Lymphatic pump technique mobilizes lymph that inhibits the inflammatory response of macrophages in vitro

Purpose: The lymphatic system maintains tissue fluid homeostasis by returning excess fluid, known as lymph, into circulation. Osteopathic physicians recognize the importance of the lymphatic system and have designed a set of lymphatic pump techniques (LPT) that enhance the flow of lymph. LPT has been used clinically to treatment respiratory tract disease, infection, and edema. LPT has also been reported to enhance mesenteric and thoracic lymphatic flow, the concentration of leukocytes, and the flux of inflammatory mediators in lymph of dogs. We propose LPT may act as an adjunctive therapy by mobilizing lymph-borne factors into circulation that protect tissues during inflammation. In this study, we propose that lymph mobilized by LPT suppresses macrophage activation in vitro. Methods: To test this hypothesis, under anesthesia, the thoracic ducts of six mongrel dogs were cannulated and thoracic duct lymph (TDL) was collected before (baseline), during, and after (recovery) LPT. TDL supernatant was collected by centrifugation and frozen. Total protein was measured in TDL using the Bradford method. Murine RAW 264.7 macrophages were cultured with baseline, LPT, or recovery TDL at 5% total volume per well with or without lipopolysaccharide (LPS) for 24 hours at 37°C and 5% CO2. After culture, cell-free supernatants were assayed for nitrite (NO2-), tumor necrosis factor-alpha (TNF-alpha), and interleukin-10 (IL-10). Macrophage viability was measured using flow cytometry and markers, annexin V and propidium iodide. Results: LPT transiently increased TDL flow and protein flux (10-fold). Baseline, LPT, or recovery TDL did not increase the production of NO2-, TNF-alpha, IL-10 or alter macrophage viability. When macrophages were activated with LPS, the addition of baseline, LPT, or recovery TDL decreased the production of NO2- (2-fold), TNF-alpha (5-fold) and IL-10 (3-fold) compared to LPS. There were no significant (p [greater than] 0.05) differences in the production of NO2-, TNF-alpha, and IL-10 by macrophages cultured with baseline, LPT, or recovery TDL and LPS. Conclusions: Our data suggests that lymph contains biological factor(s) that suppress macrophage activation without altering cell viability. The redistribution of protective lymph during LPT may provide scientific rationale for the clinical use of LPT to treat inflammation and edema. Future studies will focus on the mechanism(s) responsible for these novel findings.