Carbon monoxide suppresses bleomycin-induced lung fibrosis

Abstract

Idiopathic pulmonary fibrosis is an incurable fibrosing disorder that progresses relentlessly to respiratory failure. We hypothesized that a product of heme oxygenase activity, carbon monoxide (CO), may have anti-fibrotic effects. To test this hypothesis, mice treated with intratracheal bleomycin were exposed to low-concentration inhaled CO or ambient air. Lungs of mice treated with CO had significantly lower hydroxyproline accumulation than controls. Fibroblast proliferation, thought to play a central role in the progression of fibrosis, was suppressed by in vitro exposure to CO. CO caused increased cellular levels of p21(Cip1) and decreased levels of cyclins A and D. This effect was independent of the observed suppression of MAPK's phosphorylation by CO but was dependent on increased cGMP levels. Further, CO-exposed cells elaborated significantly less fibronectin and collagen-1 than control cells. This same effect was seen in vivo. Suppression of collagen-1 production did not depend on MAPK or guanylate cyclase signaling pathways but did depend on the transcriptional regulator Id1. Taken together, these data suggest that CO exerts an anti-fibrotic effect in the lung, and this effect may be due to suppression of fibroblast proliferation and/or suppression of matrix deposition by fibroblasts.

Figure 1

Low concentration of inhaled CO inhibits hydroxyproline deposition in the lungs of bleomycin-treated mice. A: Mice were treated with bleomycin and subsequently exposed to either room air (n = 8) or continuous inhaled CO at a concentration of 250 ppm (n = 6) for a period of 14 days. Lungs were subsequently harvested and analyzed for hydroxyproline content. Mice exposed to continuous CO had significantly lower lung hydroxyproline content than controls (P = 0.001 by analysis of variance; *, P = 0.001 versus saline using Bonferroni’s correction; #, P = 0.025 versus control using Bonferroni’s correction). Lungs of mice exposed to intratracheal saline and CO (n = 3) had equivalent hydroxyproline content to intratracheal saline controls (n = 6). B: Mice were treated with bleomycin and subsequently exposed to either room air (n = 8) or 3 hours per day inhaled CO at a concentration of 250 ppm (n = 6) for a period of 14 days. Lungs were subsequently harvested and analyzed for hydroxyproline content. Mice exposed to 3 hours per day CO had significantly lower lung hydroxyproline content than controls (P < 0.0001 by analysis of variance; *, P < 0.0001 versus saline using Bonferroni’s correction; #, P = 0.001 versus control using Bonferroni’s correction).

Figure 2

Histology of bleomycin-treated mouse lungs and Western blot for matrix components. Lungs were harvested from mice treated with bleomycin alone (A: A and B) or bleomycin with CO 250 ppm (A: C to F); H&E stainings of representative sections are shown. Although variability in degree of injury was observed in both groups, animals treated with CO had less injury on average than animals treated with bleomycin alone as assessed by an independent blinded observer. B: A summary of histology scores assigned to experimental groups is represented. Details of the scoring method are provided in the Materials and Methods section. Animals receiving bleomycin and CO had significantly lower fibrotic/reparative scores than animals receiving bleomycin alone. C: A summary of the distribution of histological scores [from 0 = no fibrosis to 4 = interstitial fibrosis affecting 76 to 100% of the high-power field (HPF)] is shown. Black bars represent bleomycin alone and gray bars represent bleomycin with CO treatment. There is a range of injury and repair in both experimental groups but the CO-treated animals are less severely affected overall. D: Western blot analysis of fibronectin and collagen-1 content of whole mouse lung exposed to bleomycin alone (Bleo) or bleomycin with CO 250 ppm (Bleo/CO). Each lane represents protein from the lung of a single animal. This blot is representative of n = 6 animals analyzed in each group. The densitometry shown in E and F quantitates the blot shown (#, P < 0.05 versus saline; #, P < 0.05 versus Bleo). Original magnifications: ×4 (A, left column); ×20 (A, right column).

Figure 3

CO suppresses fibroblast proliferation in vitro. Cultured human fetal lung fibroblasts (MRC-5) were exposed to CO (250 ppm) or control incubator air for 1 week and cells were counted daily. A: Cells exposed to CO exhibited a significantly slower rate of growth than their control counterparts (*, P < 0.005 compared with nonexposed control). B: Flow cytometry of serum-stimulated fibroblasts. Cells exposed to CO-containing atmosphere are less likely to proceed to S and G₂/M phase after serum stimulation than are control cells.

Figure 4

CO exposure augments p21^Cipl expression and suppresses cyclins A and D in vitro. Serum-starved MRC-5 fibroblasts were exposed to CO (250 ppm) or control incubator air (time 0) and subsequently serum stimulated. A: Cells were replaced in either CO-containing atmosphere or control atmosphere and harvested at 8 and 24 hours for analysis by Western blotting. Expression of cyclins A and D was decreased after exposure to CO, whereas expression of p21^Cipl was increased. B: Densitometry for the same experiment and represents an average of two to three blots for each time point. Values were normalized to the RA control for each blot to adjust for overall film lightness (*, P < 0.05 compared with RA control for same time point).

Figure 5

The role of MAPK signaling in the anti-proliferative effects of CO. Fibroblasts were stimulated with serum and exposed to CO (250 ppm) or control incubator air. A: Cells were subsequently analyzed for MAPK phosphorylation by Western blotting at time points ranging from 5 minutes to 1 hour. B: Densitometry for the same experiment is shown. Bar graphs represent an average of two to three blots for each time point and all values were normalized to RA control for each blot (*, P < 0.05 compared with RA control for same time point). To test whether deletion of select genes contributing to the MAPK pathways would affect fibroblast proliferation, lung fibroblasts were extracted from lungs of Jnk1^−/−, p38β^−/−, and MKK3^−/− mice. Early passage serum-starved fibroblasts were stimulated with serum in the presence or absence of CO. C: Deletion of these select MAPK genes did not affect the anti-proliferative effect of CO on these fibroblasts (*, P < 0.005 compared with corresponding room air control). D: To confirm this finding, the experiment was repeated using chemical inhibitors of the MAPK pathways. The black bars represent ambient air controls and white bars represent CO-exposed fibroblasts. Dimethyl sulfoxide, vehicle control; SB, p38 MAPK inhibitor SB203580; SP, JNK inhibitor SP600125; UO126, MEK1/2 inhibitor UO126; mix, all three inhibitors combined. JNK and ERK inhibition slowed fibroblast proliferation at baseline, but none of the inhibitors abrogated the effect of CO (*, P < 0.05 compared with corresponding room air control).

Figure 6

CO treatment of fibroblasts increases the levels of intracellular cGMP, and inhibition of cGMP abolishes the anti-proliferative effect of CO. A: MRC-5 fibroblasts were exposed to CO (250 ppm) and assessed for changes in intracellular cGMP by enzyme-linked immunosorbent assay at times ranging from 15 minutes to 24 hours. There was a statistically significant increase in cGMP peaking at 1 hour and returning to baseline by 4 hours (*, P < 0.05 compared with room air control). B: Fibroblasts were exposed to CO (250 ppm) for 1 hour in the presence or absence of an inhibitor of guanylate cyclase, ODQ. The rise in intracellular cGMP stimulated by CO was inhibited by ODQ (*, P < 0.05 compared with room air control; #, P < 0.05 compared with CO 1 hour). C: Fibroblasts were again exposed to CO after serum stimulation in the presence of ODQ. The anti-proliferative effect of CO was completely abolished by treatment with ODQ, whereas treatment with vehicle alone (dimethyl sulfoxide) had no effect on the fibroblast response to CO (*, P < 0.05 compared with room air control; #, P < 0.005 compared with room air control). D: Western blotting of fibroblasts similarly exposed to CO (250 ppm) with or without ODQ for a period of 8 hours. The effects of CO on cyclins A and D and p21^Cipl were completely abolished by treatment with ODQ.

Figure 7

CO suppresses TGF-β-induced fibronectin and type I collagen production by fibroblasts. MRC-5 fibroblasts were treated with TGF-β (2 ng/ml) in the presence or absence of CO (250 ppm); protein from cell lysate and supernatant was collected at 2 days, 4 days, and 6 days for analysis of matrix components by Western blotting. A: Intracellular fibronectin was dramatically decreased by exposure to CO at all time points. B: Similarly, there was a time-dependent increase in extracellular collagen type I produced by control cells, but this was inhibited at all time points by exposure to CO. Densitometry for both experiments is shown in C; each graph represents an average of three blots (*, P < 0.05 relative to RA value for same time point). Values were normalized to the RA control for each blot to adjust for overall differences in intensity among films.

Figure 8

Suppression of fibronectin production by CO does not depend on the MAPK pathways or cGMP. A: MRC-5 fibroblasts were treated with chemical inhibitors of the MAPK pathways or cGMP in the presence or absence of CO (250 ppm); protein from cell lysate was collected at 48 hours for analysis of fibronectin production by Western blotting. Intracellular fibronectin was decreased by exposure to CO even in the presence of signaling pathway inhibitors. C, control; SB, p38 MAPK inhibitor SB203580; SP, JNK inhibitor SP600125; UO, MEK1/2 inhibitor UO126; ODQ, guanylate cyclase inhibitor ODQ. B: Mkk3^−/−, p38^−/−, and Jnk1^−/− mouse lung fibroblasts along with their wild-type controls were treated with TGF-β (2 ng/ml) in the presence or absence of CO (250 ppm); protein from cell lysates was collected at 0 and 4 days for Western blot analysis of fibronectin production. Intracellular fibronectin was decreased by exposure to CO in all fibroblast lines.

Figure 9

Suppression of type I collagen production by CO depends on the presence of Id1, but suppression of proliferation by CO is independent of Id1. A: Both Id1^−/− and wild-type mouse lung fibroblasts were treated with TGF-β (2 ng/ml) in the presence or absence of CO (250 ppm); supernatant protein was collected at 0 and 4 days for analysis of collagen-1 production by Western blotting. Collagen-1 was significantly decreased by exposure to CO for 4 days in wild-type fibroblasts; but in Id1^−/− fibroblasts, the effect of CO on collagen-1 production was reversed. Bar graph shows densitometry for the same experiment and represents an average of three blots. Values were normalized to RA control (*, P < 0.05 compared with RA value for same time point). B: Growth curve for Id1^−/− fibroblasts without CO exposure (filled triangles) and exposed to CO (open triangles) and wild-type fibroblasts without CO exposure (filled diamonds) and exposed to CO (open diamonds). The absence of Id1 did not prevent the growth-inhibitory effect of CO. *, P < 0.005 compared with non-CO treated control.