Roflumilast-N-oxide Induces Surfactant Protein Expression in Human Alveolar Epithelial Cells Type II

<div><p>Surfactant proteins (SPs) are important lipoprotein complex components, expressed in alveolar epithelial cells type II (AEC-II), and playing an essential role in maintenance of alveolar integrity and host defence. Because expressions of SPs are regulated by cyclic adenosine monophosphate (cAMP), we hypothesized that phosphodiesterase (PDE) inhibitors, influence SP expression and release. Analysis of PDE activity of our AEC-II preparations revealed that PDE4 is the major cAMP hydrolysing PDE in human adult AEC-II. Thus, freshly isolated human AEC-II were stimulated with two different concentrations of the PDE4 inhibitor roflumilast-N-oxide (3 nM and 1 µM) to investigate the effect on SP expression. SP mRNA levels disclosed a large inter-individual variation. Therefore, the experiments were grouped by the basal SP expression in low and high expressing donors. AEC-II stimulated with Roflumilast-N-oxide showed a minor increase in SP-A1, SP-C and SP-D mRNA mainly in low expressing preparations. To overcome the effects of different basal levels of intracellular cAMP, cyclooxygenase was blocked by indomethacin and cAMP production was reconstituted by prostaglandin E2 (PGE2). Under these conditions SP-A1, SP-A2, SP-B and SP-D are increased by roflumilast-N-oxide in low expressing preparations. Roflumilast-N-oxide fosters the expression of SPs in human AEC-II via increase of intracellular cAMP levels potentially contributing to improved alveolar host defence and enhanced resolution of inflammation.</p> </div

mRNA levels of surfactant proteins in human AEC-II.

<p>SP-A1, SP-A2, SP-B, SP-C and SP-D mRNA expression level of human AEC-II (freshly isolated or cultured for 24 h) were measured by real-time PCR. Bar charts show mean ± SD (n = 5, native = freshly isolated non-cultured cells; 24 h culture  =  isolated cells cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038369#s2" target="_blank">Material and Methods</a>).</p

SP-B, C, and D mRNA level in presence of indomethacin and PGE2.

<p>Freshly isolated AEC-II were cultured for 24 h with indomethacin/PGE2 and the indicated substances. After the culture period SP-B (A), SP-C (B) and SP-D (C) mRNA expression was measured by real-time PCR. Due to the large variation in surfactant protein mRNA expression cultures were separated according their basal median expression of the respective surfactant protein (low level SP-B<1088, SP-C<970059, SP-D<11515 black bars; high level SP-B>1088, SP-C>970059, SP-D>11515 hatched bars; A, B, C). Bar charts show means ± SD (n_low = 6, n_high = 5, A, B,; n_low = 6, n_high =6, C; c = non-stimulated control; dbcAMP = dibutyryl-cAMP; RNO = roflumilast-N-oxide; PGE2 = prostaglandin E2).</p

Western blot analysis of total cellular lysate of SP-A protein.

<p>Equal numbers of freshly isolated AEC-II (2×10⁶cells/well) were incubated with Brefeldin A 1 M for 15 min and stimulated with dbcAMP (1 mM), roflumilast-N-oxide at 1 µM or 3 nM. Total cell lysates were separated at reducing conditions using 12% SDS-PAGE and stained with anti-SP-A, anti-SP-B and SP-C. A: representative blot for low (left panel) and high (right panel) SP-A producer, purified SP-A from a patient with alveolar proteinosis was used as positive control, B: increase in SP-A protein expression in low SPA producers measured by densitometric analysis of the blots. Values are expressed as percentage of non-stimulated cells (n = 6), C: representative blot for low (left panel) and high (right panel) SP-B and SP-C producer. (c = non-stimulated control; dbcAMP = dibutyryl-cAMP; RNO = roflumilast-N-oxide; + = positive control, purified SP-A).</p

Surfactant proteins in absence or presence of indomethacin alone or in combination with PGE2.

<p>A) AECII were cultured without or with the cyclooxygenase inhibitor indomethacin (1 µM) and SP-A1, SP-A2; SP-B; SP-C and SP-D mRNA were measured by real-time PCR after culture. Relative Expression of surfactant proteins in cultures without indomethacin (C, hatched bar) were used to calculate the percentage of remaining surfactant expression after cyclooxygenase inhibition (indomethacin, n = 5)). B) Change of relative expression of surfactant proteins in cultures without (C; left panel) and with inhibition of cyclooxygenase by indomethacin (1 µM) and substitution with external PGE2 (10 nM, right panel) measured by real-time PCR. Bar charts show mean ± SD (surfactant proteins n = 8; rE = relative expression; c = control; PGE2 = prostaglandin E2).</p

SPA-1 and SP-A2 mRNA level.

<p>Freshly isolated AEC-II were cultured for 24 h with or without the indicated substances. After the culture period expression of SP-A1 (A) and SP-A2 (B) mRNA was measured by real-time PCR. Due to the large variation in SP-A expression cultures were grouped according their basal median expression of SP-A1/SP-A2 (low level SP-A1<1436, SP-A2<2500, black bars; high level SP-A1>1436 and SP-A2>2500, hatched bars; C, D). Bar charts show mean±SD (n = 13, A; n = 13, B; n_high = 6, n_low = 7, C; n_high = 7, n_low = 6, D; rE = relative expression; c = non-stimulated control; dbcAMP = dibutyryl-cAMP; RNO = roflumilast-N-oxide).</p

Primer sequences.

<p>Primer sequences.</p

PDE1-5 activities in human AEC-II cells.

<p>Cell lysates of human AEC-II probed for PDE1-5 activities at 0.5 µM cAMP or cGMP substrate concentrations as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038369#s2" target="_blank">Methods</a>. PDE1 was measured with cAMP or cGMP as substrate. Results are shown as mean ±SEM from three different donors.</p

SP-A1 and SP-A2 mRNA level with indomethacin and PGE2.

<p>Cells were cultured for 24 h in presence of indomethacin (1 µM), PGE2 (10 nM) and stimulated with dbcAMP (1 mM) or roflumilast-N-oxide (3 nM or 1 µM; A, B). Cultures were split according their basal median expression of SP-A1/SP-A2 (low level SP-A1<136, SP-A2<474, black bars; high level SP-A1>136, SP-A2>474, hatched bars; C, D). Values presented are means ± SD (n = 19; A,B; n_high = 9, n_low = 10, C, D; rE = relative expression; c = non-stimulated control; dbcAMP = dibutyryl-cAMP; RNO = roflumilast-N-oxide, PGE2 = prostaglandin E2).</p

Effects of CSE (0.1%–10%) and LPS (10 ng/ml–10 µg/ml) on chemokine release and cell viability.

<p>Serum-starved A549 cells were incubated with medium alone (control) or with different concentration of CSE (0.1%–10%) and LPS (10 ng/ml–10 µg/ml) for 24 h. The culture supernatants were collected and the concentrations of IL-8/CXCL8, Gro-α/CXCL1 and MCP-1/CCL2 were measured by ELISA (A, B, C and D) and viability was determined by MTT test (E). The data represent the mean ± SEM of 3 experiments. * p<0.05 compared to control.</p