TNF-α plus IL-5 enhanced CD40 expression on eosinophils depending on the activation of the p38 MAPK pathway
Finally, we investigated the possible inflammatory mediators involved in enhanced CD40 and ICOSL expression on eosinophils in patients with CRS. As CRS is characterized by the increased local tissue levels of TNF-α and IL-5, especially in ECRS33-35. Previous studies have reported that TNF-α induces the expression of CD40 on epithelial and endothelial cells as well as the expression of ICOSL expression on fibroblasts, endothelial cells and B cells, monocytes36-40. IL-5 is the most potent activator of eosinophils41-43. Thus, we investigated the effect of TNF-α and IL-5 on CD40 and ICOSL expression on human eosinophils. At the baseline, purified eosinophils from healthy human peripheral blood (purity>95%) have no CD40 or ICOSL expression (Supplementary Fig. 2B). As shown in Fig. 8, the expression of CD40 was markedly upregulated on eosinophils after rTNF-ɑ (50ng/mL) stimulation (P=0.0014) for 24h, not rIL-5 (50ng/mL). Furthermore, TNF-α plus IL-5 further markedly enhanced CD40 expression on eosinophils compared with TNF-α incubation (p<0.0001). However, no time-dependent effect of TNF-α or TNF-α plus IL-5 induced CD40 expression on eosinophils were observed. Whereas, TNF-α alone or TNF-α combined with IL-5 has no significant effect on ICOSL expression on eosinophils (Supplementary Fig. 2C-D).
Activation of p38 MAPK has been shown to partly mediated TNF-α-induced anti-apoptotic signals in human eosinophils44. Finally, we sought to determine whether p38 MAPK pathway mediates the up-expression of CD40 here. Purified eosinophils were treated with the specific p38 MAPK inhibitor SB203580 before TNF-ɑ plus IL-5 stimulation. We found that SB203580 highly suppressed the TNF-ɑ+IL-5-induced CD40 expression on eosinophils (p<0.0001, Fig. 8).