Fig 1.
Time optimum for the administration of rolipram (A) and roflumilast (B).
The PDE4-inhibitors were given at indicated time points and 24h after LPS inhalation, migration of PMNs into the alveolar space (BAL) was evaluated. Data are presented as mean ± SD; n = 6; *P < 0.05 vs. control without LPS; #P < 0.05 vs. control with LPS; °P < 0.05 vs. 3 and 6h after LPS.
Fig 2.
Impact of PDE-inhibitors on PMN infiltration into the lungs identified by immunohistochemistry.
Neutrophils were stained with a specific marker and appear brown in histology (rat anti-mouse neutrophil, clone 7/4) (original magnification, x63). Images are representatives of n = 4 experiments. Alveolar septa of the different conditions were measured. Data are presented as mean ± SD; n = 6; *P < 0.05 vs. control without LPS; #P < 0.05 vs. control with LPS; °P < 0.05 vs. rolipram.
Fig 3.
Effect of rolipram and roflumilast on PMN migration into the lung.
PDE4-inhibitors were injected and migration of PMNs into the different compartments of the lung without and with LPS (IV = intravascular; IS = intersitital; BAL = alveolar space) quantified (A). In additional experiments, the effects of nebulization the PDE4-inhibitors on PMN migration were analyzed (ip = intraperitoneally, neb = nebulized) (B). Data are presented as mean ± SD; figure A: n = 4 without LPS; n = 8 with LPS; figure B: n≥4; *P < 0.05 vs. control without LPS; #P < 0.05 vs. control with LPS inhalation; °P < 0.05 vs. rolipram ip.
Fig 4.
Chemokine levels in the BAL are reduced by rolipram and roflumilast.
LPS inhalation increased all chemokine levels and the administration of the PDE4-inhibitors decreased this effect. Data are presented as mean ± SD; n = 4 without LPS; n≥6 with LPS; *P < 0.05 vs. control without LPS; #P < 0.05 vs. control with LPS inhalation; °P < 0.05 vs. rolipram.
Fig 5.
Microvascular permeability was attenuated by PDE4-inhibitors.
6h after LPS inhalation, the capillary leakage was assessed by Evans blue extravasation. Data are presented as mean ± SD; n = 6; *P < 0.05 vs. control without LPS; #P < 0.05 vs. control with LPS inhalation; °P < 0.05 vs. rolipram.
Fig 6.
Gene expression and protein levels of PDE4B and PDE4D in lungs of mice.
The impact of rolipram and roflumilast on transcription and translation of PDE4B (A) and PDE4D (B) was determined in the lungs of mice. Data are presented as mean ± SD; n = 6 without LPS; n≥7 with LPS; representative Western blot analyses of four independent experiments (for whole western blots see S1 Fig); *P < 0.05 vs. control without LPS; #P < 0.05 vs. control with LPS inhalation.
Fig 7.
The effect of inflammation and rolipram/roflumilast on PDE activity (A).
Mice were treated with roflumilast or rolipram and inflammation induced by LPS inhalation. Lung homogenates were prepared for PDE4 activity. PDE4-inhibitors decreased the activity of the enzyme even without inflammation. Data are presented as mean ± SD; groups without LPS n≥4; groups with LPS n = 6; *P < 0.05 vs. control without LPS; #P < 0.05 vs. control with LPS inhalation. The impact of the unspecific PDE-inhibitor 3-isobutyl-1-methylxanthine (IBMX) on the PDE activity compared to the PDE4-inhibiors (B). Mice inhaled LPS and were treated with PDE4-inhibiors or IBMX. Data are presented as mean ± SD; n = 5; *P < 0.05 vs. IBMX group.
Fig 8.
In vitro transmigration assay of human PMNs through a pulmonary epithelial monolayer.
PMNs or epithelium were treated with rolipram/roflumilast and migration of PMNs through human epithelium measured. Migration of PMNs was initiated through the chemokine CXCL2/3 in all wells. Data are presented as mean ± SD; n≥4; *P < 0.05 vs. control.
Fig 9.
The impact of PDE4-inhibitors on cytoskeletal remodeling.
LPS-induced formation of F-actin (green) in human pulmonary epithelial cells. Rolipram and roflumilast reduced cytoskeletal remodeling, whereas the effect was more intense in roflumilast treated cells. Images are representatives of three experiments with similar results (original magnification, x63).
Fig 10.
The effect of LPS and rolipram/roflumilast on PDE4B protein expression in epithelial cells.
Human pulmonary epithelial cells were pretreated with rolipram/roflumilast and the influence of LPS on the expression of PDE4B (green) (A) investigated. Images are representatives of four experiments with similar results (original magnification, x63). Data are presented as mean ± SD; n = 3; *P < 0.05 vs. control group without LPS inhalation, #P < 0.05 vs. control with LPS; °P < 0.05 vs. the rolipram group.
Fig 11.
PDE4B protein expression in epithelial cells after LPS and rolipram/roflumilast treatment.
To further confirm results from microscopy, we additionally performed western blots of the PDE4B of H441 cells. Images are representatives of four experiments with similar results (original magnification, x63). Data are presented as mean ± SD; n = 3; *P < 0.05 vs. control group without LPS inhalation, #P < 0.05 vs. control with LPS; °P < 0.05 vs. the rolipram group.
Fig 12.
The effect of LPS and rolipram/roflumilast on PDE4D protein expression in epithelial cells.
Human pulmonary epithelial cells were pretreated with rolipram/roflumilast and the influence of LPS on the expression of PDE4D (green) (B.1) evaluated. Images are representatives of four experiments with similar results (original magnification, x63). Data are presented as mean ± SD; n = 3; *P < 0.05 vs. control group without LPS inhalation, #P < 0.05 vs. control with LPS.
Fig 13.
PDE4D protein expression in epithelial cells after LPS and rolipram/roflumilast treatment.
To further confirm results from microscopy, we additionally performed western blots of the PDE4B of H441 cells. Images are representatives of four experiments with similar results (original magnification, x63). Data are presented as mean ± SD; n = 3; *P < 0.05 vs. control group without LPS inhalation, #P < 0.05 vs. control with LPS.
Fig 14.
The distribution of PDE4B and PDE4D protein in epithelial cells.
PDE4B and PDE4D were stained in one sample. Without LPS, PDE4D (green) is dominant. Both subtypes were found coexistent around the nucleus and also in the cytoplasm. Images are representatives of four experiments with similar results (original magnification, x63).
Fig 15.
The influence of hyperinflammation on PDE4B and PDE4D protein expression in epithelial cells.
PDE4B and PDE4D were stained in one sample to investigate the effect of LPS on the distribution of both enzymes. Even at first glance, it is striking that in the picture without LPS PDE4D (Fig 14) is dominant, whereas LPS increased predominantly PDE4B (red) (Fig 15), confirming our previous findings. Images are representatives of four experiments with similar results (original magnification, x63).