Effect of LPS and serum on cytokine release from alveolar macrophages, ATII and TT1 cells.

<p>Cells were exposed to LPS (1–100 ng/ml) in the presence and absence of serum for 24 hours. Conditioned media was then aspirated and TNFα, MCP-1 and IL-8 release measured by Luminex. Data expressed as mean ± SE (n = 6 subjects). **P<0.001, ***P<0.0002.</p

Inhibition of MALP-2-induced cytokine release from alveolar macrophages ATII and TT1 cells by MAP kinase inhibitors.

<p>Cells were pre-incubated for 30 min with 10 µM of inhibitors of p38 (SB202190), JNK (SP600125) or ERK (PD98059) before exposure to 100 ng/ml MALP-2 for 24 hours. TNFα (A), MCP-1 (B) and IL-8 (C) release were measured by Luminex. Data expressed as mean ± SE (n = 4 subjects). *P<0.05, **P<0.005, ***P<0.0001.</p

Effect of LPS on alveolar macrophage and ATII cell TLR-4 protein expression.

<p>Alveolar macrophages (A) and ATII cells (B) were exposed to LPS (100 ng/ml) over a three hour time course. Cells were fixed in methanol at 0, 15 mins, 30 mins, 1 hr, 2 hrs and 3 hrs and stained with FITC-labelled anti-TLR4 antibody. TLR4 expression was visualised using confocal microscopy. Images were taken through the centre of the cell to visualise cytosolic as well as cell surface expression. Data representative of 3 separate subjects.</p

Effect of MALP-2 on alveolar macrophage and ATII cell TLR-4 protein expression.

<p>Alveolar macrophages (A) and ATII cells (B) were exposed to MALP-2 (100 ng/ml) over a three hour time course. Cells were fixed in methanol at 0, 15 mins, 30 mins, 1 hr, 2 hrs and 3 hrs and stained with FITC-labelled anti-TLR4 antibody. TLR4 expression was visualised using confocal microscopy. Images were taken through the centre of the cell to visualise cytosolic as well as cell surface expression. Data representative of 3 separate subjects.</p

Effect of LPS and MALP-2 exposure on TLR4 expression by alveolar macrophages, ATII and TT1 cells.

<p>Using Simple PCI software analysis of images captured by confocal microscopy, the mean fluorescent intensity of staining of individual cells for TLR4 was measured over a 3 hour time course of exposure to LPS (A) or MALP-2 (B). Data expressed as mean ± SE (n = 3 subjects). *P<0.05, **P<0.002, ***P<0.0001.</p

Effect of MALP-2 and serum on cytokine release from alveolar macrophages, ATII and TT1 cells.

<p>Cells were exposed to MALP-2 (1–100 ng/ml) in the presence and absence of serum for 24 hours. Conditioned media was then aspirated and TNFα, MCP-1 and IL-8 release measured by Luminex. Data expressed as mean ± SE (n = 6 subjects). **P<0.002, ***P<0.0001.</p

Comparative expression of CD14 on primary human ATII cells, immortalised TT1 cells and A549 cells.

<p>Cells were immunostained with CD14 and cells surface expression measured using flow cytometry. The ratio of median fluorescent intensity of CD14 staining relative to IgG control was calculated for all cells. Results demonstrated that both ATII cells and TT1 cells express CD14 whereas A549 cells do not. Furthermore, ATII cells had significantly higher CD14 staining than TT1 cells. Data expressed as mean ± SE (n = 4 subjects). **P<0.007.</p

Cellular localisation of TLR4 protein in primary human alveolar macrophages and ATII cells.

<p>Under high magnification it can be seen that, in alveolar macrophages (A), there is distinct expression of TLR4 protein at the cell surface at 30 minutes with some intracellular staining. By three hours the intracellular staining has increased greatly (in particular in the perinuclear region). In contrast, in ATII cells there is strong intracellular TLR4 expression at 30 minutes which increases further by 3 hours. Similarly to the alveolar macrophages, intracellular staining is most intense in the perinuclear region. TLR4 expression could not be visualised in ATI cells due to low expression.</p