RESUMO
The aim of this work was to investigate if the low-level laser therapy (LLLT) on acute lung inflammation (ALI) induced by lipopolysaccharide (LPS) is linked to tumor necrosis factor (TNF) in alveolar macrophages (AM) from bronchoalveolar lavage fluid (BALF) of mice. LLLT has been reported to actuate positively for relieving the late and early symptoms of airway and lung inflammation. It is not known if the increased TNF mRNA expression and dysfunction of cAMP generation observed in ALI can be influenced by LLLT. For in vivo studies, Balb/c mice (n = 5 for group) received LPS inhalation or TNF intra nasal instillation and 3 h after LPS or TNF-α, leukocytes in BALF were analyzed. LLLT administered perpendicularly to a point in the middle of the dissected bronchi with a wavelength of 660 nm and a dose of 4.5 J/cm(2). The mice were irradiated 15 min after ALI induction. In vitro AM from mice were cultured for analyses of TNF mRNA expression and protein and adenosine3':5'-cyclic monophosphate (cAMP) levels. One hour after LPS, the TNF and cAMP levels in AM were measured by ELISA. RT-PCR was used to measure TNF mRNA in AM. The LLLT was inefficient in potentiating the rolipram effect in presence of a TNF synthesis inhibitor. LLLT attenuated the neutrophil influx and TNF in BALF. In AM, the laser increased the cAMP and reduced the TNF-α mRNA. LLLT increases indirectly the cAMP in AM by a TNF-dependent mechanism.
Assuntos
AMP Cíclico/metabolismo , Terapia com Luz de Baixa Intensidade , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/radioterapia , Animais , Sequência de Bases , Primers do DNA/genética , Modelos Animais de Doenças , Lipopolissacarídeos/toxicidade , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inibidores da Fosfodiesterase 4/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/genética , Rolipram/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: The aim of this work was to investigate the low-level laser therapy (LLLT) effect on alveolar macrophages (AM) activated by oxidative stress and lipopolysaccharide (LPS). BACKGROUND DATA: LLLT has been reported to actuate positively relieving the late and early symptoms of airway and lung inflammation. It is not known if the increased MIP-2 mRNA expression and intracellular reactive oxygen species (ROS) generation observed in acute lung inflammation (ALI) can be influenced by LLLT. MATERIALS AND METHODS: Rat AM cell line (AMJ2-C11) was cultured with LPS or H(2)O(2) and laser irradiated. MIP-2 mRNA and ROS production in the AM were evaluated by Real Time-PCR and the 2',7'-dichlorofluorescin diacetate (DCFH-DA) respectively. The NF-κB protein in the AM was measured by the enzyme linked immunoassay method. To investigate the antioxidant effect of laser, the AM were prebathed with N-acetylcysteine (NAC) and then irradiated with laser. LLLT was also studied in the presence of an inhibitor of NF-κB (BMS 205820). In addition, the effect of LLLT on NF-κB protein was investigated. RESULTS: LLLT attenuated the MIP-2 mRNA expression and intracellular ROS generation after LPS or H(2)O(2). When the AM were pretreated with NAC, the laser effect was potentiated. BMS 205820 suppresses the effect of LLLT on MIP-2 mRNA expression and ROS generation, stimulated by LPS or H(2)O(2). On NF-κB transcription factor, both the LLLT and NAC reduced this protein in the AM exposed to LPS or H(2)O(2). The synergistic effect between LLLT and NAC on the reduction the NF-κB was also evidenced. CONCLUSION: Results indicate that there is a synergistic action of LLLT with NAC on MIP-2 mRNA expression from LPS- or H(2)O(2)-stimulated AM, and that both ROS intracellular generation and NF-kB signaling seem to be involved.
Assuntos
Quimiocina CXCL2/genética , Terapia com Luz de Baixa Intensidade , Macrófagos Alveolares/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Células Cultivadas , Quimiocina CXCL2/metabolismo , Sequestradores de Radicais Livres/farmacologia , Macrófagos Alveolares/efeitos da radiação , RatosRESUMO
A prospective study was undertaken to assess the radiotoxicity of accelerated particles in pulmonary alveolar macrophages (AM). We evaluated the effects of a single dose (10-75 Gy) of an external low-energy (20 MeV) proton beam on cultured AM oxidative metabolism and phagocytic function. Macrophages are the first line of defense against invading pathogens and are known to generate superoxide anion (O2), nitric oxide (NO), and mediators of antimicrobial and antitumoral defense mechanisms. We obtained AM by bronchoalveolar lavage from young (1-2 month old) and aged (9-12 month old) male Wistar rats. Cell viability, phagocytosis, O2 and NO production in control and proton-irradiated cultured AM were evaluated The effect of proton irradiation on cell viability was dose-dependent The higher doses induced a dramatic decrease in viability in the aged population. Phagocytosis increased 1.3-1.4 fold inboth populations irrespective of the dose delivered. Generation of O2 was always higher in the aged population for all the doses assayed and showed no significant variation from the control values. In the young population a clear increase was observed with doses of 25 and 50 Gy. NO production in AM from young animals rose in a dose-dependent manner. Conversely, proton irradiation did not affect NO production in macrophages from aged animals. The results of this study demonstrate that AM isolated from young and aged rats are functionally different and show a distinct behavior when exposed to proton irradiation. These findings suggest that age may condition response and must be taken into account when accelerated particle-radiotherapy protocols are considered as a valid therapeutic option for the treatment of cancer. To the best of our knowledge, this is the first report comparing sham-irradiated and proton-irradiated young and aged AM.
Assuntos
Envelhecimento/fisiologia , Macrófagos Alveolares/efeitos da radiação , Prótons , Animais , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Óxido Nítrico/metabolismo , Fagocitose/efeitos da radiação , Ratos , Ratos Wistar , Superóxidos/metabolismoRESUMO
The effect of ionizing radiation on metabolic functions of alveolar macrophages (AM) have been well studied. However, variations associated to age have not been established yet. The aim of this work was to perform a comparative study on irradiated alveolar macrophages from young and aged rat lungs. Cell viability and occurrence of apoptosis as well as production of nitric oxide (NO), generation of superoxide anion (O2*-) and total antioxidant capacity were analyzed in vitro after exposure to gamma-irradiation with 10, 25, 50 and 75 Gy. Cell viability decreased only in the aged population at the higher doses. Morphological features of apoptosis were clearly evidenced in irradiated alveolar macrophages from aged animals although the DNA fragmentation assay for apoptosis showed no differences for either of the populations studied. NO production and total reactive antioxidant potential (TRAP) levels showed a dose-dependent modulation. Low radiation doses inhibited the production of NO and decreased TRAP levels whereas higher doses enhanced the NO production and increased the TRAP levels in both macrophage populations. Generation of O2*- was always higher in the aged population for all the doses assayed. We conclude that in vitro young alveolar macrophages exhibited higher radioresistance over the whole range of doses as compared to the aged macrophage population. Our results show that the aging process markedly affects the radioresistance of phagocytic cells. Therefore, immune defense and inflammatory response of lungs from aged patients should be considered when planning radiotherapy protocols.