Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages

Abstract

The enzyme heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that degrades heme to produce biliverdin/bilirubin, ferrous iron and carbon monoxide (CO). The anti-inflammatory properties of HO-1 are related to inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous CO gas treatment decreases the production of inflammatory mediators such as cytokines and nitric oxide (NO). CO-releasing molecules (CO-RMs) are a novel group of substances identified by our group that are capable of modulating physiological functions via the liberation of CO. We aimed in this study to examine the potential anti-inflammatory characteristics of CORM-2 and CORM-3 in an in vitro model of lipopolysaccharide (LPS)-stimulated murine macrophages. Stimulation of RAW264.7 macrophages with LPS resulted in increased expression of inducible NO synthase (iNOS) and production of nitrite. CORM-2 or CORM-3 (10-100 microM) reduced nitrite generation in a concentration-dependent manner but did not affect the protein levels of iNOS. CORM-3 also decreased nitrite levels when added 3 or 6 h after LPS exposure. CORM-2 or CORM-3 did not cause any evident cytotoxicity and produced an increase in HO-1 expression and heme oxygenase activity; this effect was completely prevented by the thiol donor N-acetylcysteine. CORM-3 also considerably reduced the levels of tumor necrosis factor-alpha, another mediator of the inflammatory response. The inhibitory effects of CORM-2 and CORM-3 were not observed when the inactive compounds, which do not release CO, were coincubated with LPS. These results indicate that CO liberated by CORM-2 and CORM-3 significantly suppresses the inflammatory response elicited by LPS in cultured macrophages and suggest that CO carriers can be used as an effective strategy to modulate inflammation.

Figure 1

Chemical structures of CORM-2 and CORM-3. See Introduction and Methods for detailed information on these two compounds.

Figure 2

Effect of CORM-3 on LPS-stimulated nitrite production and cell viability. (a) RAW264.7 macrophages were exposed to 1 μg ml⁻¹ LPS in the presence or absence of CORM-3 (10–100 μM) and nitrite production was assessed at 24 h. The inactive compound iCORM-3 (100 μM) was also used to determine the contribution of CO released by CORM-3 to the observed effect. Control cells were incubated with medium alone. (b) Cell viability was assessed 24 h after exposure of macrophages to 1 μg ml⁻¹ LPS in the presence or absence of CORM-3 (10–100 μM) or iCORM-3 (100 μM). Viability was expressed as percentage of control. Data represent the mean±s.e.m. of six independent experiments. ^*Indicates P<0.05 vs control; ^†indicates P<0.05 vs LPS alone.

Figure 3

Effect of CORM-2 on LPS-stimulated nitrite production and cell viability. (a) RAW264.7 macrophages were exposed to 1 μg ml⁻¹ LPS in the presence or absence of CORM-2 (10–100 μM) and nitrite production was assessed at 24 h. iCORM-2 (10–100 μM), an inactive compound that does not release CO, was also used as a negative control for CORM-2. Control cells were incubated with medium alone. (b) Cell viability was assessed 24 h after exposure of macrophages to 1 μg ml⁻¹ LPS in the presence or absence of CORM-2 (10–100 μM) or iCORM-2 (10–100 μM). Viability was expressed as percentage of control. Data represent the mean±s.e.m. of six independent experiments. ^*Indicates P<0.05 vs control; ^†indicates P<0.05 vs LPS alone.

Figure 4

Multiple additions of CORM-3 or its delivery after the LPS challenge decrease nitrite production. (a) Nitrite production was measured at 24 h in macrophages incubated with LPS alone or LPS followed 6 h later by a single addition of CORM-3 at 50 or 100 μM. Data represent the mean±s.e.m. of six independent experiments. (b) RAW264.7 macrophages were exposed to 1 μg ml⁻¹ LPS and nitrite production was assessed at 24 h. CORM-3 (10, 50 or 100 μM) was added simultaneously with LPS in all groups. In additional experiments, CORM-3 was added also at 3 h after LPS (0+3 h) or at 3 and 6 h after LPS (0+3+6 h). Data represent the mean±s.e.m. of five independent experiments. ^*Indicates P<0.05 vs LPS alone; ^†indicates P<0.05 vs CORM-3 added simultaneously with LPS (0 h).

Figure 5

Effect of CORM-3 and CORM-2 on heme oxygenase activity. (a) RAW264.7 macrophages were incubated with increasing concentrations of CORM-3 (10–100 μM) or iCORM-3 (100 μM). Heme oxygenase activity was determined 6 h after exposure to the different compounds as described in Methods. (b) Heme oxygenase activity was measured in cells exposed to 100 μM CORM-2 or iCORM-2 for 6 h. Data represent the mean±s.e.m. of six independent experiments. ^*Indicates P<0.05 vs control.

Figure 6

Effect of CORM-3 on HO-1 protein expression. RAW264.7 macrophages were incubated with increasing concentrations of CORM-3 (10–100 μM) or 100 μM iCORM-3 and HO-1 protein levels were determined by Western blot analysis. The graph shows the average expression of HO-1 following densitometric analysis of three different blots from three independent experiments and one representative image is reported. β-Actin was used as an internal control for equal loading. ^*Indicates P<0.05 vs control.

Figure 7

N-acetylcysteine (NAC) abolishes the increase in heme oxygenase activity and HO-1 expression elicited by CORM-3. RAW264.7 macrophages were incubated with increasing concentrations of 100 μM CORM-3 in the presence or absence of 1 mM NAC. Heme oxygenase activity (a) and HO-1 expression (b) were assessed after 6 h incubation. Data represent the mean±s.e.m. of six independent experiments. Western blot image is representative of three different blots. β-Actin was used as an internal control for equal loading. ^*Indicates P<0.05 vs control.

Figure 8

Effect of CORM-3 on LPS-stimulated nitrite production in the presence of SnPPIX, an inhibitor of heme oxygenase activity. RAW264.7 macrophages were exposed to 1 μg ml⁻¹ LPS in the presence or absence of CORM-3 (10–100 μM) and SnPPIX (10 μM) and nitrite production was assessed after 24 h. Control cells were incubated with medium alone. Data represent the mean±s.e.m. of six independent experiments. ^*Indicates P<0.05 vs control; ^†indicates P<0.05 vs LPS alone.

Figure 9

Effect of CORM-3 on LPS-stimulated expression of inducible NO synthase (iNOS). Macrophages were exposed to 1 μg ml⁻¹ LPS in the presence or absence of CORM-3 (10–100 μM) and iNOS protein expression was examined by Western blot analysis. The graph shows the average expression of iNOS protein following densitometric analysis of three different blots from three independent experiments and one representative image is reported. β-Actin was used as an internal control for equal loading. ^*Indicates P<0.05 vs control.

Figure 10

CORM-3 mitigates the production of TNF-α stimulated by LPS. (a) Macrophages were exposed to 1 ng ml⁻¹ LPS in the presence or absence of 10 μM CORM-3 or iCORM-3 and the level of TNF-α in the culture medium was measured at 24 h by a commercially available immunoassay (see Methods for more details). (b) TNF-α production was measured in macrophages stimulated by LPS in the presence or absence of CORM-3 and SnPPIX (10 μM). Data represent the mean±s.e.m. of six independent experiments. ^*Indicates P<0.05 compared to control; ^†indicates P<0.05 vs LPS alone.

Figure 11

Preincubation with hemin increases heme oxygenase activity and reduces LPS-stimulated nitrite production. (a) Macrophages were treated with hemin (10–100 μM) for 6 h and heme oxygenase activity was measured at the end of the incubation as described. (b) Pretreatment of macrophages with hemin (6 h) was followed by exposure to 1 μg ml⁻¹ LPS and nitrite production was assessed after 24 h. Data represent the mean±s.e.m. of six independent experiments. ^*Indicates P<0.05 vs control; ^†indicates P<0.05 vs LPS alone.

Figure 12

Heme oxygenase activity, but not biliverdin, contributes to the reduction in nitrite production elicited by hemin. (a) Macrophages were stimulated with 1 μg ml⁻¹ LPS in the presence or absence of SnPPIX (10 μM) and nitrite production was measured at 24 h. In some experiments, nitrite levels in the medium of macrophages preincubated with 10 μM hemin and SnPPIX prior to exposure to LPS were determined. (b) Nitrite levels were measured in medium of macrophages preincubated with biliverdin (1–20 μM) prior to exposure to LPS. Data represent the mean±s.e.m. of six independent experiments. ^*Indicates P<0.05 vs control; ^†indicates P<0.05 vs LPS alone.