Molecular mechanisms underlying the pro-inflammatory synergistic effect of tumor necrosis factor α and interferon γ in human microvascular endothelium

Tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are among the most potent cytokines involved in orchestrating the inflammation response. The molecular mechanisms implicated in the synergism between cytokines are still poorly characterized. We demonstrate that both cytokines dose-dependently stimulate IFNγ-inducible-protein-of-10-kDa (IP-10) secretion in human microvascular endothelial cells (HMEC-1), showing a potent synergism which is not restricted to IP-10, but is also evident for monokine-induced-by-IFNγ (MIG) and IL-6 secretion. Immunofluorescence analysis reveals that TNFα and IFNγ converge on a rapid phosphorylation of ERK, which however results in a different subcellular compartmentalization of the activated enzyme in response to the two cytokines. Differences in the subcellular recruitment of ERK in response to IFNγ and TNFα are responsible for generating different ERK downstream signaling, which can thus synergize on the secretion of IP-10 as well as of other cytokines/chemokines. The importance of ERK activation in mediating the synergism of the two cytokines is further confirmed by the inhibitory effect of the anti-diabetic drug rosiglitazone and ERK blockers on IP-10, MIG and IL-6 secretion. A further mechanism of synergism involving the reciprocal upregulation of TNFα-RII and of IFNγ-R, in response to IFNγ and TNFα, respectively, was revealed by flow cytometry and quantitative real time RT-PCR analysis.