1-Methylnicotinamide and nicotinamide: two related anti-inflammatory agents that differentially affect the functions of activated macrophages

Abstract

Introduction: 1-Methylnicotinamide (MNA), a major metabolite of nicotinamide (NA), is known to exert anti-inflammatory effects in vivo. Treatment of inflammatory skin diseases by topical application of MNA provides certain advantages over the use of NA. However, in contrast to NA, the molecular mechanisms of the anti-inflammatory properties of MNA are not well known. In this study the influence of exogenous MNA and NA in vivo on the generation of inflammatory mediators by macrophages (Mvarphi) was investigated.

Materials and methods: Peritoneal Mvarphi of CBA/J mice were activated in vitro with lipopolysaccharide and incubated with MNA or NA. The effect of these compounds on biological functions of Mvarphi was measured by evaluation of the production of reactive oxygen species (ROS) by luminol-dependent chemiluminescence, cytokines and prostaglandin E(2) (PGE(2)) by ELISA, and nitric oxide (NO) by the Griess method. Moreover, the expressions of inducible NO synthase and cyclooxygenase-2 were measured by Western blotting.

Results: It was shown that at non-cytotoxic concentrations, NA inhibits the production of a variety of pro-inflammatory agents, such as tumor necrosis factor alpha, interleukin 6, NO, PGE(2), and the generation of ROS. In contrast to NA, exogenous MNA inhibited only the generation of ROS, while its effect on the synthesis of other mediators was negligible.

Conclusions: These results indicate that the anti-inflammatory properties of MNA demonstrated previously in vivo do not depend on its capacity to suppress the functions of immune cells, but more likely may be related to its action on vascular endothelium. The authors suggest that the limited permeability for exogenous MNA, in contrast to that for NA, may be responsible for its lack of suppressor activity against Mvarphi.

Fig. 1

Chemical structure of NA and MNA.

Fig. 2

Scavenging properties of MNA and NA. Mϕ (1×10⁶ cells/well) in Hank’s solution were preincubated (60 min at 37°C in an atmosphere of 5% CO₂) on 96-well flat-bottom black plate with luminol sodium salt (0.4 mg/ml) and different concentrations of (A) MNA and (B) NA (1–30 mM). After incubation the cells were stimulated with opsonized zymosan (0.4 mg/ml) and photon emission was measured. Results are expressed as the percentage of ROS production by OZ-stimulated Mϕ ±SEM diminished by that of non-stimulated cells. Each bar represents the mean (±SEM) of three individual experiments. *p<0.05.

Fig. 3

Effect of MNA and NA on the production of cytokines by LPS-stimulated Mϕ. Cells stimulated with LPS (100 ng/ml) were exposed to MNA and NA in the culture medium for 24 h and then the content of cytokines in the medium was measured by ELISA. A — TNF-α, B — IL-6, C — IL-12p40, D — IL-10. Results are expressed as the percentage of the cytokine production by control Mϕ ±SEM. Data were calculated 5–8 separate experiments. *p<0.05.

Fig. 4

Effect of MNA and NA on the production of NO and iNOS expression by LPS plus IFN-γ-stimulated Mϕ. Cells stimulated with LPS (100 ng/ml) and IFN-γ (50 U/ml) were exposed to MNA and NA in the culture medium for 24 h and then both the medium and the cells were collected for further assay. A — the release of nitrites (NO₂⁻); the results are expressed as the percentage of the (NO₂⁻) production by control Mϕ ±SEM. Data were calculated from three separate experiments. B — the expression of iNOS; the data are normalized to constitutively expressed β-actin protein ±SEM. Densitometric analysis of bands from two independent experiments. *p<0.05.

Fig. 5

Effect of MNA and NA on the production of PGE2 and COX-2 expression by LPS-stimulated macrophages. Cells stimulated with LPS (100 ng/ml) were exposed to MNA and NA in the culture medium for 24 h and then both the medium and the cells were collected for further assays. A — the release of PGE₂; results are expressed as the percentage of the PGE₂ production by control Mϕ ±SEM. Data were calculated from two separate experiments. B — the expression of COX-2; the data are normalized to constitutively expressed β-actin protein ±SEM. Densitometric analysis of bands from two independent experiments. *p<0.05.