Poster Session 1 - R9
1,2Tali Farine, 2,3Oksana Shynlova, 1,2,3,4Stephen Lye
1 Department of Physiology, University of Toronto, ON, Canada; 2 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, ON, Canada; 3 Department of Obstetrics, University of Toronto, ON, Canada; 4 Department of Gynecology, University of Toronto, ON, Canada
Objectives: During human labour, infiltration of peripheral maternal leukocytes into the uterine tissues is required for labour induction and a successful post-partum uterine involution. In this study we examined the differences between labouring and non-labouring term decidual primary cells with respect to their (1) cytokine/chemokine secretion profiles, (2) effect on endothelial cell activation, and (3) effect on maternal peripheral leukocyte activation and migratory abilities.
Methods: This study was approved by MSH REB and all women participants provided an informed consent. Decidua samples were collected from the fetal membranes of placentae obtained from elective caesarean section of term not in labour (TNL) or term labour (TL) deliveries. Tissue underwent enzymatic digestion to obtain primary cells. To generate decidual conditioned media (DCM), cells were cultured in serum-free media for 48 hours. Simultaneous detection of multiple cytokines was achieved using the 45-plex human chemokine Luminex assay. Flow cytometric analysis of peripheral maternal blood treated with TNL DCM and TL DCM was performed to determine activation of different leukocyte sub-types (monocytes, lymphocytes and granulocytes). Fluorophore-conjugated antibodies targeting leukocyte sub-types and activation markers CD11b, CD44 and CD55 were used. Aortic endothelial cells were treated with TNL DCM and TL DCM and endothelial activation was assessed by flow cytometry using fluorophore-conjugated antibodies specific for cell adhesion molecules (CAMs) ICAM1, VCAM1, PECAM1 and E-selectin. To determine the effect of DCM on the migratory capabilities of peripheral maternal leukocytes, a transendothelial migration assay was performed on neutrophils and a migration assay on peripheral blood mononuclear cells (PBMCs) with TNL DCM and TL DCM acting as the chemoattractant in both assays.
Results: Four cytokines (G-CSF, IP-10, MCP-1, IFNg) were significantly upregulated in TL DCM compared to TNL DCM. After stimulation with TL DCM activation marker CD11b shows a trend of increase on monocytes and granulocytes as compared to TNL DCM treatment. Cell adhesion molecules VCAM1 show a significant increase in expression on endothelial cells after stimulation with TL DCM compared to TNL DCM. A significant increase in the number of migrated maternal blood peripheral leukocytes (neutrophils and PBMCs) is found in response to TL DCM compared to TNL DCM.
Conclusion: Four major cytokines were significantly upregulated in media conditioned by primary decidual cells isolated from women in active labour compared to cells isolated from decidua of non-labouring women. Changes in cytokine secretion induced endothelial-leukocyte activation and trans-endothelial leukocyte migration, implicating their role in the inflammatory pathway responsible for the infiltration of maternal leukocytes into the uterine tissues during labour. Further experiments will aim to delineate the specific role that these cytokines play as well as focus on myometrial conditioned media in order to gain a better understanding of the interactions between the myometrium and decidua during labour.