IL-6, IL-8 And Elastin Systhesis In COPD Lung Fibroblasts And Study On The Response Of Primary Human Lung Fibroblasts To Inflammatory Stimuli

OBJECTIVEFibroblasts are major cells responsible for the production and maintenance of extracellular matrix. Apart from the functions in tissue repair and reconstruction, recent research has demonstrated that fibroblasts participate in the modulation of local inflammation in diseases associated with chronic inflammation such as rheumatic arthritis. Regarding the relationship between the inflammatory response and injury repair capacity of human lung fibroblasts, data is limited. The current study therefore was designed to investigate the cytokine secretion, cellular proliferation and elatin synthesis of lung fibroblasts from subjects with chronic obstructive pulmonary disease (COPD). Furthermore, the response of primary human lung fibroblasts to the common inflammatory stimuli including lipopolysaccharide (LPS), tumor necrosis factor-a (TNF-a), apoptosis cells and necrotic cells were studied. In addition, the effect of low-dose theophylline on the LPS initiated alterations in lung fibroblasts was analyzed.METHODS(1) Primary human lung fibroblasts were isolated and cultured from surgical peripheral lesion free lung tissue for further in vitro investigation. Samples were collected after written informed consent for the acquisition of material for research was obtained according to the ethnical principles. Based on preoperative lung function, the subjects were divided into non-COPD, COPD stageⅠ-Ⅲ. The mRNA and protein levels of IL-6 and IL-8 as well as proliferatory fuction and elatin systhesis of COPD lung fibroblasts were analysed using case-control design.(2) For the evaluation of the response to inflammatory stimuli, the primary human lung fibroblasts were co-cultured with 1μg/ml LPS, 1ng/ml TNF-α, apoptosis cells or necrotic cells for 24h resrespectively, and then the supernatants and RNA were obtained and kept for further analysis.(3) Multiple fluorescent staining and confocal microscopic technique was used to evaluate the phagocytic function of primary human lung fibroblasts.(4) The concentration of TNF-α, interleukin (IL)-6, IL-8, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, IL-12p70, IL-1βand IL-10 in supernatants were examined by enzyme linked immunosorbent assay (ELISA). (5) The mRNA transcription of IL-6, IL-8, histone deacetylase (HDAC) 2 and elastin was analyzed using reverse-transcription real-time quantitative polymerase chain reaction (qRT-PCR).(6) Insoluble and soluble elastin in cell culture was quantitied by Fastin Elastin Assay.(7) The AlamarBlue(?) (AB) assay was used for measuring cellular proliferation. The alterations in human lung fibroblast proliferation were examined after exposure to LPS, TNF-α, IL-8, IL-6 as well as conditioned medium from cultured macrophages that had been pretreated with LPS.(8) The effect of theophylline at the concentration of 5μg/ml on the IL-6 and IL-8 secretion, HDAC2 mRNA regulation and proliferation alteration induced by LPS was analyzed.(9) Statistics were performed using Prism 5 for windows. For normally distributed data, results are expressed as mean±SD. For abnormally distributed data, results are expressed as medians (range), with the exception of some proliferation data which are expressed as medians (25th percentile,75th percentile). Statistical methods were used for relavant kind of data. Differences were considered significant at the level of P<0.05.RESULTS(1) The primary cultured cells appeared to have typical morphology of fibroblast and immunostaining showed that>99% of the cells were vimentin positive. Staining with antibodies to cytokeratin, vonWillebrand factor, and desmin was negative, indicating that the cultures did not contain significant numbers of epithelial or mesothelial cells, endothelial cells, or smooth muscle cells.(2) Totally 22 subjects were included and divided into two groups, i.e. Controls (10 cases, including non COPD and GOLD stage 1 COPD) and COPDs (12 cases, including GOLD stage 2 and 3). The two groups were similar in age, gender and smoking status. The two groups differed significantly in lung function. As expected, the subjects in COPDs group had lower FEV1% predicted and FEV1/FVC% (p<0.001), with values as (59.8±9.5)% vs (92.3±8.4)% and (54.9±8.3)% vs (73.6±10.8)% respectively.(3) There was a significant increase in the expression of mRNA for IL-6 and IL-8 for human fibroblasts in COPDs group. In addition, protein levels were increased in the supernatants from the human fibroblasts in COPDs group compared with controls for IL-6 [(701.6±288.0) pg/ml vs (355.7±330.8) pg/ml, p=0.02] and for IL-8 [(754.3±297.4)pg/ml vs (373.4±243.1) pg/ml, p=0.02]. The mRNA expression and protein production of IL-6 and IL-8 was negatively correlated with FEV1 pred% and FEV1/FVC%.(4) The proliferation was lower in the human lung fibroblasts from COPDs group (1.18±0.32) than that from controls group (1.63±0.22) (p=0.004).(5) A significantly higher mRNA expression for elastin and protein level for soluble elatin was observed in human lung fibroblasts from COPDs group, however, no change was seen for insoluble elastin.(6) After exposure to 1μg/ml LPS for 24h, the concentration of IL-6 in human lung fibroblast supernatants increased from (1156±455) pg/ml to (1535±439) pg/ml (p=0.0046). A similar increase was observed for IL-8 [from (510.5±170.6) pg/ml to (856.5±418.8) pg/ml, p=0.0122] and TGF-β1 [(1188±623) pg/ml to (1773±847) pg/ml, p=0.0038]. The constitutional secretion of IL-1β,TNF-α,IL-12p70,IL-10 and TGF-β2 was low and TGF-β3 was undetectable. The level of TGF-β2 also increased by almost 50% with p value being 0.0274, while a small increase for IL-1β(p=0.0027). In contrast, IL-10, an anti-inflammatory cytokine, decreased from (37.6±17.9) pg/ml to (25.1±7.5) pg/ml (p=0.0382). No alteration was observed for TNF-a and IL-12p70.(7) In response to 24-hour incubation of 1ng/ml TNF-α, the IL-8 release by human lung fibroblasts increased by 2 folds from (532.3±71.7) pg/ml to (1660.0±389.2) pg/ml (p=0.0009). The concentration of IL-6 and TGF-β1 also went up.(8) A LPS induced down-regulation of HDAC2 mRNA in human lung fibroblast was shown by qRT-PCR, with the relative expression from 3.546 (0.511,8.886) to 1.793 (0.174,3.284) (p=0.0355). Atrend of down-regulation by TNF-αtreatment was also observed without statistical significance (p=0.0625).(9) A process of phagocytosis with apoptosis cells and necrotic cells was observed under confocal microscope. The apoptosis cells led to an increase of IL-6 in human lung fibroblasts, with the values from (991.9±463.8) pg/ml to (1616.0±216.5) pg/ml (p<0.01). For necrotic cells, a different cytokine profile was presented. Briefly, IL-8 and IL-1βwas increased and IL-10 was decreased.(10) LPS appeared to have an inhibitory effect on the human lung fibroblast proliferation. The inhibition was dose-dependent and time-dependent. The inhibition became significant (p<0.05) when the concentration of LPS reached 0.1μg/ml or incubation period was longer than 24h. The proliferation was inhibited by about 8%(p=0.0112) with the concentration at 1μg/ml for 24-48h. TNF-α, IL-6 and IL-8 had a significant dose-dependent inhibitory effect on the proliferation of human lung fibroblasts. The proliferation of human lung fibroblasts was consistently inhibited by LPS-pretreated macrophage conditioned medium (p<0.05).(11)Human lung fibroblasts were treated with 1μg/ml LPS or 1μg/ml LPS+5μg/ml theophylline or without treatment. The concentration of IL-6 for control, LPS treatment and LPS+theophylline treatment was (1150±426) pg/ml, (1559±406) pg/ml and (1336±400) pg/ml respectively (p<0.05). For IL-8, the value for different treatment was (560±205) pg/ml, (1087±359) pg/ml and (933±338) pg/ml (p<0.05). However, the HDAC2 mRNA was not altered by low-dose theophylline. Theophylline did not affect the fibroblast proliferation at the concentration of 0.1-10μg/ml. The theophylline of 5μg/ml alleviated the inhibition by LPS on proliferation (P<0.05).CONCLUSIONS(1) Primary human lung fibroblasts from subjects with COPD have an enhanced production of IL-6 and IL-8, associated with a profile of impaired proliferation and transformation from soluable elastin to insoluble elatin. This indicates that the up-regulated inflammation in lung fibroblasts is related to its insufficiency in injury repair function.(2) The current data demonstrate that human lung fibroblasts have increased IL-6, IL-8 and TGF-β1 release in response to LPS and TNF-α, accompanied with a down-regulation of HDAC2 transcription. In addition, the fibroblasts responses differently in cytokines production to different inflammatory stimulus, indicating that the fibroblasts could participate in the modulation of pulmonary inflammation by producing relevant cytokines.(3) LPS, TNF-α, IL-6, IL-8 and conditioned medium from LPS-pretreated macrophages can inhibit the proliferation of in vitro human lung fibroblasts, suggesting that the inflammation in milieu exert an effect on the function of lung fibroblasts.(4) Low-dose theophylline can partly block the increased production of IL-6 and IL-8 as well as inhibited proliferation initiated by LPS.