Fig 1.
DEX reduced LPS-induced lung inflammation in the presence of G-CSF blockade.
(A) A diagram depicting the experimental regimen. DEX (2.5 mg/kg, i.p.) and control antibodies or anti-G-CSF (250 μg/kg, i.p.) were given 6 h post LPS (1 mg/kg, i.t., 24 h) challenge. (B) BAL neutrophil numbers. ✽, significantly different, two-way ANOVA followed by Bonferroni post-hoc tests. P<0.05 (N = 4–8). Comparisons indicated are among LPS and LPS+DEX groups in the presence of control or anti-G-CSF antibodies. (D) BAL G-CSF, TNF, and IL-6 protein levels. ✽, significantly different, two-way ANOVA followed by Bonferroni post-hoc tests. P<0.05 (N = 4–8). (E) BAL total protein levels and lung injury scores. ✽, significantly different, two-way ANOVA followed by Bonferroni post-hoc tests. P<0.05 (N = 4–6).
Fig 2.
Representative histology of lung sections from different treatment groups.
Scale bar, 2 mm.
Fig 3.
Representative in situ hybridization results on lung sections.
In situ hybridization using a mouse G-CSF anti-sense RNA probe detected G-CSF production locally in lungs of animals treated with vehicle (CON), dexamethasone (DEX, 2.5 mg/kg, i.p., 18 h), LPS (1 mg/kg, i.t., 24 h), and LPS+DEX (6 h post LPS). Cell types with positive signals include smooth muscle cells, epithelial cells, and infiltrated leukocytes (arrows). Control hybridization slides were processed using the sense strand of the RNA probe. Scale bar, 1 mm.
Fig 4.
DEX and proinflammatory molecules synergistically induced G-CSF.
(A) Time course experiment determining IL-1β (10 ng/ml) ± DEX (100 nM) induction of G-CSF mRNA in the immortalized human bronchial epithelial cell line BEAS 2B. Cells were treated in cell culture media supplemented with charcoal-stripped FBS that was devoid of endogenous glucocorticoids. Levels of mRNAs were quantified using realtime RT-PCR and normalized to the level of the housekeeping gene RPLP0. ✽, significantly different, two-way ANOVA followed by Bonferroni post-hoc tests. P<0.05 (N = 6–10). (B) Time course experiment as in (A) using TNF (1 ng/ml) ± DEX (100 nM). ✽, significantly different, two-way ANOVA followed by Bonferroni post-hoc tests. P<0.05 (N = 6–10). (C) DEX (100 nM, 48 h) enhanced IL-1β (3, 10, 30, and 100 ng/ml, 48 h) induction of G-CSF protein in BEAS 2B cell culture supernatant, which was confirmed using various doses of DEX (1, 10, 100, and 1000 nM) with 100 ng/ml IL-1β. ✽, significantly different from the groups indicated by the arrow, one-way ANOVA followed by Newman-Keuls post-hoc tests. P<0.05 (N = 3). (D) Results from dose-response experiments as in (C) using DEX (100 nM) and TNF (0.3, 1, 3, and 10 ng/ml). When using various doses of DEX, TNF was 10 ng/ml. ✽, significantly different from the groups indicated by the arrow, one-way ANOVA followed by Newman-Keuls post-hoc tests. P<0.05 (N = 3).
Fig 5.
G-CSF induced by glucocorticoid and IL-1β prevents neutrophil apoptosis.
(A) A representative dot plot of flow cytometric analysis of human neutrophil spontaneous apoptosis 20 h post harvest. Live (annexin V-DAPI-), early apoptotic (annexin-V+DAPI-), and late apoptotic (annexin V+DAPI+) cells were over 97% of the total cells. Necrotic cells (annexin V-DAPI+) were negligible. (B). BEAS 2B cells expressing control shRNA (shCON), but not those expressing shRNA for hG-CSF (shG-CSF), protected human neutrophils from spontaneous apoptosis in coculture experiments. BEAS 2B cells or culture media were incubated with vehicle (CON), DEX (10 nM), IL-1β (0.3 ng/ml), or IL-1β+DEX for 24 h before the addition of neutrophils, which were cultured for another 20 h. (C). G-CSF levels in the cell culture media from (B). ✽, significantly different, two-way ANOVA followed by Bonferroni post-hoc tests. P<0.05 (N = 5). The comparisons shown are between the IL-1β+DEX treated cultures indicated.