| Chapter 1Effect of Erythromycin on the release of proinflammatory cytokines induced by Cigarette Smoke in Human MacrophagesObjectives:The aim of this study was to investigate the molecular mechanism of inflammatory responses caused by cigarette smoke and the effect of erythromycin (EM) on proinflammatory cytokine induced by CSE in human macrophages.Methods:The Aqueous cigarette smoke extract (CSE) was always prepared fresh on the day of the experiment. The human monocytic cell line (U937), obtained from the ATCC, were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. The U937 monocytic cells were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA) according to standard procedures. The U937 differentiated cells were treated with either CSE (1%) or EM (1μg/ml) pretreatment for 24 h at 37°C with 5% CO2. The levels of interleukin-8 (IL-8) and tumor necrosis factor-а(TNF-а) release in the supernatant were determined by sandwich ELISA, using the respective dual antibody kits (R&D Systems) according to the manufacturer's instructions.Results:CSE 1% treatment significantly increased IL-8 release (ng/L: CSE1% 281.53±89.98, controls 217.82±83.01; n=7, P<0.05) and TNF-аrelease(pg/ml: CSE1% 729.25±444.17, controls 316.60±224.89; n=6, P<0.05) from human macrophages cells at 24h of treatment. To find out the role of EM on CSE-mediated proinflammatory cytokine release, we pretreated macrophages with EM for 24 h and then washed cells, then incubated with CSE 1% for 24 h. EM significantly inhibited CSE-mediated IL-8 release (ng/L: CSE1% 281.53±89.98, CSE1% + EM1μg/ml 203.144±95.77, n=7, P<0.05) and TNF-аrelease (pg/ml: CSE1% 729.25±444.17, CSE1% + EM1μg/ml 510.29±309.83, n=6, P<0.05).Conclusions:Our studies show that cigarette smoke induces an inflammatory response by driving proinflammatory gene transcription and stimulating the release of proinflammatory cytokines in human macrophages. EM is able to decrease CSE-induced release of proinflammatory cytokines, such as IL-8 and TNF-а. This suggests that EM protects against the proinflammatory effects of CSE in macrophages cells. Chapter 2Effect of Erythromycin on the transcriptional activity of NF-КB activated by Cigarette Smoke in Human MacrophagesObjectives:The aim of this study was to investigate the molecular mechanism of inflammatory responses caused by cigarette smoke and the effect of erythromycin on the transcriptional activity of nuclear factor-КB (NF-КB) activated by cigarette smoke in human macrophages cells.Methods:The Aqueous CSE was always prepared fresh on the day of the experiment. The human monocytic cell line (U937), obtained from the ATCC, were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. The U937 monocytic cells were differentiated into macrophages using PMA according to standard procedures. The U937 differentiated cells were treated with either CSE (1%) or EM (1μg/ml) pretreatment , and NF-КB inhibitor pyrrolidine dithiocarbamate (PDTC) for 24 h at 37°C with 5% CO2. NF-КB was assessed by electrophoretic mobility-shift assay(EMSA). Briefly, nuclear extracts were prepared according to a method described previously. Protein concentration was determined by the Bradford method. EMSA was done with the Gel-Shift Assay System following the manufacturer′s instructions. The levels of IL-8 release in the supernatant were determined by sandwich ELISA, using the respective dual antibody kits (R&D Systems) according to the manufacturer's instructions.Results:Treatment with CSE1% for 24h increased NF-КB nuclear binding and IL-8 release in human macrophage cells. EM pretreatment has inhibitory effect on CSE-induced NF-КB transcription activity and IL-8 release in human macrophage cells. As a negative control, PTDC inhibit NF-КB activation and IL-8 release.Conclusions:Our data show that cigarette smoke induces an inflammatory response by activating transcription factor NF-КB in human macrophages cells, which maybe results in increasing IL-8 release. EM has an anti-inflammatory action, presumably via an interaction with the NF-КB signaling pathway. Our data suggest that Erythromycin inhibited NF-КB transcriptional activity resulting in decreasing cigarette smoke-induced IL-8 release to exert anti-inflammatory effect.Chapter 3Effect of Erythromycin on Histone Deacetylase Activity Decreased by Cigarette Smoke in Human MacrophagesObjectives:The aim of this study was to investigate the molecular mechanism of inflammatory responses caused by cigarette smoke and the effect of erythromycin on histone deacetylase (HDAC) activity and HDAC1, HDAC2, and HDAC3 protein expression, correlation with NF-КB expression and inflammatory mediators release in human macrophages cells. Methods:The Aqueous CSE was always prepared fresh on the day of the experiment. The human monocytic cell line (U937), obtained from the ATCC, were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. The U937 monocytic cells were differentiated into macrophages using PMA according to standard procedures. The U937 differentiated cells were treated with either CSE (1%) or EM (1μg/ml) pretreatment , and HDAC inhibitor trichostatin A (TSA; 100 ng/ml) for 24 h at 37°C with 5% CO2. The levels of IL-8 and TNF-аrelease in the supernatant were determined by sandwich ELISA, using the respective dual antibody kits (R&D Systems) according to the manufacturer's instructions. And the HDAC activity was measured with a colorimetric assay kit and Western Blot was used for HDAC1, 2, 3 and NF-КB protein assays.Results:1. CSE 1% treatment significantly increased IL-8 release (ng/L: CSE1% 281.53±89.98, controls 217.82±83.01; n=7, P<0.05) and TNF-аrelease(pg/ml: CSE1% 729.25±444.17, controls 316.60±224.89; n=6, P<0.05) from human macrophages cells at 24h of treatment. Pretreatment with EM significantly inhibited CSE-mediated IL-8 release (ng/L: CSE1% 281.53±89.98, CSE1% + EM1μg/ml 203.144±95.77, n=7, P<0.05) and TNF-аrelease (pg/ml: CSE1% 729.25±444.17, CSE1% + EM1μg/ml 510.29±309.83, n=6, P<0.05). Further, TSA treatment significantly increased IL-8 release (ng/L: TSA 232.21±109.67, controls217.82±83.01; n=7, P<0.05) and TNF-аrelease(pg/ml: TSA 428.71±371.00, controls 316.60±224.89; n=6, P<0.05) from human macrophages cells at 24h of treatment. 2. CSE 1% significantly decreased HDAC activity and HDAC1, 2, 3 protein levels at 24h, which were associated with the increased protein expression of NF-КB. We also showed that CSE-mediated inhibition of HDAC activity and HDAC1, 2, 3 protein levels and increase of NF-КB protein expression were restored by EM pretreatment at 24h . We further showed that CSE particularly decreased HDAC2 protein level, which was significantly restored by EM pretreatment. As a positive control, TSA decreased HDAC activity and HDAC1, -2, and -3 protein levels and increased NF-КB protein expression.Conclusions:Cigarette smoke reduced HDAC expression and decreased the protein levels of HDAC1, HDAC2 and HDAC3 in human macrophages cells. Particularly, cigarette smoke caused a decline in HDAC2 protein expression. The decrease of HDAC activity and HDAC1, 2, 3 expression may be responsible for the increased inflammatory gene transcription. EM is able to restore HDAC activity and HDAC1, HDAC2, HDAC3 levels decreased by cigarette smoke and inhibite NF-КB activity resulting in decreasing CSE-mediated IL-8 and TNF-аrelease, which has shown an important explanation that EM possess the anti-inflammatory effect induced by cigarette smoke. |
PDF Full Text Request