RESUMO
The aim of this work was to measure HMGB1, TNF-alpha, and IL-8 in bronchoalveolar lavage (BAL), serum and TLR2 and TLR4mRNA expression in lung tissue of rabbits with two grades of acute lung injury (ALI). The animals were randomly assigned to groups with severe (S) and mild/moderate (MM) ALI, induced with warm saline, and a control group. HMGB1, TNF-alpha, IL-8, TLR2mRNA and TLR4mRNA were measured after ALI induction. The results showed increased levels of IL-8, TNF-alpha, HMGB1 and TLR4mRNA in the ALI groups. HMGB1, IL-8 and TNF-alpha concentrations in BAL were higher in S compared MM. Increased TLR4mRNA was observed in S and MM versus control. The results suggest an early participation of HMGB1 in ALI together with IL-8 and TNF-alpha and association with severity. TLR4 has early expression and role in ALI pathophysiology but is not associated with severity.(AU)
O objetivo deste trabalho é determinar os níveis de HMGB1, TNF-alfa e IL-8 no lavado broncoalveolar (BAL), bem como quantificar a expressão sérica de TLR2 e TLR4 mRNA em tecido pulmonar de coelhos com dois graus de lesão pulmonar aguda (LPA). Os animais foram distribuídos aleatoriamente em grupos com LPA grave (S) e leve / moderada (MM), induzidas com solução salina morna, e um grupo controle. HMGB1, TNF-alfa, IL-8, TLR2mRNA e TLR4mRNA foram medidos após a indução de LPA e quatro horas de ventilação mecânica. Os resultados mostraram níveis aumentados de IL-8, TNF-alfa, HMGB1 e TLR4mRNA nos grupos com LPA. As concentrações de HMGB1, IL-8 e TNF-alfa no LBA foram maiores no S comparado ao MM. Aumento de TLR4mRNA foi observado em S e MM versus controle. Os resultados sugerem uma participação precoce da HMGB1 na LPA em conjunto com IL-8 e TNF-alfa e associação com a gravidade da LPA. O TLR4 foi expresso na ALI e possivelmente possui papel precoce na fisiopatologia da LPA, mas sem associação com a gravidade.(AU)
Assuntos
Animais , Coelhos , Citocinas , Proteína HMGB1 , Lesão Pulmonar Aguda , RNA Mensageiro , Interleucina-8 , Fator de Necrose Tumoral alfa , Receptor 2 Toll-Like , Receptor 4 Toll-LikeRESUMO
The aim of this work was to measure HMGB1, TNF-alpha, and IL-8 in bronchoalveolar lavage (BAL), serum and TLR2 and TLR4mRNA expression in lung tissue of rabbits with two grades of acute lung injury (ALI). The animals were randomly assigned to groups with severe (S) and mild/moderate (MM) ALI, induced with warm saline, and a control group. HMGB1, TNF-alpha, IL-8, TLR2mRNA and TLR4mRNA were measured after ALI induction. The results showed increased levels of IL-8, TNF-alpha, HMGB1 and TLR4mRNA in the ALI groups. HMGB1, IL-8 and TNF-alpha concentrations in BAL were higher in S compared MM. Increased TLR4mRNA was observed in S and MM versus control. The results suggest an early participation of HMGB1 in ALI together with IL-8 and TNF-alpha and association with severity. TLR4 has early expression and role in ALI pathophysiology but is not associated with severity.(AU)
O objetivo deste trabalho é determinar os níveis de HMGB1, TNF-alfa e IL-8 no lavado broncoalveolar (BAL), bem como quantificar a expressão sérica de TLR2 e TLR4 mRNA em tecido pulmonar de coelhos com dois graus de lesão pulmonar aguda (LPA). Os animais foram distribuídos aleatoriamente em grupos com LPA grave (S) e leve / moderada (MM), induzidas com solução salina morna, e um grupo controle. HMGB1, TNF-alfa, IL-8, TLR2mRNA e TLR4mRNA foram medidos após a indução de LPA e quatro horas de ventilação mecânica. Os resultados mostraram níveis aumentados de IL-8, TNF-alfa, HMGB1 e TLR4mRNA nos grupos com LPA. As concentrações de HMGB1, IL-8 e TNF-alfa no LBA foram maiores no S comparado ao MM. Aumento de TLR4mRNA foi observado em S e MM versus controle. Os resultados sugerem uma participação precoce da HMGB1 na LPA em conjunto com IL-8 e TNF-alfa e associação com a gravidade da LPA. O TLR4 foi expresso na ALI e possivelmente possui papel precoce na fisiopatologia da LPA, mas sem associação com a gravidade.(AU)
Assuntos
Animais , Coelhos , Citocinas , Proteína HMGB1 , Lesão Pulmonar Aguda , RNA Mensageiro , Interleucina-8 , Fator de Necrose Tumoral alfa , Receptor 2 Toll-Like , Receptor 4 Toll-LikeRESUMO
INTRODUCTION: Toll-like receptor (TLR)-4 and TLR-2 are involved in inflammatory response of monocytes. These cells are activated in pregnant women with preeclampsia (PE), and over-produce inflammatory cytokines. TLR4 may recognize endogenous ligands, the so-called danger signals released by damaged cells, leading to production of pro-inflammatory cytokines. OBJECTIVES: This study investigated TLR2 and TLR4 expression and cytokine production by monocytes from women with PE before and after stimulation with TLR ligands. METHODS: Monocytes (5×10(5)cell/mL) were obtained from 32 preeclamptic (PE) and 20 normotensive (NT) pregnant women in the last trimester of pregnancy. TLR2 and TLR4 expression on monocyte surface was determined by flow cytometry in non-stimulated cells, and after 18h of culture with lipopysaccharide (LPS) and peptidoglycan (PG). TNF-α, IL-10 and IL-12p70 production by these cells stimulated or not with LPS or PG was evaluated by enzyme immunoassay. Results were analyzed by non-parametric tests with significance level set at 5%. RESULTS: In the absence of stimulation, the basal TLR4 expression by monocytes detected by the median fluorescence intensity (MFI) was significantly higher in the PE group than in the NT group while no significant differences were observed between groups in relation to endogenous TLR2 expression. An increase in TLR4 MFIs was detected after monocytes from NT pregnant women were stimulated with LPS while TLR2 expression was increased after PG-stimulation. No alterations in TLR expression was detected after LPS or PG-stimulation in monocytes from patients with PE. Evaluation of endogenous cytokine levels in supernatant culture of monocytes showed higher concentrations of TNF-α and IL-12p70 in preeclamptic women in comparison with the NT group, whereas IL-10 values were significantly higher in NT pregnant women than in the PE group. In contrast, when monocytes were stimulated with the TLRs ligands LPS and PG, the release of TNF-α was significantly reduced, while IL-12p70 levels were significantly higher in women with PE compared to NT group. IL-10 production was similar in both groups studied. CONCLUSION: The basal up-regulation of TLR4 expression associated with endogenous high TNF-α and IL-12p70 production by monocytes from preeclamptic women confirms the activated profile of these cells by the disease process. These findings provide new insights into possible roles for TLRs in the pathogenesis of systemic inflammation detected in PE. FINANCIAL SUPPORT: FAPESP 2009/11924-3 and 2010/20207-0.
RESUMO
INTRODUCTION: Monocytes from peripheral blood of pregnant women with preeclampsia are endogenously activated and secrete high levels of free radicals and inflammatory cytokines. OBJECTIVES: This study aimed at evaluating whether the inflammatory state of monocytes observed in preeclampsia is associated with the polarization of monocyte to M1 profile in peripheral blood, correlating the expression of surface receptors CD64, TLR2, TLR4, and CD163 and CD206 with cytokine production. METHODS: We studied 90 pregnant women, 30 normotensive and 60 with preeclampsia, matched for gestational age. Peripheral blood monocytes obtained from normotensive pregnant or preeclamptic pregnant women were cultured for 18h, and the expression of surface receptors on M1 inflammatory monocyte subpopulation (TLR2, TLR4 and CD64) and M2 suppressor monocyte subpopulation (CD163 and CD206) were evaluated by flow cytometry, using specific monoclonal antibodies, labeled with fluorochromes. The values were expressed as ??the mean fluorescence intensity. Moreover, the production of proinflammatory cytokines associated with M1 profile (TNF-α, IL-12p70 and IL-23) and the anti-inflammatory cytokine associated with M2 profile (IL-10) were evaluated in the monocyte supernatant of culture by enzyme immunoassay. Results were analyzed using nonparametric tests with significance level set at 5%. RESULTS: The expression of CD4 and TLR4 on monocyte surface, from women with preeclampsia was significantly higher, while the expression of CD163 and CD206 was significantly decreased compared with normotensive pregnant women, suggesting the predominance of monocyte M1 profile. Endogenous production of TNF-α, IL-12p70 and IL-23 by monocytes was increased, while synthesis of IL-10 was lower in women with preeclampsia compared with normotensive pregnant women. Positive correlations between TLR4 and CD64 (r=0.5849), TLR4 and TNF-α (r=0.5126) or TLR4 and IL-23 (r=0.8095), as well as between CD64 and TNF-α (r = 0.7133) or CD64 and IL-23 (r = 0.6051) were observed in the preeclamptic group. The results confirm the activated state of monocytes from women with preeclampsia by increased production of proinflammatory cytokines and the expression of receptors characteristic of the M1 subpopulation. CONCLUSION: This study provides evidence that monocytes from women with preeclampsia are classically activated and the systemic inflammatory environment may differentiate and polarize these cells to the M1 profile. FINANCIAL SUPPORT: CNPq, FAPESP 2009/11924-3 and 2010/20207-0.