Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages

Abstract

Background: 2-Chloroethyl ethyl sulphide (CEES) is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD). Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS) enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO) production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS) activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing.

Results: We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours) in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA) or dichlorofluorescin diacetate (DCFH-DA). Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity

Conclusion: CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-kappaB) signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS-stimulated macrophages could elevate oxidative stress. Since macrophage generated NO is known to play a key role in cutaneous wound healing, it is possible that this work has physiological relevance with respect to the healing of HD induced skin blisters.

Figure 1

CEES inhibits NO production and iNOS expression in LPS stimulated RAW264.7 macrophages. Panel A: Macrophages were simultaneously treated with various levels of CEES (as indicated) and low doses of LPS (as indicated). NO production was monitored as the concentration of nitrite in the culture medium after 24 h. Panel B: Cells were treated similarly as for Panel A; LPS, 10 ng/ml; CEES, 100, 200, or 300 μM (as indicated). Means not sharing a common letter are significantly different (p < 0.05). Nitrite levels in the culture medium (OD at 532 nm) were normalized to the amount of viable cells (OD of the MTT product at 580 nm). Panel C: Western blot analysis of iNOS protein from cells simultaneously incubated with 300 μM CEES and/or 10 ng/ml LPS for 24 h; cell lysates were prepared as described in Materials and Methods: Con, control cells; Pos, iNOS protein for positive control; Veh, vehicle; L, 10 ng/ml LPS stimulated cells; C, 300 μM CEES treated cells; L+C, LPS/CEES treated cells.

Figure 2

Time course of NO production and iNOS expression in LPS stimulated RAW264.7 macrophages incubated with CEES. Panel A: Macrophages were incubated with 10 ng/ml LPS alone, 300 μM CEES alone or simultaneously with both 300 μM CEES 10 ng/ml LPS for various time intervals (as indicated). NO production measured as concentration of nitrite in culture medium. Panel B: Western blot analysis of iNOS protein from the cells incubated with 300 μM CEES with or without 10 ng/ml LPS; cell lysates were prepared after 3, 6, 12, or 24 hour incubation (as indicated) as described in Materials and Methods; L, LPS; C, CEES.

Figure 3

CEES reduces intracellular NO in LPS stimulated RAW264.7 macrophages. Panel A: Intracellular DCFH (20 μM) oxidation in LPS stimulated macrophages (as indicated) incubated for 2 h. Fluorescence (excitation 485 nm, emission 520 nm) was measured in Relative Fluorescence Units (RFU); the oxidation rate was expressed as RFU/min. Panel B: Macrophages stimulated with 20 ng/ml LPS, were incubated in the presence or absence of 500 μM CEES (as indicated) for 24 h. Post, CEES was applied after the 24 hours of LPS stimulation; Sim, CEES was applied simultaneously with LPS. Panel C: LPS stimulated cells were incubated in the presence or absence of 25 μM ebselen, a selective iNOS inhibitor (as indicated). 10, 10 ng/ml LPS; 20, 20 ng/ml LPS. Mean values not sharing a common letter are significantly different (p < 0.05).