| Objective To investigate effects of macrophages toll-like receptor4(TLR4) in acute exacerbation of chronic obstructive pulmonary diseases (COPD)Method Peripheral blood mononuclear cells were isolated from30COPD patients and24healthy persons, and were transformed into monocyte-derived macrophages(MDM) in vitro. MDM were identified by observing cell morphology under the microscope and detecting CD14by flow cytometer. The MDM from COPD patients were divided into3groups:control group(group A), LPS group(group B), LPS and TLR4McAb group (group C).The MDM from healthy controls were also divided into3groups:control group(groupD), LPS group(group E), LPS and TLR4McAb group(group F). Real time PCR was used to measure the level of TLR4and NF-κB mRNA.The expression of TLR4and NF-κB protein were detected using immunofluorescence with laser scanning confocal microscopy. The contents of tumor necrosis factor-a(TNF-a) in the cell cultured supernatants were tested by ELISA. One way ANOVA was used for difference comparison during groups and Pearson correlation was used for correlation analysis.Results①MDM formation:when mononcyte were cultured till12days, cell size significantly increased, most cells were round or oval, the minority were polygonal or long spindle, the platy pseudopods or protuberance could be seen around cells, monocyte have been turned into MDM. CD14positive rate of monocyte was (90.88±6.3)%, but CD14positive rate of MDM decreased to (43.35±9.5)%, the difference has statistics meaning (t=14.03, P<0.01).②The mRNA and protein levels of TLR4and NF-κB were higher in group A(3.83±0.27,34.08±1.62)(3.71±0.16,58.05±4.66) than that in group D(1,14.03±1.63)(1,32.46±1.87), TNF-a level was also significantly higher in group A [(43.42±2.68)ug/L] than that in group D[(21.22±0.70)μg/L]. After stimulating with LPS, the mRNA and protein levels of TLR4and NF-κB were both increased in group B and group E, group B were (4.89±0.12,67.12±1.54)(4.59±0.25,83.09±5.16)[(51.19±1.09)μg/L] respectively, but group E were (4.19±0.29.75.72±3.59)(2.22±0.24,93.69±3.61)[(43.19±2.67)μg/L] respectively, the increased range of group B was significantly lower than group E. When pretreated with TLR4McAb before stimulating with LPS, the mRNA and protein levels of TLR4and NF-κB, and TNF-a level were both decreased in group C (4.37±0.41,46.21±2.32)(4.14±0.21,65.32±3.85)[(47.45±1.03)μg/L)] and group F(2.53±0.24, 33.14±1.62)(1.75±0.32,61.20±2.18)[(31.45±1.11)μg/L] compared to LPS alone, but still higher than resting state (F=91.94～334.02, all P<0.01).③Under resting state, the postive correlations were respectively existed between TLR4protein and NF-κB protein, TNF-a level (r=0.443,0.502, P<0.05,0.001), and between the NF-κB protein and TNF-a level (r=0.515, P<0.01) in COPD patients. After stimulating with LPS, the postive correlations were respectively existed between TLR4protein and NF-κB protein, TNF-a level (r=0.624,0.438, P<0.01、0.05), and between the NF-κB protein and TNF-a level (r=0.453, P<0.05) in COPD patients.Conclusion COPD patients are in inflammation activation state, the mRNA and protein levels of TLR4and NF-κB were high in macrophage of COPD patients, and the secretion of TNF-a was also increased in COPD patients. The response to LPS stimulation of macrophage from COPD patients was decreased, it may be connected with the impaired function of recognizing bacteria of COPD patients and repeated acute exacerbations of COPD. NF-κB activation and TNF-a secretion which caused by LPS stimulation were partly achieved through TLR4pathway. |
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