Serotonin Induces Inflammatory Cytokine Production and Regulates Lymphatic Endothelial Cell Function

Abstract

Serotonin or 5-hydroxytryptamine (5-HT) is a neuromodulator, with both neuroendocrine and neurotransmitter functions, which is synthesized in serotonergic neurons in the central nervous system and enterochromaffin cells throughout the gastrointestinal tract. The lymphatics play a central role in inflammatory and immune response and lymphatic endothelial cells (LECs) are active participants in these processes. Several immune cells store or respond to 5-HT thereby implicating it in inflammatory pathways and it is also known as a growth factor for several types of non-tumoral cells. Lymphatic muscle has been shown to be significantly responsive to 5-HT however the effects of 5-HT on modulation of lymphatic endothelial phenotype or inflammatory function is not well understood. In this study, we hypothesized that increased levels of 5-HT modulate LEC morphological and phenotypic behavior and also induces cytokine and chemokine production that contribute to inflammation. LECs were initially evaluated for the expression of specific receptors by real time PCR. HDLECs were treated with 5-HT and its receptor antagonist and the effects of 5-HT on LEC proliferation, migration and tube formation was evaluated. Real time PCR was carried out to evaluate the alterations in specific cytokines, chemokines and growth factors produced by LECs in response to 5-HT treatment. In addition, conditioned medium was collected from LECs treated with 5-HT and/or its receptor inhibitor and subjected to cytokine and chemokine array analysis. Our real time PCR analysis show that LECs show varying basal level expression of the 5-HT receptors (5-HTR1-7) with 5-HTR7 showing the highest level of expression. Serotonin treatment of the LECs significantly induces LEC proliferation and migration shown by XTT and Boyden chamber assays. Further, LECs treated with 5-HT for 24hrs show significant induction of several growth factors, inflammatory and lymphangiogenic molecules including angiogenin, endoglin and IL23 indicating the activation of key inflammatory or lymphangiogenic pathways. In keeping with these findings, our results also show that 5-HT treatment enhances lymphatic tube formation that is abrogated by 5-HTR inhibition. Further, 5-HT also activates the PI3K/AKT and ERK MAPK key lymphangiogenic and growth promoting pathways in the LECs. Taken together, we show that 5-HT alters phenotypic and morphological behavior of LECs and activate several growth factors and signaling molecules that are pro-lymphangiogenic and thus can be a viable therapeutic strategy for modulation of lymphangiogenesis and LEC function during inflammation.