Characteristics of alveolar macrophages from murine models of OVA-induced allergic airway inflammation and LPS-induced acute airway inflammation

<div><p>ABSTRACT</p><p><i>Background</i>: Macrophages include the classically activated pro-inflammatory M1 macrophages (M1s) and alternatively activated anti-inflammatory M2 macrophages (M2s). The M1s are activated by both interferon-γ and Toll-like receptor ligands, including lipopolysaccharide (LPS), and have potent pro-inflammatory activity. In contrast, Th2 cytokines activate the M2s, which are involved in the immune response to parasites, promotion of tissue remodeling, and immune regulatory functions. Although alveolar macrophages (AMs) play an essential role in the pulmonary immune system, little is known about their phenotypes. <i>Methods</i>: Quantitative reverse transcription polymerase chain reaction and flow cytometry were used to define the characteristics of alveolar macrophages derived from untreated naïve mice and from murine models of both ovalbumin (OVA)-induced allergic airway inflammation and LPS-induced acute airway inflammation. AMs were co-cultured with CD4⁺ T cells and were pulsed with tritiated thymidine to assess proliferative responses. <i>Results</i>: We characterized in detail murine AMs and found that these cells were not completely consistent with the current M1 versus M2-polarization model. OVA-induced allergic and LPS-induced acute airway inflammation promoted the polarization of AMs towards the current M2-skewed and M1-skewed phenotypes, respectively. Moreover, our data also show that CD11c⁺ CD11b⁺ AMs from the LPS-treated mice play a regulatory role in antigen-specific T-cell proliferation in vitro. <i>Conclusions</i>: These characteristics of AMs depend on the incoming pathogens they encounter and on the phase of inflammation and do not correspond to the current M1 versus M2-polarization model. These findings may facilitate an understanding of their contributions to the pulmonary immune system in airway inflammation.</p></div

MiR-503 expression in COPD and control lung fibroblasts in the absence or presence of IL-1ß and TNF-α.

<p>Control (White square, n = 19) and COPD (Black triangle, n = 18) lung fibroblasts were cultured with 10% FCS containing DMEM for 2 days, after which the medium was changed to DMEM in the absence and presence of IL-1ß and TNF-α (1 ng/ml). After 1 day, total RNA was extracted from the cultured cells. MiR-503 expression was examined by real time qPCR. Vertical axis: level of miR-503 expression, expressed as fold of 18s-rRNA values in the same sample. Horizontal axis: culture condition. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Correlations between lung function and miR-503 expression in control and COPD lung fibroblasts cultured with or without IL-1ß and TNF-α.

<p>Control (n = 19) and COPD (n = 18) lung fibroblasts were cultured with 10% FCS containing DMEM for 2 days, after which the medium was changed to DMEM in the absence (baseline) or presence of IL-1ß/TNF-α (1 ng/ml). After 1 day, total RNA was extracted from the cultured cells. Correlation between miR-503 expression in lung fibroblasts and %FVC (Control (n = 6) and COPD (n = 17)) ((A) baseline, (B) IL-1ß/TNF-α), þV1 (Control (n = 6) and COPD (n = 18)) ((C) baseline, (D) IL-1ß/TNF-α), % DLco (Control (n = 5) and COPD (n = 14)) ((E) baseline, (F) IL-1ß/TNF-α) were shown. White square: control, Black triangle: COPD. Horizontal axis: level of miR-503 expression, expressed as fold of 18s-rRNA values. The correlation was calculated by Spearman’s correlation test.</p

Clinical features of subjects.

<p>Clinical features of subjects.</p

MiR-503 inhibits VEGF protein release and mRNA expression of human lung fibroblasts by direct binding to the 3’ untranslated region (UTR) of VEGF mRNA.

<p>(A-D) Primary human fetal lung fibroblasts (HFL-1 cells) cultured in monolayer were transfected with miR-503 mimic (Black bar) or control miRNA (White bar) transfection reagent, as described in Materials and Methods. 24hr after transfection, the medium was changed to DMEM containing 0.2% FCS, with or without IL-1ß and TNF-α (1 ng/ml), TGF-ß1 (1 ng/ml), or PGE₂ (1 x 10⁻⁷ M). (A, B) 3 days after transfection, the culture medium was harvested and assayed for (A) VEGF or (B) IL-8 (with or without IL-1ß and TNF-α) by ELISA. (A) Vertical axis: VEGF release (pg per 10⁵ cells per 2 days). (B) Vertical axis: IL-8 release (pg per 10⁵ cells per 2 days). Horizontal axis: culture condition. (C) 1 day after transfection, RNA was isolated and endogenous miR-503 expression was analyzed in the presence of control miRNA or miR-503 mimic by real-time qPCR. Vertical axis: level of miR503 expression, expressed as fold of 18s-rRNA values. (D) 2 days after transfection, RNA was isolated and assayed for VEGF mRNA by real-time qPCR. Vertical axis: level of VEGF mRNA expression, expressed as fold of 18s-rRNA values. Horizontal axis: culture conditions. *<i>p</i> < 0.05, ***<i>p</i> < 0.001. (E) Diagrams showing luciferase reporter constructs containing UTRs of human VEGF-A gene and miR-503 targeting site, and seed and full sequences of miR-503. (F) HFL-1 cells cultured in monolayer were co-transfected with a VEGF 3’ UTR-LUC construct, control vector, miR-503 mimic, and control miRNA. Cell layers were harvested 48hr after transfection, and luciferase activity was analyzed by dual luciferase assay. Vertical axis: Firefly luciferase activity, normalized to Renilla Luciferase activity expressed as a relative value to control (indicated as an open bar). Horizontal axis: culture conditions. *<i>p</i> < 0.05. The data presented are means ± SE from 3 separate experiments.</p

Endogenous miR-503 and VEGF production with various stimulations.

<p>HFL-1 cells were cultured in monolayer for 2 days, after which the medium was changed to DMEM containing 0.2% FCS, with or without IL-1ß and TNF-α (1 ng/ml), TGF-ß1 (1 ng/ml) or PGE₂ (1 x 10⁻⁷ M). (A) 1 day after stimulation, RNA was isolated and miR-503 expression was analyzed by real-time qPCR. MiR-503 expression was calculated as fold of 18s-rRNA expression. Vertical axis: level of miR-503 expression, expressed as fold of control medium alone. Horizontal axis: culture conditions. *<i>p</i> < 0.05 compared with the values of control medium alone. (B) HFL-1 cells cultured in monolayer were transfected with miR-503 inhibitor (Black bar) or control miRNA (White bar) transfection reagent, as described in Materials and Methods. 24hr after transfection, the medium was changed to DMEM containing 0.2% FCS, with or without TGF-ß1 (1 ng/ml), IL-1ß and TNF-α (1 ng/ml) or PGE₂ (1 x 10⁻⁷ M). 3 days after transfection, the culture medium was harvested and assayed for VEGF by ELISA. Vertical axis: VEGF release (pg per 10⁵ cells per 2 days). *<i>p</i> < 0.05, ***<i>p</i> < 0.001 compared with the values of control medium alone. The data represent the means ± SE of 3 separate experiments.</p

Cytokines, growth factors, lipid mediator and fibronectin release in COPD and control lung fibroblasts.

<p>Control (n = 13) and COPD (n = 13) lung fibroblasts were cultured with 10% FCS containing DMEM for 2 days, after which the medium was changed to DMEM in the absence and presence of IL-1ß and TNF-α (1 ng/ml). (A) IL-6, (B) IL-8, (C) PGE₂, (D) HGF, (E) KGF, (F) VEGF, and (G) fibronectin release in the cultured medium were examined by ELISA or EIA. Vertical axis: (A) IL-6, (B) IL-8, (C) PGE2, (D) HGF, (E) KGF, (F) VEGF release (pg per 10⁵ cells per 1 day) and (G) fibronectin release (ng per 10⁵ cells per 1 day). Horizontal axis: culture condition. White bar: control, Black bar: COPD. *<i>p</i> < 0.05, **p < 0.01, ***<i>p</i> < 0.001.</p