RESUMO
Pleural organization may occur after empyema or complicated parapneumonic effusion and can result in restrictive lung disease with pleural fibrosis (PF). Pleural mesothelial cells (PMCs) may contribute to PF through acquisition of a profibrotic phenotype, mesothelial-mesenchymal transition (MesoMT), which is characterized by increased expression of α-SMA (α-smooth muscle actin) and other myofibroblast markers. Although MesoMT has been implicated in the pathogenesis of PF, the role of the reactive oxygen species and the NOX (nicotinamide adenine dinucleotide phosphate oxidase) family in pleural remodeling remains unclear. Here, we show that NOX1 expression is enhanced in nonspecific human pleuritis and is induced in PMCs by THB (thrombin). 4-Hydroxy-2-nonenal, an indicator of reactive oxygen species damage, was likewise increased in our mouse model of pleural injury. NOX1 downregulation blocked THB- and Xa (factor Xa)-mediated MesoMT, as did pharmacologic inhibition of NOX1 with ML-171. NOX1 inhibition also reduced phosphorylation of Akt, p65, and tyrosine 216-GSK-3ß, signaling molecules previously shown to be implicated in MesoMT. Conversely, ML-171 did not reverse established MesoMT. NOX4 downregulation attenuated TGF-ß- and THB-mediated MesoMT. However, NOX1 downregulation did not affect NOX4 expression. NOX1- and NOX4-deficient mice were also protected in our mouse model of Streptococcus pneumoniae-mediated PF. These data show that NOX1 and NOX4 are critical determinants of MesoMT.
Assuntos
Transição Epitelial-Mesenquimal , NADPH Oxidase 1/metabolismo , Pleura/enzimologia , Pleurisia/enzimologia , Pneumonia Pneumocócica/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniae/patogenicidade , Animais , Células Cultivadas , Modelos Animais de Doenças , Fator Xa/metabolismo , Fibrose , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 1/deficiência , NADPH Oxidase 1/genética , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Pleura/microbiologia , Pleura/patologia , Pleurisia/microbiologia , Pleurisia/patologia , Pleurisia/fisiopatologia , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/patologia , Transdução de Sinais , Trombina/metabolismoRESUMO
BACKGROUND AND PURPOSE: Leukotrienes (LTs) are inflammatory mediators produced via the 5-lipoxygenase (5-LOX) pathway and are linked to diverse disorders, including asthma, allergic rhinitis and cardiovascular diseases. We recently identified the benzimidazole derivative BRP-7 as chemotype for anti-LT agents by virtual screening targeting 5-LOX-activating protein (FLAP). Here, we aimed to reveal the in vitro and in vivo pharmacology of BRP-7 as an inhibitor of LT biosynthesis. EXPERIMENTAL APPROACH: We analysed LT formation and performed mechanistic studies in human neutrophils and monocytes, in human whole blood (HWB) and in cell-free assays. The effectiveness of BRP-7 in vivo was evaluated in rat carrageenan-induced pleurisy and mouse zymosan-induced peritonitis. KEY RESULTS: BRP-7 potently suppressed LT formation in neutrophils and monocytes and this was accompanied by impaired 5-LOX co-localization with FLAP. Neither the cellular viability nor the activity of 5-LOX in cell-free assays was affected by BRP-7, indicating that a functional FLAP is needed for BRP-7 to inhibit LTs, and FLAP bound to BRP-7 linked to a solid matrix. Compared with the FLAP inhibitor MK-886, BRP-7 did not significantly inhibit COX-1 or microsomal prostaglandin E2 synthase-1, implying the selectivity of BRP-7 for FLAP. Finally, BRP-7 was effective in HWB and impaired inflammation in vivo, in rat pleurisy and mouse peritonitis, along with reducing LT levels. CONCLUSIONS AND IMPLICATIONS: BRP-7 potently suppresses LT biosynthesis by interacting with FLAP and exhibits anti-inflammatory effectiveness in vivo, with promising potential for further development.
Assuntos
Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacologia , Anti-Inflamatórios/farmacologia , Benzimidazóis/farmacologia , Antagonistas de Leucotrienos/farmacologia , Leucotrienos/biossíntese , Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Carragenina , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Masculino , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Peritonite/induzido quimicamente , Peritonite/enzimologia , Peritonite/prevenção & controle , Pleurisia/induzido quimicamente , Pleurisia/enzimologia , Pleurisia/prevenção & controle , Ratos Wistar , ZimosanRESUMO
OBJECTIVES: We have investigated the anti-inflammatory and antinociceptive effects of (E)-4-(3,7-dimethylocta-2,6-dienylamino)phenol (LQFM-015), which was designed through molecular simplification strategy from 4-nerolidylcatechol. METHODS: The possible anti-inflammatory and antinociceptive effects were assayed on carrageenan-induced paw oedema and pleurisy, acetic acid-induced abdominal writhing and formalin tests in mice. KEY FINDINGS: LQFM-015 reduced the activity of PLA2 enzyme in vitro by 18%. Docking studies into the catalytic site of PLA2 were used to identify the binding mode of the LQFM-015. LQFM-015 showed a moderate antinociceptive effect, since this compound reduced the number of writhings by approximately up to 40% in the acetic acid-induced pain model; this antinociceptive activity also emerged in the second phase of the formalin-induced pain model (58% of inhibition). The anti-inflammatory action of LQFM-015 was confirmed in acute inflammation models, in which it reduced the formation of oedema to 52.78 ± 8.6 and 46.64 ± 5.2 at the second and third hour of carrageenan-induced paw oedema, respectively. Also in the carrageenan-induced pleurisy model, LQFM-015 reduced the migration of leucocytes by 26.0% and decrease myeloperoxidase activity by 50%. LQFM-015 showed different concentrations to inhibit 50% of isoenzyme cyclooxygenase activity (IC50); COX-1 IC50 = 36 µM) and COX-2 IC50 = 28 µM. CONCLUSIONS: LQFM-015 demonstrated inhibition of both PLA2 and COX enzymes; thus, the moderate antinociceptive effect of this compound could be attributed to its anti-inflammatory activity.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Catecóis/uso terapêutico , Desenho de Fármacos , Inibidores Enzimáticos/uso terapêutico , Oxirredutases/antagonistas & inibidores , Dor Abdominal/enzimologia , Dor Abdominal/prevenção & controle , Analgésicos/administração & dosagem , Analgésicos/química , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Domínio Catalítico , Catecóis/administração & dosagem , Catecóis/química , Catecóis/farmacologia , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Edema/enzimologia , Edema/imunologia , Edema/prevenção & controle , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/imunologia , Masculino , Camundongos , Conformação Molecular , Simulação de Acoplamento Molecular , Oxirredutases/metabolismo , Inibidores de Fosfolipase A2 , Fosfolipases A2/química , Pleurisia/enzimologia , Pleurisia/imunologia , Pleurisia/prevenção & controle , EstereoisomerismoRESUMO
Lipoxygenases (LOs) are iron-containing enzymes that catalyze the conversion of arachidonic acid into hydroperoxyeicosatetraenoic acids (HPETEs) and other bioactive lipid mediators. In mammals, 5-LO, 15-LO, and 12-LO enzymes seem to have distinct roles in pathophysiological contexts, which have emphasized the need for selective inhibitors. Cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) has been proposed as potent and selective inhibitor of platelet-type 12-LO (p12-LO). Here, we re-evaluated the selectivity profile of CDC on LOs, and we show that CDC is a potent and direct inhibitor of 5-LO. CDC reduced 5-LO activity in cell-free assays (purified human recombinant enzyme or leukocyte homogenates), with IC(50) values in the low nanomolar range (9-25 nM) and a selectivity index of approximately 35 and 15 over p12-LO and 15-LO1, respectively. Likewise, CDC inhibited 5-LO product formation in intact human polymorphonuclear leukocytes and monocytes (IC(50) = 0.45-0.8 µM). A lower potency was observed for 15-LO1, whereas p12-LO activity in platelets was hardly affected. In human whole blood, CDC efficiently reduced the formation of 5-LO products, and similar effects were observed for 12(S)-H(P)ETE and 15(S)-H(P)ETE. Finally, CDC (3.5 and 7 mg/kg i.p.) was effective in vivo in the platelet-activating factor-induced shock in mice and reduced formation of the 5-LO product leukotriene B(4) in the rat carrageenan-induced pleurisy after a single oral dose of 10 mg/kg. Together, our data demonstrate that CDC is a potent inhibitor of 5-LO with efficacy in vivo and encourage further development of CDC as the lead compound.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Ácidos Cafeicos/farmacologia , Inibidores de Lipoxigenase/farmacologia , Adulto , Animais , Ácidos Cafeicos/química , Ácidos Cafeicos/uso terapêutico , Células Cultivadas , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/uso terapêutico , Camundongos , Pleurisia/tratamento farmacológico , Pleurisia/enzimologia , Ratos , Ratos WistarRESUMO
Cryptococcal infection is the 4th most common opportunistic infection in patients with acquired immune deficiency syndrome (AIDS). Although pleural effusion alone is an unusual presentation, we present a case of cryptococcal pleuritis in an AIDS patient which was initially difficult to discriminate from tuberculous pleuritis because of the high level of pleural adenosine deaminase (ADA). Cryptococcus neoformans was detected in the culture of the pleural effusion after the initiation of antituberculous treatment. High levels of ADA in the pleural fluid can be observed in patients with cryptococcal pleuritis, and longer incubation of pleural fluid should be performed in all patients who present with pleuritis associated with a high ADA level as the only significant finding.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Adenosina Desaminase/metabolismo , Criptococose/enzimologia , Pleurisia/microbiologia , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/enzimologia , Criptococose/diagnóstico , Cryptococcus neoformans/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Pleurisia/enzimologiaRESUMO
Selective inhibition of pro-inflammatory prostaglandin (PG)E(2) formation via microsomal PGE(2) synthase-1 (mPGES-1) might be superior over inhibition of all cyclooxygenase (COX)-derived products by non-steroidal anti-inflammatory drugs (NSAIDs) and coxibs. We recently showed that benzo[g]indol-3-carboxylates potently suppress leukotriene biosynthesis by inhibiting 5-lipoxygenase. Here, we describe the discovery of benzo[g]indol-3-carboxylates as a novel class of potent mPGES-1 inhibitors (IC(50)0.1 microM). Ethyl 2-(3-chlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (compound 7a) inhibits human mPGES-1 in a cell-free assay (IC(50)=0.6 microM) as well as in intact A549 cells (IC(50)=2 microM), and suppressed PGE(2) pleural levels in rat carrageenan-induced pleurisy. Inhibition of cellular COX-1/2 activity was significantly less pronounced. Compound 7a significantly reduced inflammatory reactions in the carrageenan-induced mouse paw edema and rat pleurisy. Together, based on the select and potent inhibition of mPGES-1 and 5-lipoxygenase, benzo[g]indol-3-carboxylates possess potential as novel anti-inflammatory drugs with a valuable pharmacological profile.
Assuntos
Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Indóis/síntese química , Indóis/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Ácidos Carboxílicos/química , Linhagem Celular Tumoral , Edema/tratamento farmacológico , Edema/enzimologia , Inibidores Enzimáticos/química , Humanos , Indóis/química , Concentração Inibidora 50 , Oxirredutases Intramoleculares/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Microssomos , Pleurisia/tratamento farmacológico , Pleurisia/enzimologia , Prostaglandina-E Sintases , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Several studies have shown that the anti-inflammatory effect of Pioglitazone extends beyond the cardiovascular system. This study examines the anti-inflammatory effect of Pioglitazone in comparison to reference drugs (Dexamethasone and Indomethacin) in the mouse model of pleurisy induced by carrageenan which is characterized by two distinct phases (4 and 48 h) of inflammation. Pioglitazone (20 and 50 mg/kg, i.p., 0.5 h before pleurisy) inhibited both neutrophil (4 h) and mononuclears (48 h) influxes (P<0.01), but not exudation (P>0.05). While one dose of Pioglitazone was effective in inhibiting inflammation at 4 h, additional doses (10 or 20 mg/kg, i.p., 0.5 h before pleurisy induction followed by either a second dose at 24 h after the first one or two further doses at 12 h of time interval after the first one) were necessary to elicit inhibition of the second (48 h) inflammation phase. These effects were associated with a marked decrease in adenosine-deaminase (ADA) activity, tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta) levels (P<0.01). Myeloperoxidade (MPO) activity was inhibited only at 4 h (P<0.05). By contrast, reference drugs were able to inhibit all the studied inflammatory parameters (P<0.05). These results demonstrated an interesting anti-inflammatory property of this thiazolidinedione class and strengthen prior evidence that PPAR pathways constitute another important route of inflammatory process inhibition of this pleurisy model.
Assuntos
Anti-Inflamatórios/administração & dosagem , Interleucina-1beta/metabolismo , Pleurisia/tratamento farmacológico , Tiazolidinedionas/administração & dosagem , Fator de Necrose Tumoral alfa/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Adenosina Desaminase/metabolismo , Animais , Carragenina/administração & dosagem , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Terapia de Imunossupressão , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Peroxidase/genética , Peroxidase/imunologia , Peroxidase/metabolismo , Pioglitazona , Pleurisia/induzido quimicamente , Pleurisia/enzimologia , Pleurisia/imunologia , Pleurisia/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Abscess formation is a classic host response to infection by many pathogenic microorganisms. Here, we studied the role of prostaglandins (PGs) and their signal transduction in abscess formation. Zymosan was injected into the pleural cavity of rats. Expression of enzymes involved in PG synthesis, their receptors, and cytokines in exudate leukocytes and abscesses were analyzed by polymerase chain reaction, Western blotting, and immunohistochemistry. Treatment with ketorolac, a cyclooxygenase (COX)-1 inhibitor, or N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide (NS-398), a COX-2 inhibitor, reduced the size of abscesses and the number of cells recovered from the abscess. COX-2 was detected in leukocytes of the exudate and a marginal area of abscesses. Among detected terminal PG synthases, the major one was cytosolic PGE synthase. Membrane-bound PGE synthase (mPGES)-1 was detected in cells that were similar to the COX-2-expressing cells in morphology and localization. A high level of the E-prostanoid (EP)(2) receptor and a low level of the EP(4) receptor were detected. The expression pattern of the EP(2) receptor paralleled that of COX-2 and mPGES-1. 11,15-O-Dimethyl PGE(2) (ONO-AE1-259), an EP(2) receptor agonist, and rolipram, a phosphodiesterase type-4 inhibitor, reversed the effects of COX inhibitors on abscess formation. In contrast, 16-(3-methoxymethyl) phenyl-omega-tetranor-3,7-dithia PGE(1) (ONO-AE1-329), an EP(4) receptor agonist, did not reverse the effects of NS-398. Moreover, NS-398 reduced the mRNA levels in exudate leukocytes of some proinflammatory and fibrogenic cytokines, which was reversed by ONO-AE1-259. These results suggest that PGE(2) generated via COX-1 and COX-2 may interact with the EP(2) receptor and may up-regulate in cAMP-dependent fashion the production of cytokines that promote abscess formation.
Assuntos
Abscesso/etiologia , Dinoprostona/biossíntese , Pleurisia/complicações , Receptores de Prostaglandina E/biossíntese , Transdução de Sinais , Abscesso/enzimologia , Abscesso/metabolismo , Abscesso/prevenção & controle , Animais , Western Blotting , Ciclo-Oxigenase 2/biossíntese , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Imuno-Histoquímica , Masculino , Pleurisia/induzido quimicamente , Pleurisia/enzimologia , Pleurisia/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E Subtipo EP2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , ZimosanRESUMO
Cathepsins S and H are present in immune cells and tissues and may play a role in the activation of an adoptive immune response. Our goal was to assess their protein levels in pleural fluids from 82 patients who underwent thoracentesis or thoracoscopy for therapeutic or diagnostic reasons and to relate them to an inflammatory, neoplastic or hemodynamic origin. Pleural effusions were also analyzed for a panel of 13 inflammatory or proliferative markers to test possible links to a nonspecific host reaction. Increased levels of cathepsin S were found in parainflammatory and cancer-related effusions compared to transudates. Cathepsin H levels were elevated only in parainflammatory effusions, whereas the levels in cancer-related effusions were comparable to transudates. Cathepsin S values significantly correlated with LDH, alpha-1-AT, VEGF, sICAM, sVCAM, MPO, uPA, MMP-9/TIMP-1, IL-8 and MCP-1, but not with CRP, IL-10 or cathepsin H. In contrast to cathepsin S, cathepsin H values did not correlate with markers of inflammation, indicating a specific role for cathepsin H in the pleural host response. In conclusion, the estimation of cathepsin S and cathepsin H may help to distinguish between effusions of different etiology.
Assuntos
Biomarcadores Tumorais/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Derrame Pleural Maligno/enzimologia , Derrame Pleural/enzimologia , Idoso , Biomarcadores/sangue , Biomarcadores/metabolismo , Biomarcadores Tumorais/sangue , Catepsina H , Catepsinas/sangue , Cisteína Endopeptidases/sangue , Feminino , Humanos , Inflamação/enzimologia , Neoplasias Pulmonares/enzimologia , Masculino , Pessoa de Meia-Idade , Síndromes Paraneoplásicas/enzimologia , Pleurisia/enzimologiaRESUMO
UNLABELLED: Lung cancer remains the leading cause of cancer death over the world. Although new diagnostic methods have been discovered, new biomarkers of the cancer are still under studying. A human chitinolytic enzyme called chitotriosidase hydrolyzes chitin and chitotrioside substrates. It is specifically expressed by activating macrophages and seems to play a role in the defense against chitinous human pathogens. Recently it has been shown that chitotriosidase may also attend to the inflammatory process. The aim of the study was to determine chitotriosidase activity in serum of patients with lung cancer and patients with inflammatory exudate. We studied the usefulness of the above parameter determination in differentiation between lung cancer and inflammation. In addition, serum activity of lysozyme and cathepsin H was determined. MATERIALS AND METHODS: The study included 17 patients with inflammatory pleural exudate--group 1., 40 lung cancer patients with malignant pleural effusion--group 2. and 37 healthy subjects. All the patients of group 2. were divided into 2 subgroups: 2A without metastases (n = 23) and 2B with metastases (n = 17). Chitotriosidase and cathepsin H activity was determined in serum by a fluorometric methods. Serum lysozyme activity was measured by turbidimetric method with Micrococcus luteus as substrate. RESULTS: We observed an increase of the chitotriosidase activity in serum patients of both studied group in comparison with the control. The activity of the chitotriosidase in lung cancer patients was significantly higher than in the control (36.7 vs 68.1 nmol/ml/h; p < 0.01). There were no significant differences in serum lysozyme and cathepsin H activity in patients in comparison to healthy subjects. CONCLUSION: The results suggest that activity of the chitotriosidase can not be used to differentiation between inflammation and cancer in lung.
Assuntos
Biomarcadores Tumorais/sangue , Catepsinas/sangue , Cisteína Endopeptidases/sangue , Hexosaminidases/sangue , Neoplasias Pulmonares/enzimologia , Muramidase/sangue , Derrame Pleural/enzimologia , Pleurisia/enzimologia , Idoso , Catepsina H , Diagnóstico Diferencial , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Derrame Pleural/diagnóstico , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/enzimologia , Pleurisia/diagnósticoRESUMO
SETTING: Pleural adenosine deaminase (ADA) levels have been found to be useful in diagnosing tuberculous pleuritis. Elevated ADA levels have been attributed to ADA2 isoenzyme, although no comprehensive studies have evaluated ADA2 as a diagnostic test. OBJECTIVE: To estimate the diagnostic accuracy of ADA and ADA2 in diagnosing tuberculous pleurisy. METHOD: A 3-year retrospective study was carried out. ADA and ADA2 were determined on patients diagnosed according to predetermined criteria. RESULTS: A total of 951 samples were received, including 387 patients with tuberculosis (TB). ADA values>or=52.4 U/l yielded a sensitivity, specificity and positive (PPV) and negative predictive value (NPV) respectively of 93.7% (95%CI 90.0-96.0), 88.7% (95%CI 85.7-91.3), 85.5% (95%CI 81.7-88.8) and 95.2% (95%CI 92.9-96.9). ADA2 values>or=40.6 U/l yielded a sensitivity, specificity and PPV and NPV of respectively 97.2% (95%CI 95.0-98.7), 94.2% (95%CI 91.8-96.0), 92.2% (95%CI 89.1-94.7) and 98.0% (95%CI 96.3-99.0). The chi2 and McNemar tests proved the superiority of ADA2 statistically. CONCLUSION: ADA2 is superior to ADA in the diagnosis of tuberculous pleuritis and should be used as a routine test in the diagnostic work-up of patients with pleural effusions in areas with high TB prevalence.
Assuntos
Adenosina Desaminase/metabolismo , Derrame Pleural/diagnóstico , Derrame Pleural/enzimologia , Tuberculose Pleural/diagnóstico , Tuberculose Pleural/enzimologia , Adulto , Feminino , Humanos , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Derrame Pleural/microbiologia , Pleurisia/diagnóstico , Pleurisia/enzimologia , Pleurisia/microbiologia , Valor Preditivo dos Testes , Estudos Retrospectivos , Tuberculose Pleural/complicaçõesRESUMO
The aim of the present study is to evaluate the contribution of mitogen-activated protein kinase 1-3 MAPK3/MAPK1) in a model of acute lung inflammation in mice. Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by: accumulation of fluid containing a large number of neutrophils (PMNs) in the pleural cavity, infiltration of PMNs in lung tissues and subsequent adhesion molecule expression (I-CAM and P-selectin), lipid peroxidation, and increased production of tumour necrosis factor-alpha, (TNF-alpha) and interleukin-1beta (IL-1beta). Furthermore, carrageenan induced lung apoptosis (Bax and Bcl-2 expression) as well as nitrotyrosine formation, NF-kB activation, and pJNK expression, as determined by immunohistochemical analysis of lung tissues and the degree of lung inflammation and tissue injury (histological score). Administration of PD98059, an inhibitor of MAPK3/MAPK1 (10 mg/kg) 1 h after carrageenan caused a reduction in all the parameters of inflammation measured. Thus, based on these findings we propose that inhibitors of the MAPK3/MAPK1 signaling pathways, such as PD98059, may be useful in the treatment of various inflammatory diseases.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Flavonoides/farmacologia , Pulmão/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Pleurisia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Carragenina , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas I-kappa B/metabolismo , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Inibidor de NF-kappaB alfa , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Selectina-P/metabolismo , Fosforilação , Pleurisia/induzido quimicamente , Pleurisia/enzimologia , Pleurisia/imunologia , Pleurisia/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O2.- dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O2.- related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO2 adduct. The log kcat (O2.-) value for MnTBAP is estimated to be about 3.16, which is approximately 5 and approximately 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only approximately 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k red (ONOO-) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O2.-, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO-/CO3.- from O2.- pathways.
Assuntos
Escherichia coli/enzimologia , Sequestradores de Radicais Livres/metabolismo , Metaloporfirinas/metabolismo , Ácido Peroxinitroso/metabolismo , Superóxidos/metabolismo , Animais , Carragenina/efeitos adversos , Escherichia coli/genética , Metaloporfirinas/química , Metaloporfirinas/genética , Camundongos , Modelos Animais , Infiltração de Neutrófilos , Estresse Oxidativo , Ácido Peroxinitroso/química , Derrame Pleural/metabolismo , Pleurisia/induzido quimicamente , Pleurisia/enzimologia , Transdução de Sinais , Especificidade por Substrato , Superóxido Dismutase/deficiência , Superóxidos/químicaRESUMO
Prostanoid production depends on the activity of two cyclooxygenase (COX) isoforms. It is appreciated that COX-1 plays a role in physiological processes, whereas COX-2 acts in pathological conditions. However their roles, particularly roles of COX-1, have not yet been fully established in inflammation. Here, we examined the effects of COX inhibitors, having differential isoform selectivity, on the late phase of rat carrageenin-induced pleurisy to elucidate the role of COX-2 expressed in the draining lymph nodes and found substantial contribution of COX-1-product(s). Protein and mRNA of COX-2 were detectable with Western blotting analysis and reverse-transcription polymerase chain reaction (RT-PCR) analysis in parathymic lymph nodes, peaking at 48 h after induction of pleurisy. Microsomal prostaglandin E synthase (mPGES)-1 was detectable by immunohistochemical analysis in cells with dendritic processes, a morphological characteristic similar to that of COX-2 expressing cells. Although aspirin, indomethacin and a COX-1 inhibitor, ketorolac, significantly decreased the volume of pleural exudate, they did not affect the levels of COX-2 and mPGES-1 in the lymph node 24 h after induction of pleurisy. In contrast, COX-2 inhibitors, nimesulide and NS-398, had no effect on the exudate volume, but they increased the number of COX-2- and mPGES-1-expressing cells and extension of their dendritic processes with significant increase in the COX-2 level, which were antagonised by ketorolac. These results suggest that COX-2-expressing cells may negatively self-regulate their functions by producing PGE2 via mPGES-1: migration into the draining lymph node and their differentiation. Moreover, COX-1- and COX-2-derived prostanoids may play differential or sometimes antagonistic roles in the late phase of acute inflammation.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/biossíntese , Linfonodos/enzimologia , Proteínas de Membrana/metabolismo , Derrame Pleural/enzimologia , Pleurisia/enzimologia , Prostaglandinas/metabolismo , Doença Aguda , Animais , Western Blotting , Carragenina , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Dinoprostona/metabolismo , Modelos Animais de Doenças , Indução Enzimática/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Linfonodos/patologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Derrame Pleural/induzido quimicamente , Derrame Pleural/tratamento farmacológico , Derrame Pleural/patologia , Pleurisia/induzido quimicamente , Pleurisia/tratamento farmacológico , Pleurisia/patologia , Prostaglandina-E Sintases , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
OBJECTIVE: Progelatinase B/proMMP-9 has recently been identified as an indicator of pleural inflammation, presumably originating from granulocytes. The aim of this study was to verify the origin of progelatinase B by simultaneous estimation of specific markers of neutrophil recruitment and activation in pleural effusions following induced pleurisy and pleural injury. MATERIAL AND METHODS: Sixty-three samples of pleural fluid from patients undergoing therapeutic talc pleurodesis (n = 8) and explorative thoracoscopy (n = 3) collected before and at different time intervals after the intervention were analyzed for progelatinase B and neutrophil gelatinase-associated lipocalin (NGAL)-gelatinase complex by substrate electrophoresis, for myeloperoxidase (MPO) and interleukin-8 (IL-8) by immunoadsorbent sandwich assay, as well as for leukocyte count, C-reactive protein (CRP) and total protein (TP). RESULTS: A significant increase in free and NGAL-complexed progelatinase B, MPO and IL-8 was recorded within 48 h following treatment in all subjects. Progelatinase B was strongly correlated with NGAL-gelatinase complex (r = 0.88, p = 0.001), MPO (r = 0.81, p = 0.001), neutrophil count (r = 0.75, p = 0.01) and IL-8 (r = 0.71, p = 0.001), but not with CRP and TP. CONCLUSIONS: The results support the neutrophil origin of the proenzyme, which confirms progelatinase B as an indicator of a local inflammatory reaction. Quantifying the inflammatory reaction may be helpful in the evaluation of both the technical variants of therapeutic pleurodesis and finer discrimination of paraneoplastic effusions.
Assuntos
Degranulação Celular/fisiologia , Colagenases/biossíntese , Precursores Enzimáticos/biossíntese , Gelatinases/biossíntese , Metaloendopeptidases/biossíntese , Neutrófilos/fisiologia , Pleura/enzimologia , Pleura/lesões , Pleurisia/enzimologia , Proteínas de Fase Aguda/metabolismo , Idoso , Biomarcadores/metabolismo , Feminino , Humanos , Interleucina-8/metabolismo , Lipocalina-2 , Lipocalinas , Masculino , Metaloproteinase 9 da Matriz , Pessoa de Meia-Idade , Neutrófilos/enzimologia , Neutrófilos/patologia , Peroxidase/metabolismo , Pleura/patologia , Derrame Pleural/enzimologia , Derrame Pleural/patologia , Pleurisia/patologia , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Glucocorticoid-induced TNFR-related gene (GITR) participates in the immune/inflammatory response. Because GITR expression has been described in cells other than T lymphocytes, we investigated whether it also modulates acute inflammatory response. Using GITR-deficient (GITR(-/-)) mice, we analyzed the role of GITR in the development of carrageenan-induced lung inflammation (pleurisy) by studying several proinflammatory markers 2-8 h after carrageenan injection. When compared with GITR(+/+), GITR(-/-) mice exhibited decreased production of turbid exudate containing a lower number of leukocytes. This was correlated with the reduction of inflammatory markers (including TNF-alpha, IL-1beta, myeloperoxidase, inducible NO synthase, and cyclooxygenase 2) in the pleural exudate and/or in the lung. Moreover, endothelial cells expressed lower levels of adhesion molecules. In lungs of GITR(+/+) mice, GITR ligand expression was not modulated during pleurisy, while that of GITR increased, as a consequence of increased infiltration by GITR-expressing cells and of GITR up-regulation in macrophages and endothelial cells. Finally, cotreatment of GITR(+/+) mice with carrageenan and Fc-GITR fusion protein decreased the number of inflammatory cells (pleural macrophages and lung neutrophils) as compared with carrageenan treatment alone, confirming that GITR plays a role in the modulation of pleurisy.
Assuntos
Glucocorticoides/fisiologia , Mediadores da Inflamação/fisiologia , Pulmão/imunologia , Pulmão/patologia , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/fisiologia , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Doença Aguda , Animais , Carragenina/administração & dosagem , Permeabilidade da Membrana Celular/genética , Permeabilidade da Membrana Celular/imunologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Inflamação/induzido quimicamente , Inflamação/enzimologia , Inflamação/genética , Inflamação/prevenção & controle , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Contagem de Leucócitos , Leucócitos/patologia , Ligantes , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Derrame Pleural/induzido quimicamente , Derrame Pleural/enzimologia , Derrame Pleural/genética , Derrame Pleural/prevenção & controle , Pleurisia/induzido quimicamente , Pleurisia/enzimologia , Pleurisia/genética , Pleurisia/prevenção & controle , Receptores de Fator de Crescimento Neural/antagonistas & inibidores , Receptores de Fator de Crescimento Neural/deficiência , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores do Fator de Necrose Tumoral/deficiência , Fatores de Necrose Tumoral/biossínteseRESUMO
The phosphatidylinositol-3 kinase (PI3K) family of signaling enzymes plays a crucial role in leukocyte recruitment and activation and hence, likely regulates the induction and propagation phases of inflammation. However, little data have emerged showing a role for these processes in the resolution phase in models of in vivo inflammation. Here, we have evaluated the role of PI3K for the migration and survival of eosinophils in a model of allergic pleurisy in mice. Eosinophil accumulation in PI3Kgamma-deficient mice was inhibited at 48 h, as compared with wild-type mice but not at earlier time-points (6 and 24 h). Experiments with adoptive transfer of bone marrow showed that PI3Kgamma in eosinophils but not in non-bone marrow-derived cells was required for their accumulation. Systemic treatment with PI3K inhibitors before antigen challenge prevented the recruitment of eosinophils. This was associated with decreased Akt phosphorylation, interleukin-5 production, and eosinophil release from the bone marrow. Treatment with PI3K inhibitors 24 h after antigen challenge markedly cleared the accumulated eosinophils, an effect associated with inhibition of Akt phosphorylation and an increased number of apoptotic events. Altogether, our data demonstrate an important role of PI3Kgamma for the maintenance of eosinophilic inflammation in vivo, whereas other isoforms of PI3K may be relevant for the recruitment process.
Assuntos
Eosinófilos/imunologia , Hipersensibilidade/enzimologia , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Pleurisia/enzimologia , Androstadienos/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Cromonas/farmacologia , Classe Ib de Fosfatidilinositol 3-Quinase , Eosinófilos/patologia , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Inflamação , Isoenzimas/antagonistas & inibidores , Isoenzimas/imunologia , Camundongos , Morfolinas/farmacologia , Ovalbumina , Fosfatidilinositol 3-Quinases/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Pleurisia/imunologia , Pleurisia/patologia , WortmaninaRESUMO
Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) is up-regulated in response to inflammatory stimuli. To evaluate the extent to which local pleural inflammation involves additional site in the pleural cavity and elsewhere, we investigated the time course of the levels of iNOS and its product in the inflammatory and other sites, and compared those with a level of COX-2 in rat carrageenin-induced pleurisy. The exudate and plasma NOx levels rose, reaching peaks at 9 and 14 h, respectively. Both COX-2 and iNOS became detectable in exudate leukocytes, their levels reaching peaks at 3 and 9 h after irritation, respectively. COX-2 was detectable mainly in neutrophils, but iNOS was detectable in both neutrophils and mononuclear leukocytes. Furthermore, iNOS became detectable in neutrophils and mononuclear leukocytes in enlarged parathymic lymph nodes from 3h in addition to those in peripheral blood and Kupffer cells from 3 to 14 h, respectively. The gene product is also detectable in thymic large dendritic cells of pleurisy-induced rats as well as normal control rats. COX-2 became detectable in stellar dendritic cells of the enlarged draining lymph nodes from 14 h. Thus, these gene products were induced in the immediate proximity of regional lymph nodes, and even at a considerable distance of liver by the local inflammatory stimulus. Although their expression pattern was quite different from each other, these gene products were detectable in phagocytic cells.
Assuntos
Carragenina/química , Óxido Nítrico Sintase/biossíntese , Pleurisia/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Animais , Carragenina/farmacologia , Contagem de Células , Ciclo-Oxigenase 2 , Modelos Animais de Doenças , Imuno-Histoquímica , Leucócitos/efeitos dos fármacos , Leucócitos/ultraestrutura , Masculino , Nitratos/metabolismo , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , Pleurisia/induzido quimicamente , Pleurisia/patologia , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Acute inflammation can be considered in terms of a series of checkpoints where each phase of cellular influx, persistence, and clearance is controlled by endogenous stop and go signals. It is becoming increasingly apparent that in addition to initiating the inflammatory response, eicosanoids may also mediate resolution. This suggests there are two phases of arachidonic acid release: one at onset for the generation of proinflammatory eicosanoids and one at resolution for the synthesis of proresolving eicosanoids. What is unclear is the identity of the phospholipase (PLA2) isoforms involved in this biphasic release of arachidonic acid. We show here that type VI iPLA2 drives the onset of acute pleurisy through the synthesis of PGE2, LTB4, PAF, and IL-1beta. However, during resolution there is a switch to a sequential induction of first sPLA2 (types IIa and V) that mediates the release of PAF and lipoxin A4, which, in turn, are responsible for the subsequent induction of type IV cPLA2 that mediates the release of arachidonic acid for the synthesis of proresolving prostaglandins. This study is the first of its kind to address the respective roles of PLA2 isoforms in acute resolving inflammation and to identify type VI iPLA2 as a potentially selective target for the treatment of inflammatory diseases.
Assuntos
Fosfolipases A/fisiologia , Pleurisia/enzimologia , Doença Aguda , Animais , Ácido Araquidônico/fisiologia , Carragenina/toxicidade , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Convalescença , Corticosterona/sangue , Ciclo-Oxigenase 2 , Progressão da Doença , Indução Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fosfolipases A2 do Grupo II , Fosfolipases A2 do Grupo IV , Fosfolipases A2 do Grupo V , Fosfolipases A2 do Grupo VI , Interleucina-1/biossíntese , Interleucina-1/genética , Isoenzimas/fisiologia , Leucotrieno B4/biossíntese , Leucotrieno B4/genética , Lipoxinas/biossíntese , Lipoxinas/genética , Lipoxinas/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Fosfolipases A2 , Fator de Ativação de Plaquetas/biossíntese , Fator de Ativação de Plaquetas/genética , Fator de Ativação de Plaquetas/fisiologia , Pleurisia/sangue , Pleurisia/induzido quimicamente , Pleurisia/fisiopatologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Ratos , Ratos WistarRESUMO
In vitro and in vivo studies have demonstrated that methylguanidine, an inhibitor of nitric oxide synthase (NOS), is also able to reduce tumour necrosis factor-alpha (TNF-alpha) release. In the present study, we evaluated the anti-inflammatory potential of methylguanidine treatment in two models of acute inflammation (carrageenan-induced paw edema and pleurisy) where oxyradical, nitric oxide (NO) and prostaglandins play a crucial role in the inflammatory processes. Our data show that methylguanidine, given intraperitoneally at the dose of 30 mg/kg, inhibits the inflammatory response reducing significantly (P<0.05) paw swelling, pleural exudates formation, mononuclear cell infiltration and histological injury. Furthermore, our data suggests that there is a significant (P<0.05) reduction in the activity and expression both of the inducible NOS (iNOS) and of cyclooxygenase-2 in lung tissue of pleurisy model. Methylguanidine is also able to reduce the appearance of nitrotyrosine and of the nuclear enzyme poly(adenosine diphosphate [ADP]-ribose) synthase immunoreactivity in the inflamed lung tissues. Treatment with aminoguanidine, the reference drug, significantly reduced all the evaluated pro-inflammatory parameters in carrageenan-treated rats. Taken together, the present results demonstrate that methylguanidine exerts potent anti-inflammatory effects that could be, in part, related to an inhibition of the expression/activity of the iNOS and cyclooxygenase-2 and, another part, may be related to a reduction of TNF-alpha release.