Nitric oxide and tumor necrosis factor-⍺ levels are negatively correlated in endotoxin tolerance recovery in vitro

Abstract

Endotoxin tolerance (ET) is the hyporesponsiveness to lipopolysaccharide (LPS) after prior exposure. It is characterized by the downregulation of pro-inflammatory cytokine levels. Although ET protects against inflammation, its abolishment or recovery is critical for immunity. Nitric oxide (NO) plays various roles in the development of ET; however, its specific role in ET recovery remains unknown. To induce ET, RAW264.7 cells (a murine macrophage cell line) were pre-exposed to LPS (LPS₁, 100 ng/mL for 24 h) and subsequently re-stimulated with LPS (LPS₂, 100 ng/mL for 24 h). Expression of cytokines, NO, nitrite and inducible NO synthase (iNOS) were measured after 0, 12, 24, and 36 h of resting after LPS₁ treatment with or without the iNOS-specific inhibitor, 1400W. LPS₂-induced tumor necrosis factor-⍺ (TNF-⍺) and interleukin-6 (IL-6) were downregulated after LPS₁ treatment, confirming the development of ET. Notably, TNF-⍺ and IL-6 levels spontaneously rebounded after 12–24 h of resting following LPS₁ treatment. In contrast, levles of NO, nitrite and iNOS increased during ET development and decreased during ET recovery. Moreover, 1400W inhibited ET development and blocked the early production of NO (<12 h) during ET recovery. Our findings suggest a negative correlation between iNOS-induced NO and cytokine levels in the abolishment of ET.