Figure 1.
Expression of inflammatory mediators in TI and TII cells.
RT2 Profiler Array for rat inflammatory cytokines and receptors was performed with mRNA from TI and TII cells isolated from control and LPS-injured rats (LPS 10 mg/kg). Results are expressed as fold changes of the various genes as illustrated of control TI cells over control TII cells (Figure 1A) and of LPS-stimulated TI cells over LPS-stimulated TII cells (Figure 1B). The top 10 differentially expressed genes are listed for control TI versus TII cells and the top 12 are listed for LPS-stimulated TI versus TII cells. For each condition, n = 6 and *p<0.05.
Figure 2.
Validating enhanced expression of inflammatory mediators.
A. qPCR assay: IL6r, IL10ra, CCL21, CCR5 and SPP1 expression in TI and TII cells isolated from control (n = 4) and LPS-injured (LPS 10 mg/kg) animals (n = 4) was measured using qPCR employing TaqMan probes and primers. Relative to the expression of the above genes in TII cells of control animals, uninjured TI cells had higher levels of expression of all inflammatory mediators studied. LPS injury increased cytokine transcripts of all genes compared to controls for TI and TII cells, but the only in the case of IL10ra and CCL21 in injured TI cells was the fold-increase significant over uninjured TI cells (*p<0.05). Data is expressed as fold expression of inflammatory mediator transcript over TII cell controls ± SEM; all values were normalized to 18S. B. Western blot analysis: Western blots of cell lysates from TI and TII cells of control and LPS-injured (LPS 10 mg/kg) rats were probed with antibodies against CCR5 and SPP1. β-actin staining is shown to exhibit equalization of protein loading. Blots represent 3 separate experiments. Densitometry measurements suggest that control TI cells contain ∼20% more SPP1 than control TII cells, and injured TI cells contain ∼30% more SPP1 protein than control TI cells. Both differences are statistically significant. Furthermore, SPP1 protein expression from injured TI cells was significantly greater than from control TI cells. Control TI cells contain only ∼5% more CCR5 than control TII cells and injured TI cells express ∼15% more CCR5 than injured TII cells, but the differences were not statistically significant. (*p<0.05).
Figure 3.
Cytokine response in cultured TI and TII cells stimulated with LPS.
TNF-α, IL-6 and IL-1β were detected in the supernatants of cultured TI and TII cells isolated via FACS both at baseline and after in vitro LPS (10 µg/ml) stimulation. TNF-α, IL-6 and IL-1β production in LPS-stimulated cells was significantly increased over baseline for TI cells (n = 5 for each cell type, *p<0.05). Levels of IL-6 in LPS-stimulated TII cells were significantly greater than in control TII cells. Results are expressed as mean level of cytokine ± SEM; *p <0.05 for control TI vs. LPS-treated TI cells, **p<0.05 for TII control vs. LPS-treated TII cells.
Figure 4.
A. Cytokine expression in non-FACS TII cells is greater than in FACS TII cells. TNF-α and IL-6 levels were higher in TII cells isolated in the conventional manner (non-FACS) than in FACS-isolated TII cells after treatment with LPS (10 µg/ml). TNF-α levels in FACS TII cells were 97% lower than in non-FACS TII cells (n = 7, *p<0.05). Similarly, IL-6 levels in FACS TII cells were 77% lower than in non-FACS TII cells (n = 6, *p<0.05). Results are expressed as mean level of cytokine ± SEM. B. Immunohistochemistry of FACS TII cells. A cytocentrifuged preparation of FACS TII cells was double-stained with a marker for TII cells, RTII70 (green), and a marker for TI cells, RTI40 (red). The white arrow points to the lone TI cell in the field. The corresponding phase contrast image is shown. Magnification is 20×.
Figure 5.
Cytokine production in LPS-stimulated macrophages and alveolar epithelial cells.
TNF-α (Figure 5A) or IL-6 (Figure 5B) cytokine expression in TI cells co-cultured with progressively higher concentrations of macrophages significantly increased TNF-α and IL-6 cytokine production in a dose-dependent manner after stimulation with LPS (10 µg/ml) (n = 4 for each set of experiments). Addition of macrophages to TII cells did not significantly alter TNF-α or IL-6 production from results obtained examining LPS-treated macrophages alone, except in TII cells co-cultured with 20% macrophages, where IL-6 levels were significantly lower. Results are expressed as mean level of cytokine in pg/ml ± SEM, *p<0.05.
Figure 6.
Effect of conditioned media on TI cell and macrophage cytokine production.
Conditioned media from LPS-stimulated macrophages (Mac CM, n = 4, LPS 10 µg/ml) significantly increased TI cell TNF-α production but decreased IL-6 production when compared to TI cells stimulated with LPS alone (n = 6, *p<0.05). Addition of conditioned media from LPS-stimulated TI cells (TI CM, n = 4, LPS 10 µg/ml) to macrophages increased both TNF-α and IL-6 expression compared to macrophages stimulated with LPS alone (*p<0.05 for TNF-α). Results are expressed as mean level of cytokine in pg/ml ± SEM.
Figure 7.
Effect of surfactant on LPS-stimulated cytokine response in TI cells.
Surfactant significantly decreased LPS-induced IL-6 production in TI cells by 43% with 5 µg/ml surfactant (n = 4) and 35% with 10 µg/ml surfactant (n = 3) when compared to IL-6 production in TI cells stimulated with LPS alone (LPS 10 µg/ml, n = 4, *p<0.05). Surfactant also decreased the level of TNF-α production in TI cells stimulated with LPS by ∼11% at both concentrations, but the reduction was not significant. Surfactant alone did not alter TNF-α or IL-6 expression in the absence of LPS (n = 3).
Figure 8.
LPS may activate cytokines in TI cells via the NF-kB/IκB pathway.
A. LPS stimulates IKKα/β expression in TI cells. Cytocentrifuged mixed cell preparations from LPS-treated (10 µg/ml) TI cells were double-stained with an antibody to phospho-IKKα/β and RTI40, a TI cell marker. A, D, G) Mixed lung cell preparation stained with phospho-IKKα/β at various timepoints after LPS stimulation, demonstrating decreasing staining intensity of phospho-IKKα/β over time; B, E, H) cells stained with RTI40 to display TI cells; C, F, I) merged images demonstrating colocalization of phospho-IKKα/β and a TI cell-specific marker. J) Control image showing lack of staining in the absence of a specific primary antibody; K) phase contrast image of the control slide. Image magnification is 40×. B. LPS induces NF-κB transcript in cultured TI cells. Expression of NF-κB transcript in cultured TI cells increased after LPS stimulation (LPS 10 µg/ml). Relative to the amount of transcript found in TI cells at baseline (n = 4), LPS injury increased NF-κB levels by 9-fold in TI cells (n = 4, *p<0.05). LPS also increased NF-κB transcripts 8-fold in TII cells (n = 5) over TII controls (n = 5, ** p<0.05). Data is expressed as fold expression of NF-κB mRNA over control ± SEM and all values were normalized to 18S.