Abstract Title

Thoracic Duct Lymph Suppresses the Inflammatory Response of Macrophages in vitro

Presenter Name

Rudy Castillo

RAD Assignment Number

1400

Abstract

Purpose: The gastrointestinal lymphatic vessels redistribute a large pool of lymph rich in immune cells, inflammatory mediators, and lipids. Recent literature suggests that during gastrointestinal injury, soluble factors released from the mesentery redistribute duct to the lung via the thoracic duct where they initiate inflammation and contribute to multiple organ dysfunction syndrome (MODS). Alternatively, normal mesenteric lymph has been shown to suppress inflammation in vivo and in vitro. Specifically, under inflammatory conditions, normal mesenteric lymph reduced the expression of cell adhesion molecules and myeloperoxidase in pulmonary tissue and reduced expression of cell adhesion molecules on pulmonary endothelial cells. However, the role of normal lymph on phagocyte function remains unknown. Importantly, macrophages have been to shown to contribute to MODS. The aim of this study was to investigate the effect of normal lymph on macrophage function. Specifically, we hypothesized that normal thoracic duct lymph (TDL) would suppress the release of inflammatory mediators by LPS-activated macrophages.

Methods: To test this hypothesis, under anesthesia the thoracic ducts of eight mongrel dogs were cannulated and lymph was collected. The TDL was centrifuged to remove cells and the TDL supernatant was frozen and stored at -80oC. Murine RAW 264.7 macrophages were cultured in vitro with TDL at 0.5, 1, 2, 5 or 10% total volume per well or phosphate-buffered saline at 5 or 10% total volume per well with or without lipopolysaccharide (LPS) for 24 hours at 37°C with 5% CO2. After incubation, cell cultures were centrifuged to remove cells and the supernatants were assayed for nitric oxide (NO) and tumor necrosis factor-alpha (TNF-a) production. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide to distinguish live cells from apoptotic cells.

Results: TDL did not augment the production of NO2-, TNF-a or alter cell viability by macrophages cultured in media alone. However, when macrophages were activated with LPS, TDL suppressed the release of NO and TNF-a. Specifically, the addition of TDL at 5% total volume per well suppressed NO2- production (15±0.6 uM) and TNF-a production (5016±425 pg/mL) compared to LPS. Culture with LPS and/or TDL did not alter cell viability.

Conclusions: Our data suggests that during stimulation with LPS, a biological factor in lymph suppressed the release of inflammatory mediators by macrophages. Furthermore, cell viability was unaltered, suggesting that that TDL altered macrophage function. Future studies will focus on the ability of lymph to suppress the inflammatory response in disease models.

Research Area

Immunology

Presentation Type

Poster

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Thoracic Duct Lymph Suppresses the Inflammatory Response of Macrophages in vitro

Purpose: The gastrointestinal lymphatic vessels redistribute a large pool of lymph rich in immune cells, inflammatory mediators, and lipids. Recent literature suggests that during gastrointestinal injury, soluble factors released from the mesentery redistribute duct to the lung via the thoracic duct where they initiate inflammation and contribute to multiple organ dysfunction syndrome (MODS). Alternatively, normal mesenteric lymph has been shown to suppress inflammation in vivo and in vitro. Specifically, under inflammatory conditions, normal mesenteric lymph reduced the expression of cell adhesion molecules and myeloperoxidase in pulmonary tissue and reduced expression of cell adhesion molecules on pulmonary endothelial cells. However, the role of normal lymph on phagocyte function remains unknown. Importantly, macrophages have been to shown to contribute to MODS. The aim of this study was to investigate the effect of normal lymph on macrophage function. Specifically, we hypothesized that normal thoracic duct lymph (TDL) would suppress the release of inflammatory mediators by LPS-activated macrophages.

Methods: To test this hypothesis, under anesthesia the thoracic ducts of eight mongrel dogs were cannulated and lymph was collected. The TDL was centrifuged to remove cells and the TDL supernatant was frozen and stored at -80oC. Murine RAW 264.7 macrophages were cultured in vitro with TDL at 0.5, 1, 2, 5 or 10% total volume per well or phosphate-buffered saline at 5 or 10% total volume per well with or without lipopolysaccharide (LPS) for 24 hours at 37°C with 5% CO2. After incubation, cell cultures were centrifuged to remove cells and the supernatants were assayed for nitric oxide (NO) and tumor necrosis factor-alpha (TNF-a) production. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide to distinguish live cells from apoptotic cells.

Results: TDL did not augment the production of NO2-, TNF-a or alter cell viability by macrophages cultured in media alone. However, when macrophages were activated with LPS, TDL suppressed the release of NO and TNF-a. Specifically, the addition of TDL at 5% total volume per well suppressed NO2- production (15±0.6 uM) and TNF-a production (5016±425 pg/mL) compared to LPS. Culture with LPS and/or TDL did not alter cell viability.

Conclusions: Our data suggests that during stimulation with LPS, a biological factor in lymph suppressed the release of inflammatory mediators by macrophages. Furthermore, cell viability was unaltered, suggesting that that TDL altered macrophage function. Future studies will focus on the ability of lymph to suppress the inflammatory response in disease models.