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

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.