Abstract
Context:
By promoting the recirculation of tissue fluid, the lymphatic system preserves tissue health, aids in the absorption of gastrointestinal lipids, and supports immune surveillance. Failure of the lymphatic system has been implicated in the pathogenesis of several infectious and inflammatory diseases. Thus, interventions that enhance lymphatic circulation, such as osteopathic lymphatic pump treatment (LPT), should aid in the management of these diseases.
Objective:
To determine whether thoracic duct lymph (TDL) mobilized during LPT would alter the function of macrophages in vitro.
Methods:
The thoracic ducts of 6 mongrel dogs were cannulated, and TDL samples were collected before (baseline), during, and 10 minutes after LPT. Thoracic duct lymph flow was measured, and TDL samples were analyzed for protein concentration. To measure the effect of TDL on macrophage activity, RAW 264.7 macrophages were cultured for 1 hour to acclimate. After 1 hour, cell-free TDL collected at baseline, during LPT, and after TDL was added at 5% total volume per well and co-cultured with or without 500 ng per well of lipopolysaccharide (LPS) for 24 hours. As a control for the addition of 5% TDL, macrophages were cultured with phosphate-buffered saline (PBS) at 5% total volume per well and co-cultured with or without 500 ng per well of LPS for 24 hours. After culture, cell-free supernatants were assayed for nitrite (NO2−), tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10). Macrophage viability was measured using flow cytometry.
Results:
Lymphatic pump treatment significantly increased TDL flow and the flux of protein in TDL (P<.001). After culture, macrophage viability was approximately 90%. During activation with LPS, baseline TDL, TDL during LPT, and TDL after LPT significantly decreased the production of NO2−, TNF-α, and IL-10 by macrophages (P<.05). However, no significant differences were found in viability or the production of NO2−, TNF-α, or IL-10 between macrophages cultured with LPS plus TDL taken before, during, and after LPT (P>.05).
Conclusion:
The redistribution of protective lymph during LPT may provide scientific rationale for the clinical use of LPT to reduce inflammation and manage edema.
The lymphatic system maintains tissue fluid homeostasis by returning excess interstitial fluid to the blood circulation. Lymph can contain apoptotic or necrotic cells, immune cells, soluble antigens, microbes, toxins, proteins, and lipids. By promoting the recirculation of lymph, the lymphatic system preserves tissue health, aids in the absorption of gastrointestinal lipids, and promotes immune surveillance. Failure of the lymphatic system has been implicated in the pathogenesis of cardiovascular disease, inflammation, and edema.
1,2 Therefore, interventions that promote lymphatic circulation should promote tissue health and aid in the management of infectious and inflammatory diseases.
Since its inception, the osteopathic medical profession has emphasized the importance of the lymphatic system in maintaining health. Andrew Taylor Still, MD, DO, asserted that stimulating lymph flow would facilitate the removal of blood cells, particulate matter, exudates, toxins, and bacteria that may adversely affect cellular activity and predispose tissue to dysfunction and disease.
3 Many osteopathic manipulative treatment techniques were designed to promote lymph circulation.
4-6 Lymphatic pump treatment (LPT) is used to manage congestive heart failure, upper and lower gastrointestinal tract dysfunction, respiratory tract disease, infection, and edema.
4 Although the mechanisms of action of LPT are still under investigation, it has been proposed that LPT can improve health by promoting circulation, stimulating immunity, and enhancing the efficacy of vaccines and medications.
6
Previous studies
7-10 have demonstrated that abdominal LPT can significantly enhance thoracic and mesenteric lymphatic flow and the concentration of leukocytes in lymph in dogs. Additionally, abdominal LPT can significantly increase the lymphatic flux of inflammatory cytokines, chemokines, and reactive oxygen and nitrogen species in thoracic and mesenteric lymph.
9,10 Collectively, results of these studies
7-10 suggest that abdominal LPT can enhance the lymphatic redistribution of cells and inflammatory mediators, which may protect against a variety of infectious and inflammatory diseases.
Lymph has been reported to suppress inflammation,
11-13 blunt the pulmonary inflammatory response to endotoxins,
12 inhibit neutrophil apoptosis in vitro
,11,12 increase endothelial cell permeability,
14,15 and redistribute leukocytes, cytokines, and chemokines to distant organs.
15 However, the effect of lymph on macrophage function has not been described. Macrophages reside in the tissue and alert the immune system to tissue injury, infection, and inflammation. During infection, macrophages phagocytose microorganisms; release antibacterial factors, such as nitric oxide; and produce inflammatory cytokines and chemokines, such as tumor necrosis factor α (TNF-α), which recruit monocytes and neutrophils to the site of infection.
16 Macrophages also aid wound healing and tissue repair by producing anti-inflammatory cytokines and growth factors, such as interleukin 10 (IL-10), that dampen leukocyte activity and prevent immunopathologic reactions.
16
The purpose of the current study was to determine whether the lymph mobilized during abdominal LPT would suppress macrophage activity in vitro. Specifically, we hypothesized that thoracic duct lymph (TDL) collected during LPT would suppress macrophage activation in vitro. If distant organs are inflamed, promoting lymph output using treatments such as LPT may redistribute lymph-borne factors to the affected tissue and enhance protection against disease.
The dogs were fasted overnight and anesthetized with intravenous sodium pentobarbital (30 mg/kg) before the surgical procedure. After endotracheal intubation, the dogs were ventilated with room air and supplemented with oxygen to maintain normal arterial blood gases. Arterial blood pressure was monitored via a femoral artery pressure monitoring catheter connected to a pressure transducer (Hewlett-Packard Pressure Monitor, 78354A) to ensure that arterial blood pressure remained within normal limits. The chest was opened by a left lateral thoracotomy in the fourth intercostal space, and the thoracic duct was isolated from connective tissue and ligated. Caudal to the ligation, a PE60 catheter (0.76-mm inner diameter; 1.22-mm outer diameter), whose outflow tip was positioned 8 cm below heart level to compensate for the hydraulic resistance of the catheter, was inserted into the duct and secured with a ligature. The TDL was drained at atmospheric pressure through the catheter. Thoracic duct lymph was continuously collected for 4 minutes before LPT (baseline), for 4 minutes during LPT, and for 10 minutes after the cessation of LPT.