Figure 7. p21 regulates hyporesponsiveness of human monocytes from healthy donors and sepsis patients.

(A) Total RNA was extracted from monocytes isolated from sepsis patients and healthy volunteers and analyzed by RT-PCR. Top, P21 mRNA levels were significantly higher in monocytes from sepsis patients than those from controls. The median is shown (n = 7), *P < 0.05, 2-tailed Mann-Whitney U test. Bottom, Correlation between P21 and Ifnb levels in monocytes from sepsis patients. Results were normalized to β-actin and log2 values were used to calculate correlation; n = 7, 2-tailed Pearson’s test r = –0.7944, *P < 0.05. (B) Monocytes from healthy volunteers and sepsis patients were challenged ex vivo with 10 ng/ml LPS (1 hour). RT-PCR analysis showed Ifnb upregulation in monocytes from healthy donors but not from sepsis patients (top). High P21 expression was detected in monocytes from sepsis patients before and after LPS treatment (bottom); (n = 5), 1-tailed Student’s t test. NS, not significant. (C) Cultured human monocytes from healthy volunteers were tolerized with LPS, washed, and restimulated with LPS. RT-PCR analysis showed high P21 expression in tolerant human monocytes (top). Ifnb expression was downregulated in tolerant cells; (n = 4), 1-way ANOVA. (D) Cultured human monocytes from healthy volunteers were transfected with p21 siRNA or control siRNA. Cells were then LPS tolerized and rechallenged with LPS. After LPS rechallenge, RT-PCR showed that P21 mRNA was abolished in cultures transfected with P21-specific siRNA, and Ifnb expression was upregulated compared with control siRNA–transfected cells; (n = 4), 2-tailed Student’s t test. In all RT-PCR experiments, results were normalized to β-actin. Data shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.