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A7269 - Modulation of Airway Smooth Muscle Phenotype Through Physical Contact with CD4+ T-Cells
Author Block: J. H. Jang1, S. Farahnak1, R. Sharan1, A. Wang1, J. G. Martin2; 1Meakins Christie Laboratories, McGill University, Montreal, QC, Canada, 2Research Institute of the MUHC, McGill University, Montreal, QC, Canada.
Background: Remodeling of airway smooth muscle (ASM) is a plausible driver of airway hyperresponsiveness in obstructive airway diseases, such as asthma. Hyperproliferative and/or hypercontractile alterations in the ASM phenotype seem to be features of the remodeling but it is unclear why these alterations take place. CD4+ T-cells are potential mediators of ASM phenotype alterations due to their known interactions with ASM. Th2 cytokines, such as IL-4 and 13, are known to induce airway hyperresponsiveness in mouse models. CD4+ T-cells are also known to physically interact with ASM cells through integrins, MHC-TCR interactions, and membrane nanotubes. In this study, we aim to study the influence that paracrine interactions and physical contact with CD4+ T-cells have on ASM phenotype.
Methods: Human ASM cells were isolated from airway tissues from donors with no history of airway diseases. Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood collected from healthy volunteers after informed consent was given. Jurkat cells, a T lymphocyte cell line, were bought from ATCC. PBMCs and Jurkat cells were activated with PMA and ionomycin for 48 hours and then purified for CD4+ T-cells by magnetic activated cell sorting (MACS) before co-culture. ASM cells were starved in 0.1% FBS in DMEM for 24 hours prior to co-culture. Cells were seeded at a 5:1 ratio (CD4+ T-cells/Jurkat:ASM cells) and cultured for 24 or 48h before harvest. A Transwell™ system was also used to separate the two cell types and study the significance of physical contact in T-cell-ASM interactions. To assess proliferation, the thymidine analogue, bromodeoxyuridine (BrdU), was added 18hours before harvest. Cells were stained using viability dye eflour780, anti-BrdU and anti-CD4 (for T-cell negative selection) antibodies. Incorporation of BrdU was assessed in ASM cells by flow cytometry. mRNA was harvested 24 and 48hours after co-culture and expression of ASM specific contractile genes were measured by qPCR. Cells were loaded with Fura-2 AM to assess calcium responses to histamine by fluorescent imaging.
Results: ASM cells incorporated more BrdU after co-culture with CD4+ T-cells. Transcription of ASM specific contractile genes trended downward after 48hours of co-culture with CD4+ T-cells or Jurkat cells. Peak calcium responses to histamine in ASM cells were also reduced after co-culture with Jurkat cells. These results were not observed in the Transwell™ system.
Conclusions: These results suggest that CD4+ T-cells induce a proliferative phenotype in ASM cells that is dependent on physical contact between the two cell types.