NF-κB translocation and endothelial cell activation is potentiated by macrophage-released signals co-secreted with TNF-α and IL-1β

Objective and Methods: Over-expression of the immune response can lead to pathological conditions such as septic shock or chronic inflammation. Endothelial cell activation by pro-inflammatory products of activated macrophages plays a key role in these conditions. Here we examine the response of primary human endothelial cells (HUVEC) to conditioned media (CM) obtained from LPS-activated macrophages. We further characterized the translocation of NF-kappaB in the presence of CM by studying the degradation rate of individual IkappaB isoforms. Results: We show that, as expected, CM induced NF-kappaB translocation, as well as adhesion capacity in HUVEC. We further show that this response is critically dependent on TNF-alpha and IL1beta naturally present in the CM. However, both the amplitude of NF-kappaB translocation and adhesiveness observed with CM were well beyond the saturation levels attained after the sole stimulation with recombinant TNF-alpha and IL-1beta, either separately or together. Our results show that CM induced a faster degradation of the IkappaB-beta and IkappaB-epsilon isoforms than the recombinant cytokines, leading to an enhanced recruitment of NF-kappaB activity. Conclusions: The above results suggest that the physiological context of factors co-secreted by LPS-activated macrophages enhances TNF-alpha mediated endothelial activation.