immunohistochemistry. Cells were treated with ATRA ± 5-AZA-CdR, and the effects on the cell cycle and senescence were assessed. In PMOL tissues, RARß expression was variable, but lower in biopsies which gave rise to immortal cell cultures. Treatment of iPMOL cells with ATRA resulted in little change in RARß expression, but the addition of 5-AZA-CdR resulted in significant increases. The effects on the cell cycle and senescence were variable and may be related to 5-AZA-CdR, as this has wider effects on the cell cycle. Overall, the response of iPMOL cells to ATRA and 5-AZA-CdR treatment was variable and is dependent on several factors, including RARß-promoter methylation. These findings may help to explain the lack of consistent effect of retinoids in PMOLs seen in chemoprevention trials.
RESUMO
Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD(3)) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD(3) and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells.