The tumor necrosis factor-alpha (TNF)-induced inflammatory response in human lung microvascular endothelial cells (MVECs) is an early event in acute lung injury. Studies have shown that p38 mitogen-activated protein kinase (MAPK), MAPK-activated protein kinase 2 (MK2) and heat shock protein 27 (HSP27) are involved in the expression of pro-inflammatory mediators in other cell types. However, their role in the TNF-induced inflammatory response in lung MVECs has not been determined. We evaluated the role of p38 MAPK, MK2 and HSP27 in regulating the TNF-induced expression of ICAM-1 and IL-8 in human lung MVECs. Inhibition of p38 MAPK reduced ICAM-1 and IL-8 expression without influencing NF-kappaB activation or ICAM-1 and IL-8 mRNA levels. TNF stimulation induced p38 MAPK-dependent phosphorylation of MK2 and HSP27. MK2 silencing reduced ICAM-1 and IL-8 expression without influencing NF-kappaB activation or ICAM-1 and IL-8 mRNA levels. HSP27 silencing reduced cellular HSP27 levels and HSP27 phosphorylation following TNF stimulation but had no effect on ICAM-1 and IL-8 expression. Our study demonstrates for the first time that MK2 mediates post-transcriptional regulation by p38 MAPK of the TNF-induced expression of ICAM-1 and IL-8 in human lung MVECs, and that this regulation by the p38 MAPK/MK2 pathway is dissociated from HSP27 phosphorylation.