LPS-induced tolerance up-regulates Siglec-1 expression. Endotoxin tolerance was induced by pretreating RAW 264.7 cells or D2SC/1 cells for 24 h with 100 ng/ml LPS, washed three times with PBS, and then challenged for 16 h with 1 μg/ml LPS. A, evaluation of Siglec expression in non-tolerant or tolerant RAW 264.7 cells by real-time PCR using Siglec-primer sets. B, immunoblot analysis of siglec-1 on tolerant RAW 264.7 cells. Evaluation of Siglec-1 expression in non-tolerant or tolerant D2SC/1 cells (C) or human peripheral blood cells (D) by real-time PCR. Flow cytometric analysis cell surface expression of Siglec-1 on RAW 264.7 cells (E), or D2SC/1 cells (F) after induction of endotoxin tolerance. G, induction of endotoxin tolerance leads to increased and maintained expression of Siglec-1. Expression of Siglec-1 mRNA in non-tolerant and tolerant RAW 264.7 cells was determined by Real-time PCR. H, induction of endotoxin tolerance does not affect the expression of CD14. Expression of CD14 mRNA in non-tolerant and tolerant RAW 264.7 cells was determined by Real-time PCR. All experiments were repeated three times. Data are shown as mean ± S.D.