Recent studies have indicated that mast cells play an intermediate role in chemokine-induced neutrophil recruitment in vivo. The aim of the present investigation was to determine the role of tumour necrosis factor-alpha (TNF-alpha) in neutrophil recruitment provoked by the CXC chemokine macrophage inflammatory protein-2 (MIP-2). For this purpose, we used mast cell- and TNF-alpha-deficient mice and studied neutrophil adhesion to endothelial cells in vitro and neutrophil recruitment in the mouse cremaster muscle in vivo. In contrast to the classical chemoattractant formyl-methionine-leucine-phenylalanin (fMLP), MIP-2 dose dependently increased neutrophil accumulation in vivo. This MIP-2-regulated neutrophil recruitment was abolished in mast cell-deficient mice. TNF-alpha increased E-selectin mRNA expression in both wild-type (WT) and mast cell-deficient mice. In contrast, MIP-2 challenge increased gene expression of E-selectin in WT but not in mast cell-deficient animals. Moreover, MIP-2-provoked extravascular accumulation of neutrophils was reduced by 78% in mice lacking TNF-alpha. In order to better define the role of mast cell-derived TNF-alpha in neutrophil responses to MIP-2, we used an in vitro endothelial cell adhesion assay with and without mast cells. Interestingly, MIP-2-induced neutrophil adhesion to endothelial cells was decreased by 58% using TNF-alpha-deficient compared to WT mast cells. Moreover, mast cell secretion of TNF-alpha increased by more than 71% in response to challenge with MIP-2. Taken together, our results suggest that MIP-2-induced neutrophil recruitment is mediated by TNF-alpha released from local mast cells. These findings help to explain the complex molecular interactions between chemokines, mast cell activation and neutrophil infiltration in vivo.