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Original Research
Lymphatic pump manipulation mobilizes inflammatory mediators
into lymphatic circulation
Artur Schander1, H Fred Downey2,3 and Lisa M Hodge1,3
1
Department of Molecular Biology and Immunology;
2
Department of Integrative Physiology;
3
Osteopathic Research Center,
University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA
Corresponding author: Lisa M Hodge. Email: lisa.hodge@unthsc.edu
Abstract
Lymph stasis can result in edema and the accumulation of particulate matter, exudates, toxins and bacteria in tissue
interstitial fluid, leading to inflammation, impaired immune cell trafficking, tissue hypoxia, tissue fibrosis and a variety of
diseases. Previously, we demonstrated that osteopathic lymphatic pump techniques (LPTs) significantly increased thoracic
and intestinal duct lymph flow. The purpose of this study was to determine if LPT would mobilize inflammatory mediators
into the lymphatic circulation. Under anesthesia, thoracic or intestinal lymph of dogs was collected at resting (pre-LPT),
during four minutes of LPT, and for 10 min following LPT (post-LPT), and the lymphatic concentrations of interleukin-2
(IL-2), IL-4, IL-6, IL-10, interferon-
g
, tissue necrosis factor
a
, monocyte chemotactic protein-1 (MCP-1), keratinocyte
chemoattractant, superoxide dismutase (SOD) and nitrotyrosine (NT) were measured. LPT significantly increased MCP-1
concentrations in thoracic duct lymph. Further, LPT increased both thoracic and intestinal duct lymph flux of cytokines
and chemokines as compared with their respective pre-LPT flux. In addition, LPT increased lymphatic flux of SOD and NT.
Ten minutes following cessation of LPT, thoracic and intestinal lymph flux of cytokines, chemokines, NT and SOD
were similar to pre-LPT, demonstrating that their flux was transient and a response to LPT. This re-distribution of
inflammatory mediators during LPT may provide scientific rationale for the clinical use of LPT to enhance immunity and
treat infection.
Keywords: lymph, lymphatic pump technique, cytokines, chemokines, inflammatory mediators, mesenteric duct lymph,
thoracic duct lymph, infection, edema, immune system, reactive nitrogen species, reactive oxygen species, immunity,
osteopathic manipulative medicine
Experimental Biology and Medicine 2012; 237: 58 – 63. DOI: 10.1258/ebm.2011.011220
Introduction
Osteopathic physicians have developed osteopathic manip-
ulations collectively known as lymphatic pump techniques
(LPTs), which are designed to enhance lymph flow.
1,2
By
increasing lymph flow, LPT is thought to aid in the
removal of metabolic wastes, toxins, exudates and cellular
debris that accumulate in the tissue interstitial fluid
during infection or edema.
3
Clinically, LPT has been
shown to enhance vaccine specific antibodies,
4,5
and
reduce the length of hospital stay and the duration of anti-
biotic use in elderly patients with pneumonia.
6
During infection and edema, inflammatory cytokines, che-
mokines, reactive oxygen species (ROS), such as superoxide
dismutase (SOD), and reactive nitrogen species (RNS), such
as nitrotyrosine (NT) are generated. The proinflammatory cyto-
kines and chemokines, interleukin-2 (IL-2), IL-4, IL-6, IL-8,
tissue necrosis factor-
a
(TNF-
a
), interferon-
g
(IFN-
g
),
monocyte chemotactic protein-1 (MCP-1) and keratinocyte che-
moattractant (KC) induce leukocyte activation, migration and
cell-mediated immune responses to pathogens,
7,8
whereas anti-
inflammatory cytokines such as IL-10 limit inflammation by
suppressing cell-mediated immune responses.
7,8
Recent use of animal models has provided insight into the
mechanisms by which LPT affects the lymphatic and
immune systems.
2,9 – 12
Previously, we reported that LPT
enhances thoracic duct lymph (TDL) and mesenteric duct
lymph (MDL) flow and leukocyte concentrations in dogs
and rats.
2,10,11,13
The purpose of this study was to determine
if LPT would mobilize inflammatory mediators into the
lymphatic circulation.
7,8
In addition, SOD and NT were
measured. The results of this study provide support for
the clinical application of LPT to enhance function of the
immune system, and may explain, in part, a mechanism
by which LPT protects against infection and edema.
ISSN: 1535-3702
Copyright #2012 by the Society for Experimental Biology and Medicine
Experimental Biology and Medicine 2012; 237:58–63
Materials and methods
Animals
This study was approved by the Institutional Animal Care
and Use Committee and was conducted in accordance
with the Guide for the Care and Use of Laboratory Animals
(NIH Publication no. 85-23, revised 1996). Twelve adult
mongrel dogs, free of clinically evident signs of disease,
were used for this study.
Surgical techniques
Dogs were anesthetized with sodium pentobarbital
(30 mg/kg, intravenously). After endotracheal intubation,
the dogs were ventilated with room air supplemented with
oxygen to maintain normal arterial blood gases. In addition,
arterial blood pressure was monitored via a femoral artery
catheter and remained within normal limits throughout the
experiment. In six dogs, the chest was opened by a thoracot-
omy in the left, fourth intercostal space. The thoracic duct
was isolated from connective tissue and ligated. Caudal to
the ligation, a PE 60 catheter (inner diameter 0.76 mm,
outer diameter 1.22 mm) was inserted into the duct and
secured with a ligature. Lymph was drained at atmospheric
pressure through a catheter whose outflow tip was posi-
tioned 8 cm below heart level to compensate for the hydrau-
lic resistance of the catheter. The outflow tip of the catheter
was maintained at this position for all experimental con-
ditions. Approximately 60 min following cannulation of the
thoracic duct, thoracic lymph was collected during 4 min
pre-LPT, during 4 min of LPT and for 10 min following cessa-
tion of LPT ( post-LPT). Lymph flow rate was computed from
the volume of lymph collected during these time intervals.
In separate experiments, mesenteric lymph was collected.
Six additional dogs were surgically prepared for experimen-
tation as described above. However, rather than opening the
chest, a midline abdominal incision was made to expose a
large mesenteric lymph duct. This duct was isolated,
ligated, and a PE 60 catheter was inserted into the duct
and secured with a ligature. The catheter was exteriorized
through the abdominal incision, which was then closed
with 2-0 silk suture. Approximately 60 min after cannula-
tion of the mesenteric lymph duct, mesenteric lymph
samples were collected, and lymph flow was measured as
described above for TDL.
Lymphatic pump technique
The anesthetized dogs were placed in a right lateral recumbent
position. To perform abdominal LPT, the operator contacted
the abdomen of the animal with the hands placed bilaterally
below the costo-diaphragmatic junction. Pressure was exerted
medially and cranially to compress the abdomen until signifi-
cant resistance was encountered, and then the pressure was
released. Abdominal compressions were administered at a
rate of approximately 1/s for a total of 4 min of LPT.
Measurements of TDL and MDL
A commercially available multiplex assay (Millipore,
Billerica, MA, USA) was used to determine the
concentrations of cytokines and chemokines in TDL and
MDL. Specifically, the cytokines IL-2, IL-4, IL-6, IL-10,
IFN-
g
and TNF-
a
, and the chemokines MCP-1 and KC
were measured. A range of standards, provided with the
multiplex assay, was used, and the assay was analyzed
using the Luminexw200 System with the xPONENT
Software Interface (Millipore). The minimum detectable
concentrations for IL-2, IL-4, IL-6, IL-8, IL-10, IFN-
g
,
TNF-
a
,MCP-1 and KC were 6.4, 28.8, 12.1, 20.3, 1.6, 4.4,
0.4, 8.6 and 1.6 pg/mL, respectively. To compute the cyto-
kine/chemokine flux in TDL and MDL, the respective con-
centration was multiplied by lymph flow during each
minute for each condition, and these values were averaged.
Thoracic lymph concentrations of SOD (Cayman
Chemicals, Ann Arbor, MI, USA) and NT (Molecular
Probes, Inc, Eugene, OR, USA) were measured using com-
mercially available kits. The SOD assay measures all three
forms of SOD by utilizing a tetrazolium salt for the detec-
tion of xanthine oxidase and hypoxanthine-derived super-
oxide radicals. One unit of SOD is defined as the amount
of enzyme necessary to cause 50% dismutation of the super-
oxide radical. The SOD minimum detectable concentration
for this assay is 0.025 U/mL. NO reacts with superoxide to
form peroxynitrite.
14
Subsequently, peroxynitrate reacts
with proteins, resulting in measurable NT. The minimum
detectable concentration for NT of this assay is 2 nmol/L
SOD and NT was measured only in TDL, since the
samples of MDL were not sufficient for both these measure-
ments and the Luminex assays. To compute SOD or NT flux
in TDL, the respective concentration was multiplied by
lymph flow during each minute for each condition, and
these values were averaged.
Statistical analysis
Data are presented as arithmetic means+standard error
(SE). Values from multiple animals at respective time
points were averaged and are shown in either tables or
plotted in figures. For statistical evaluation, data were sub-
jected to repeated measures analysis of variance or analysis
of variance followed by a Student – Newman–Keuls mul-
tiple comparisons test. Analyses were performed with
GraphPad Prism version 5.0 for Windows (GraphPad
Software, San Diego, CA, USA). Differences among mean
values with at least P0.05 were considered statistically
significant.
Results
LPT increased intestinal and TDL flow
Similar to our previous reports,
10,11
LPT enhanced TDL
and MDL flow. LPT increased TDL flow from 0.90 +
0.19 mL/min during pre-LPT to 5.65+0.93 mL/min (P,
0.001) and the flow subsequently decreased to 2.07 +
0.28 mL/min during post-LPT (P,0.01). LPT also increased
MDL flow from 0.30+0.03 mL/min during pre-LPT to
2.71 +1.01 mL/min (P,0.05) and the flow subsequently
decreased to 0.32 +0.25 mL/min during post-LPT (P,0.05).
.................................. ..... ..... ..... ..... ..... ...... ..... ..... ..... ..... ..... ..... ..... ...... .............................. ...... ..
Schander et al.LPT and lymph cytokines 59
LPT increased the concentrations of MCP-1 in TDL
Concentrations of cytokines and chemokines in TDL and in
MDL are reported in Table 1. While cytokine and chemo-
kine concentrations in both TDL and MDL tended to
increase during LPT compared with pre- and post-LPT,
the only statistically significant increase detected was
MCP-1 in TDL (P,0.05). However, during LPT, differences
were detected between MDL and TDL in the concentrations
of IL-8 and MCP-1. Specifically, the concentration of IL-8
was greater during LPT in MDL (126%; P,0.05) compared
with TDL.
Of interest, the concentration of MCP-1 was greater in
MDL compared with TDL in all samples (Table 1).
Specifically, MCP-1 was greater at pre-LPT (435%; P,
0.01), during LPT (200%; P,0.01) and post-LPT (214%;
P,0.01) when compared with respective TDL MCP-1
concentrations.
LPT increased lymphatic cytokine and chemokine flux
The effect of LPT on flux of cytokines and chemokines in
TDL is shown in Figure 1. LPT significantly increased
TDL flux of IL-6 (615%; P,0.05), IL-8 (944%; P,0.001),
IL-10 (917%; P,0.001), MCP-1 (1505%; P,0.01) and KC
(788%; P,0.001) compared with pre-LPT. Furthermore,
these concentrations decreased post-LPT by 79% in IL-6
(P,0.05), 55% in IL-8 (P,0.01), 53% in IL-10 (P,0.01),
74% in MCP-1 (P,0.05) and 57% in KC (P,0.001).
TheeffectofLPTonfluxofcytokinesandchemokinesin
MDL is shown in Figure 2. LPT significantly increased the
MDL flux of IL-6 (394%; P,0.05), IL-8 (741%; P,0.001),
IL-10 (556%; P,0.05), MCP-1 (651%; P,0.01) and KC
(496%; P,0.001). As seen in TDL, the flux of cytokines
and chemokines in MDL declined after LPT. From LPT to
post-LPT, IL-6 decreased by 67% (P,0.05), IL-8 by 82%
(P,0.001), IL-10 by 86% (P,0.05), MCP-1 by 86% (P,
0.01) and KC by 83% (P,0.001). Cytokines IL-2, IL-4,
IFN-
g
and TNF-
a
were not detectable in TDL or in MDL
at any of the time points.
Figure 2 LPT increased cytokine and chemokine flux in mesenteric
duct lymph (MDL). Data are means +SE (n¼6). Greater than respective
pre-LPT and post-LPT (P,0.05). Greater than respective pre-LPT and
post-LPT values (P,0.01). Greater than respective pre-LPT and post-LPT
values (P,0.001). Repeated measures ANOVA with Student –Newman –Keuls
post-test. LPT, lymphatic pump technique; ANOVA, analysis of variance
Figure 1 LPT increased cytokine and chemokine flux in thoracic duct lymph
(TDL). Data are means +SE (n¼6). Greater than respective pre-LPT and
post-LPT (P,0.05). Greater than respective pre-LPT and post-LPT values
(P,0.01). Greater than respective pre-LPT and post-LPT values (P,
0.001). Repeated measures ANOVA with Student – Newman – Keuls post-test.
LPT, lymphatic pump technique; ANOVA, analysis of variance
Table 1 LPT significantly altered the concentration of MCP-1, but
did not significantly alter other cytokine, chemokine and reactive
oxygen species concentrations in lymph
Pre-LPT LPT Post-LPT
Thoracic duct lymph (TDL)
IL-6 (pg/mL) 193 +65 217 +74 158 +59
IL-8 (pg/mL) 183 +25 240 +48 217 +42
IL-10 (pg/mL) 24 +931+529+5
MCP-1 (pg/mL) 578 +135 1160 +304958 +266
KC (pg/mL) 1351 +165 1501 +247 1284 +172
SOD (U/mL) 0.135 +0.038 0.176 +0.026 0.173 +0.019
NT (mmol/L/mL) 3.93 +2.54 6.79 +1.93 2.86 +0.76
Mesenteric duct lymph (MDL)
IL-6 (pg/mL) 217 +43 241 +116 333 +115
IL-8 (pg/mL) 396 +85 543 +114
†
392 +54
IL-10 (pg/mL) 51 +20 72 +24 47 +17
MCP-1 (pg/mL) 3094 +749
‡
3482 +685
‡
3007 +473
‡
KC (pg/mL) 2389 +554 2522 +506 2270 +526
LPT, lymphatic pump technique; MCP-1, monocyte chemotactic protein-1;
IL, interleukin; KC, keratinocyte chemoattractant; SOD, superoxide
dismutase; NT, nitrotyrosine
Data are means+SE (n¼6)
Greater than respective pre-LPT (P,0.05)
†
Different from respective TDL value (P,0.05)
‡
Different from respective TDL value (P,0.01)
Repeated measures analysis of variance with Student – Newman – Keuls
post-test
.................................. ..... ..... ..... ..... ..... ...... ..... ..... ..... ..... ..... ..... ..... ...... .............................. ...... ..
60 Experimental Biology and Medicine Volume 237 January 2012
LPT increased the flux of ROS and RNS in TDL
The effect of LPT on the flux of SOD in TDL is shown in
Figure 3 and the corresponding effect on NT is shown in
Figure 4. Although LPT did not significantly increase the
concentrations of SOD and NT in TDL (Table 1), LPT
increased SOD flux 367% in TDL from 0.15 +0.07 U/min
pre-LPT to 0.7 +0.1 U/min during LPT (P,0.01).
Post-LPT, SOD flux decreased 64% to 0.25 +0.08 U/min
(P,0.01; Figure 3). LPT increased NT flux in TDL, 373%
from 5.8 +mmol/L/min pre-LPT to 27.4 +10.9 mmol/L/min
during LPT (P,0.05). Post-LPT, NT flux decreased 84% to
4.4 +1.6 mmol/L/min (P,0.05; Figure 4).
IL-6 flux was greater in TDL than in MDL during LPT
Flux of cytokines and chemokines in TDL and in MDL
during LPT is compared in Figure 5. During LPT,
IL-6 flux in TDL increased 318% more than the flux in
MDL (P,0.01).
Discussion
This study is the first to report the effects of LPT on the con-
centration and flux of inflammatory mediators in the lym-
phatic system. LPT did not significantly increase cytokine,
chemokine, ROS or RNS concentrations in lymph, with
the exception of MCP-1; however, LPT increased lymphatic
flow, which significantly increased the flux of these inflam-
matory mediators from tissue to blood via the lymphatic
system. Specifically, LPT increased the flux of IL-6, IL-8,
IL-10, MCP-1 and KC in thoracic and mesenteric lymph.
While we did not measure ROS or RNS in MDL, LPT signifi-
cantly increased SOD and NT flux in TDL. Collectively,
these results suggest that by increasing lymph flow, LPT
enhances the mobilization of inflammatory mediators into
the lymphatic circulation for transport to the blood
circulation.
Cytokines, chemokines, ROS and RNS are generated
during the innate immune response to pathogens. During
infection, the cytokines IL-6, IL-8, MCP-1 and KC induce
inflammation by recruiting and activating leukocytes,
while IL-10 regulates the inflammatory response.
7,8,15 – 17
During acute inflammation, inflammatory cytokines
Figure 3 LPT increased SOD flux in thoracic duct lymph. Data are means+
SE (n¼6). Greater than respective pre-LPT and post-LPT values (P,0.01).
Repeated measures ANOVA with Student– Newman – Keuls post-test. LPT,
lymphatic pump technique; ANOVA, analysis of variance; SOD, superoxide
dismutase
Figure 5 LPT created a difference in measurable IL-6 in TDL versus MDL.
Data are means +SE (n¼6). Greater than respective MDL value (P,
0.01). ANOVA with Student– Newman – Keuls post-test. LPT, lymphatic pump
technique; ANOVA, analysis of variance; IL-6, interleukin-6; MDL, mesenteric
duct lymph; TDL, thoracic duct lymph
Figure 4 LPT increased NT flux in thoracic duct lymph. Data are means +SE
(n¼6). Greater than respective pre-LPT and post-LPT values (P,0.05).
Repeated measures ANOVA with Student– Newman – Keuls post-test. LPT,
lymphatic pump technique; ANOVA, analysis of variance; NT, nitrotyrosine
.................................. ..... ..... ..... ..... ..... ...... ..... ..... ..... ..... ..... ..... ..... ...... .............................. ...... ..
Schander et al.LPT and lymph cytokines 61
stimulate the formation of edema by accumulating in the
interstitial fluid, which initially lowers the interstitial fluid
pressure, setting the stage for the influx of proteins and
plasma fluid.
18,19
Therefore, LPT may suppress edema by
mobilizing inflammatory mediators out of interstitial fluid
into the lymphatic circulation, as well as directly increasing
lymph flow and removing excessive interstitial fluid.
2,12
LPT is used to treat infection,
4 – 6,20,21
but the mechanisms
by which LPT protects against infectious diseases are
unclear. LPT may enhance protection against infection by
increasing mesenteric-derived inflammatory mediators in
circulation, enabling the re-distribution of these mediators
to other tissues. In support of this notion, lymph has been
shown to re-distribute mesenteric-derived cytokines and
chemokines to distant organs.
22 – 25
Furthermore, it has
been shown in vitro that mesenteric lymph can activate neu-
trophils and increase endothelial cell permeability.
26
It is not
surprising, that LPT would enhance this re-distribution and
potentially enhance immune function.
Previously, we reported that LPT releases leukocytes from
mesenteric lymph nodes into TDL and enhances leukocyte
flux in MDL and TDL.
10
Following exposure to microorgan-
isms, phagocytes, such as macrophages and neutrophils,
release ROS and RNS which are bactericidal.
7
Thus, by
enhancing the lymphatic flux of leukocytes, cytokines, che-
mokines, ROS and RNS, LPT may facilitate cell-mediated
clearance of infection.
It has been hypothesized that following tissue injury,
lymph flow quickly increases and provides the earliest
signal in the lymphatic system to induce the inflammatory
response.
27
It has been documented that lymphedema
impairs immune cell trafficking and increases susceptibility
to infection.
28
Recently, transmural flow across lymphatic
endothelia was shown to regulate cell and fluid transport
functions of lymphatic endothelium.
29
Specifically, trans-
mural flow increased chemokine ligand secretion, influ-
enced dendritic cell migration into lymphatic vessels,
increased vessel permeability and upregulated cell adhesion
molecules on lymphatic vessels.
29
The resulting increase in
shear stress induces endothelial nitric oxide expression in
human lymphatic endothelial cells;
30
so elevated lymph
flow causes release of endogenous nitric oxide from lym-
phatic endothelial cells.
31,32
Therefore, in addition to releas-
ing leukocytes into lymphatic circulation, by enhancing
lymph flow and NT release into lymph, LPT may signal
the lymphatic system to increase immune cell trafficking.
We also compared the lymphatic cytokine and chemokine
composition between thoracic and mesenteric lymph. The
thoracic duct is a large vessel and transports lymph
drained from abdominal visceral organs (mainly the liver
and intestines), skin and skeletal muscle.
7,8,33
We found
that the concentrations of cytokines and chemokines were
higher in MDL (Table 1), which is consistent with the
prior report that most of the lymph and protein in the thor-
acic duct is derived from the mesenteric lymph.
34
This result
suggests that compared with mesenteric lymph, lymph
derived from the liver and other tissues contains low con-
centrations of inflammatory mediators, and thus dilutes
mesenteric-derived cytokines in TDL. It is important to
note that these were healthy animals; therefore, during
infection or inflammation, the concentrations of inflamma-
tory mediators in TDL and MDL may vary.
In conclusion, we have demonstrated that LPT transiently
increased the flux of chemokines, cytokines and reactive
oxygen and nitrogen species in lymph. These findings are
consistent with our previous reports, which demonstrated
that LPT transiently increases thoracic and mesenteric
lymph flow and leukocyte concentrations. This study was
performed in healthy animals, and the effect of LPT on
the lymphatic release of leukocytes and inflammatory
mediators may be intensified or altered during infection.
Our studies support the hypothesis that LPT may enhance
immune response by enhancing the release of leukocytes
and inflammatory mediators into lymphatic circulation.
Author contributions: AS performed the surgery, animal
instrumentation, statistical analysis and data interpretation,
provided the LPT and prepared the manuscript. HFD par-
ticipated in the study design, data interpretation and prep-
aration of the manuscript. LMH designed and provided
the oversight for the study. In addition, she reviewed and
interpreted the data and participated in the preparation of
the manuscript.
ACKNOWLEDGEMENTS
This study was funded by grants from the National
Institutes of Health, grants R01 AT004361 (LMH) and U19
AT002023 (HFD). The authors thank the Osteopathic
Heritage Foundation for their continued support of the
Basic Science Research Chair (LMH). The authors would
also like to thank Arthur Williams Jr and Linda Howard
for assistance in the animal surgery, and Jamie Huff and
Xin Zhang for help in the preparation of enzyme-linked
immunosorbent assays and multiplex assays.
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(Received June 20, 2011, Accepted September 10, 2011)
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