Download Lipopolysaccharide modulates neutrophil recruitment and

Am J Physiol Heart Circ Physiol 309: H2042–H2057, 2015.
First published October 9, 2015; doi:10.1152/ajpheart.00467.2015.
Lipopolysaccharide modulates neutrophil recruitment and macrophage
polarization on lymphatic vessels and impairs lymphatic function in rat mesentery
Sanjukta Chakraborty,1 Scott D. Zawieja,1 Wei Wang,1 Yang Lee,1 Yuan J. Wang,2
Pierre-Yves von der Weid,2 David C. Zawieja,1 and Mariappan Muthuchamy1
1
Department of Medical Physiology, Cardiovascular Research Institute, Division of Lymphatic Biology, Texas A&M Health
Science Center College of Medicine, College Station, Texas; and 2Department of Physiology and Pharmacology,
Inflammation Research Network, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary,
Calgary, Alberta, Canada
Submitted 15 June 2015; accepted in final form 5 October 2015
inflammation; lymphatic function; neutrophils; M1 and M2 macrophages; lipopolysaccharide
NEW & NOTEWORTHY
LPS causes a conducible environment in the mesentery that
decreases the neutrophils and shifts the balance toward a M2
macrophage polarization on and/or near the vicinity of lymphatics. LPS-TLR4-mediated regulation of NF-␬B, pAKT,
Address for reprint requests and other correspondence: M. Muthuchamy, Dept. of
Medical Physiology, Cardiovascular Research Institute, Div. of Lymphatic Biology,
Texas A&M Health Science Center College of Medicine, 359 Reynolds Medical
Bldg., College Station, TX 77843-1114 (e-mail: [email protected]).
H2042
pERK, and pMLC20 in lymphatic muscle cells promotes inflammation and significant impairment in mesenteric lymphatic
function.
as a central player in the process
of inflammation and play active roles in both resolution and
progression of inflammation (40, 76). Mesenteric lymphatics,
in particular, are directly exposed to both the inflammatory
activation and dyslipidemia resulting from the aberrant elevated postprandial chylomicron production in metabolic syndrome, as well as the dietary endotoxins, such as LPS associated with them (32, 37, 74). LPS, a critical cell wall component
of most gram-negative bacteria, has been identified as the
major effector of conditions, such as peritonitis and endotoxemia, that result in septic shock and multiple organ failure (6,
58, 60, 70). As lymphatic pumping is significantly affected by
mechanical and chemical stimuli, including inflammatory mediators and an increased fluid load, these stimuli significantly
affect lymphatic pumping during edemagenic conditions (30).
Previous studies in sheep and guinea pig have shown that
mesenteric lymph flow increases rapidly on LPS injection,
which may be mainly due to microvascular hyperpermeability
and plasma albumin leakage, but later affects contractility (9,
28, 56). In mouse models of LPS-induced peritonitis, which
closely replicates many features of endotoxemia and sepsis,
LPS has been shown to increase lymphatic density and lymphangiogenesis in the mouse diaphragm. These changes appeared to be mediated by investiture of myeloid-derived
CD11b⫹ cells on the peritoneal side of the diaphragmatic
lymphatic vessels that exhibited a profibrotic phenotype (47).
Another study has shown that lymph propulsion is interrupted
in mouse in acute response to LPS (3). However, no direct link
has been established between LPS-induced changes in surrounding inflammatory lymphatic microenvironment, specifically the surrounding immune cells and lymphatic contractile
function.
We have previously documented that a novel population of
antigen-presenting cells are resident within the walls of muscular, prenodal lymphatics in the normal rats (13). Chatterjee et
al. (20) and Nagai et al. (55) have shown that in older rats,
representing a chronic inflammatory condition, an increase in
the number of preactivated mast cells is associated with mesenteric lymphatics that could be one of the underlying factors
for the lymphatic contractile dysfunction observed in aged rats.
In the present study, we have raised the following questions: 1)
What are the effects of LPS on lymphatic contractile parameters and its pump function? 2) How does the profile of the
immune cells found around the lymphatics in the mesentery
THE LYMPHATICS HAVE EMERGED
0363-6135/15 Copyright © 2015 the American Physiological Society
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Chakraborty S, Zawieja SD, Wang W, Lee Y, Wang YJ, von
der Weid PY, Zawieja DC, Muthuchamy M. Lipopolysaccharide
modulates neutrophil recruitment and macrophage polarization on
lymphatic vessels and impairs lymphatic function in rat mesentery.
Am J Physiol Heart Circ Physiol 309: H2042–H2057, 2015. First
published October 9, 2015; doi:10.1152/ajpheart.00467.2015.—Impairment of the lymphatic system is apparent in multiple inflammatory pathologies connected to elevated endotoxins such as LPS.
However, the direct mechanisms by which LPS influences the
lymphatic contractility are not well understood. We hypothesized
that a dynamic modulation of innate immune cell populations in
mesentery under inflammatory conditions perturbs tissue cytokine/
chemokine homeostasis and subsequently influences lymphatic
function. We used rats that were intraperitoneally injected with
LPS (10 mg/kg) to determine the changes in the profiles of innate
immune cells in the mesentery and in the stretch-mediated contractile responses of isolated lymphatic preparations. Results demonstrated a reduction in the phasic contractile activity of mesenteric lymphatic vessels from LPS-injected rats and a severe impairment of lymphatic pump function and flow. There was a
significant reduction in the number of neutrophils and an increase
in monocytes/macrophages present on the lymphatic vessels and in
the clear mesentery of the LPS group. This population of monocytes and macrophages established a robust M2 phenotype, with
the majority showing high expression of CD163 and CD206.
Several cytokines and chemoattractants for neutrophils and macrophages were significantly changed in the mesentery of LPSinjected rats. Treatment of lymphatic muscle cells (LMCs) with
LPS showed significant changes in the expression of adhesion
molecules, VCAM1, ICAM1, CXCR2, and galectin-9. LPS-TLR4mediated regulation of pAKT, pERK pI-␬B, and pMLC20 in LMCs
promoted both contractile and inflammatory pathways. Thus, our
data provide the first evidence connecting the dynamic changes in
innate immune cells on or near the lymphatics and complex
cytokine milieu during inflammation with lymphatic dysfunction.
INFLAMMATION AND IMMUNE CELLS IMPACT LYMPHATIC FUNCTION
MATERIALS AND METHODS
LPS rat model. Male Sprague-Dawley rats weighing ⬃150 g were
intraperitoneally injected with LPS (10 mg/kg body wt) (LPS from
Escherichia coli 0127:B8, L3129; Sigma-Aldrich, St. Louis, MO). All
animals were housed in a facility accredited by the Association for the
Assessment and Accreditation of Laboratory Animal Care and were
maintained in accordance with the policies defined by the Public
Health Service Policy for the Humane Care and Use of Laboratory
Animals and the U.S. Department of Agriculture’s Animal Welfare
Regulations, and all of the protocols were approved by the Texas
A&M University Laboratory Animal Care Committee. Rats were
treated with PBS (control group) or LPS for 6 h, 24 h, or 3 days (LPS
injections were administered every 24 h).
Isolated vessel preparation and functional analyses. Control and
LPS-treated rats (6 h and 72 h) were anesthetized with a combination of a droperidol-fentanyl (0.3 ml·kg⫺1·1⫺1 im of a solution
of droperidol, 20 mg/ml fentanyl, 0.4 mg/ml), and diazepam (2.5
mg/kg im). A midline excision was made, and a loop of intestine
3– 4 cm long was carefully exteriorized. A section of the mesentery
was gently positioned over a semicircular viewing pedestal on a
vessel preparation board. Mesenteric collecting lymphatic vessels
were carefully cleaned of surrounding adipose and connective
tissues. Vessels were maintained in albumin-supplemented physiological saline solution (APSS; in mM: 145.0 NaCl, 4.7 KCl, 2
CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 sodium pyruvate, 0.02 EDTA, and 3.0 MOPS and 1% wt/vol BSA) at pH 7.4 at
38°C, as described earlier (29). The isolated lymphatic was cannulated and tied on to two glass pipettes (tip diameter: 80 –100
␮m). All of the isolated lymphatic length between the two glass
tips (for all the experiments) was ⬃1.2–1.5 mm containing one
valve. Only vessels that did not have apparent constriction sites
due to damage were used. Vessels were then allowed to equilibrate
at a transmural pressure of 1 cmH2O for ⬃30 min. After the
equilibration period, contractions of each vessel were recorded for
5 min at pressures 1 cm, 3 cm, and 5 cmH2O. Finally, the passive
diameter of the vessel at each transmural pressure was measured
after the vessel was exposed to nominally Ca2⫹-free APSS (0 mM
added Ca2⫹ and EDTA, 3.0 mM) for 15 min. Experimental data
were acquired for the last 3 min of each 5-min interval at the
different transmural pressures tested (1, 3, and 5 cmH2O). To
determine the effect of LPS on lymphatic vessel contractility,
isolated and cleaned mesenteric lymphatic vessels were immediately deposited into 3.5-mm sterile petri dishes filled with DMEM/
F12 solution without (n ⫽ 12) or with LPS (10 ␮g/ml) (n ⫽ 13).
The dishes were then placed in an incubator (5% CO2, 37°C) for a
period of 24 h. Vessels were then subsequently cannulated with
two glass pipettes, pressurized, and prepared for contractile activity measurements, as described above.
Isolated vessel video analysis. Experiments were dynamically
monitored by a microscope charge-coupled device video camera,
and the video data were recorded to a video DVD for the functional
analyses of lymphatic contractions after the experiment. Lymphatic diameter was traced for each 5 min of video capture with a
vessel wall-tracking program developed and provided by Dr.
Michael J. Davis (University of Missouri, Columbia, MO) (25).
Outer lymphatic vessel diameters were tracked 30 times/s, providing a tracing of diameter changes throughout the periods of
lymphatic systole and diastole. The following analogies to the
cardiac pump parameters were derived: lymphatic tonic index,
contraction amplitude, ejection fraction, contraction frequency,
fractional/lymph pump flow, and systolic/diastolic diameters, as
previously described (9, 29). Briefly, tonic index is the difference
between passive outer lymphatic diameter in Ca2⫹-free APSS
(normalized to 100%) and outer end-diastolic diameter, expressed
as a percentage of the passive outer lymphatic diameter, reflecting
the sustained tonic contraction of the lymphatics; fractional pump
flow ⫽ ejection fraction [(end-diastolic volume ⫺ end-systolic
volume)/end-diastolic volume] ⫻ contraction frequency, showing
the practical capacity of the lymphatic pumping in lymph transportation and lymph pump flow ⫽ stroke volume (end-diastolic
diameter ⫺ end-systolic diameter) ⫻ contraction frequency, showing the comprehensive capacity of the lymphatic pumping in lymph
transportation.
Whole-mount mesenteric preparation and imaging. Rat mesenteric
tissue was removed post mortem and pinned out into loops in
GIBCO Dulbecco’s phosphate buffered saline (DPBS) on a Sylgard-coated dish. These loops were fixed and permeabilized for 1
h with prechilled methanol at ⫺20°C and blocked in DPBS
supplemented with 5% goat serum. Following this, mesenteric
arcades were cut from the intestinal wall, and the two mesenteric
bundles were incubated in block solution (DPBS supplemented
with 5% goat serum) for 2 h at room temperature. The tissue
arcades were then incubated overnight with primary antibodies
against neutrophil elastase (NE; 1:100), CD11b (1:100), smooth
muscle ␣-actin (1:500), CD206 (1:100), MHCII (1:200), CD163
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change in acute inflammation? and 3) What are the molecular
mechanisms of LPS-induced changes in lymphatic contractions? To address these questions, we designed a series of
experiments to specifically delineate the association of two key
innate immune cell populations, neutrophils and macrophages,
with lymphatic function in a rat model of LPS-induced inflammation (42, 45).
Neutrophils, as the body’s first line of defense, dominate the
early stages of inflammation and set the stage for repair by
macrophages of tissue damage (14). Macrophages on the other
hand, play an essential role in homeostasis and defense and can
be polarized by the microenvironment to mount specific M1 or
M2 functional programs (35, 54). Classical macrophage polarization (M1) is driven in response to microbial products or Th1
cytokines, such as IFN-␥, and is characterized by an enhanced
capacity to kill intracellular microorganisms and produce generous amounts of proinflammatory mediators. Conversely, alternative macrophage polarization (M2) can be generated in
response to a variety of stimuli, such as Th2 cytokines (IL-4,
IL-13, and IL-10), glucocorticoids, or a mixture of Ig complexes and TLR ligands, producing an anti-inflammatory phenotype (33, 69). Although the role of the lymphatic network in
the recruitment and transport of activated dendritic cells has
been well established (5, 41, 65, 66), its regulation of the acute
phase of inflammatory insult, and coordination of the primary
innate responders (macrophages, monocytes, and neutrophils)
within the tissue are not clearly understood. We hypothesize
that in the presence of an inflammatory stimulus, such as LPS,
there is an impairment of lymphatic contractility that may be
associated with distinct shifts in neutrophil populations and
alterations in macrophage polarization on or near the mesenteric lymphatics. These changes would alter the milieu surrounding the mesenteric lymphatics and affect their physiological function. To test our hypothesis, we examined lymphatic
contractile parameters in isolated vessel preparations from
normal and LPS-injected rats and the effects of LPS on the
recruitment of neutrophils and macrophage polarization in
the vicinity of the collecting lymphatics. We also determined
the direct molecular mechanisms by which LPS mediates the
contractile and inflammatory signaling in the lymphatics and
the effects of key cytokines associated with neutrophils and
macrophages on lymphatics using the rat mesenteric lymphatic
muscle cell (LMC) culture model.
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INFLAMMATION AND IMMUNE CELLS IMPACT LYMPHATIC FUNCTION
secured to the stage of a multiphoton/confocal microscope (Leica
AOBS SP2) for immediate observation.
Protein isolation and Western blot analysis. Rat mesenteric LMCs
from mesenteric vessel explants were used as described earlier
(18). LMCs were treated with LPS (0 –200 ng/ml) for 0 –72 h.
Similarly treatments were also carried out with IL-8, (100 nM) in
the absence or presence of LPS. Cells were also treated with the
TLR4 inhibitor, polymixin B (10 ␮g/ml; InvivoGen, San Diego,
CA), ERK inhibitor, PD98059 (10 nM), and AKT inhibitor
LY294002 (20 ␮g/ml) in the presence or absence of LPS for 24 h.
Western blot analysis was then carried out as previously described
(18). LMCs were lysed in 1⫻ SDS buffer supplemented with
protease and phosphatase inhibitor and run on a 4 –20% precast
gradient SDS-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA). The proteins were transferred to a nitrocellulose membrane with a Bio-Rad transblot apparatus. The transfer was verified
with Ponceau-S staining. The membrane was blocked in 5% milk
in TBS/TBST and then incubated with a primary antibody followed
by incubation with the corresponding HRP-conjugated secondary
antibody. The following dilutions of the primary antibodies in TBS
were used: p-MLC20 (1:500), MLC20 and GAPDH (1:5,000),
phospho-ERK1/2, phospho-ERK1/2, AKT, p-AKT (1:1,000),
VCAM1 (1:2,000), ICAM1 (1:1,000), CXCR2 (1:1,000), galectin
9 (1:500), pI-␬B␣ and I-␬B␣ (1:1,000), and ␤-actin (1:10,000).
The immunoreactive bands were visualized using the Pierce detection system (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific, Rockford, IL). Densitometry analyses on
the resulting bands were performed using Quantity One MultiAnalyst Software (Bio-Rad Laboratories). The membranes were
stripped using ImmunoPure IgG Elution Buffer (Pierce, Rockford,
IL) and then reprobed. GAPDH was used as the loading control.
All experiments were done in triplicate, and data are presented as
means ⫾ SE.
Protein cytokine array analysis. Rat proteome profiler cytokine
arrays were obtained from R&D Systems (Minneapolis, MN). For
parallel detection of a panel of 29 proinflammatory cytokines and
chemokines, protein cytokine array profiling was carried out according to manufacturer’s instructions. Briefly, lymphatic mesenteric tissue arcades were isolated from untreated control and
LPS-treated rats (10 mg/kg body wt) for 24 h. Proteins were
isolated from these tissue arcades and mixed with a cocktail of
biotinylated detection antibodies and streptavidin-HRP, and
chemiluminescent detection was carried out as per the manufacturer’s instructions. The images were captured on Fuji ImageQuantLAS 4000 detection (GE Healthcare, Piscataway, NJ) and
processed by National Institutes of Health’s ImageJ software.
Statistical analyses. Statistical significance in the pressurized
vessel experiments was determined through two-way ANOVA
with Fisher’s post hoc analysis by the Statplus (Analystsoft)
statistical software package. All other data were analyzed by
Student’s t-test or one-way or two-way ANOVA, as applicable.
Data are expressed as means ⫾ SE, and P values of ⱕ0.05 were
considered as significant. All experiments were carried out at
minimum in triplicate.
RESULTS
LPS induces inflammation and recruits large numbers of
CD11b⫹ cells in mesenteric bed and near the lymphatic collecting vessels. Masson Trichrome and MPO staining was
significantly increased in the lymph node and liver sections
from the LPS-injected rats, indicating fibrosis and onset of
acute-phase inflammatory cascades compared with the sections from untreated controls (Fig. 1, A and B). In addition,
LPS recruited significantly large numbers of CD11b⫹ cells
to the vicinity of and on the walls of the lymphatics and in
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(1:100), substance P, and SP (1:200) overnight. Controls were
prepared with normal mouse, rabbit, or hamster IgG. Goat antimouse/anti-rabbit Alexa Fluor 488 or anti-mouse/anti rabbit Alexa
Fluor 647 (Life Technologies, Carlsbad CA) was applied for 2 h.
Images were taken using confocal microscope (Leica) at 488 nm or
647 nm. Tissues were then mounted between two coverslips with
antifade mounting reagent (Life Technologies). Images were collected at ⫻20, ⫻40 and ⫻63 magnifications using a Leica scanning
confocal microscope with lymphatic vessels centered in the field of
view (FOV). At multiple sites within each sample, 0.5-␮m z-axis
steps were taken. One to three lymphatic vessels were “tracked”
from the intestinal wall toward the root of the mesentery per
staining combination. For each animal, around five to eight separate ⫻20, ⫻40, or ⫻63 magnification image stacks were acquired
and quantified, and average cell counts were determined. The
images were focused around mesenteric collecting lymphatics and
used to determine the changes in the number of immune cells and
lymphatic vessel association. Lymphatic vessels were determined
by their morphology, and the presence of bulbar valve regions after
smooth muscle actin-positive vessels were traced within the mesentery. Image reconstruction and orthogonal viewing on the image
stacks were performed using the Leica confocal software and
ImageJ. The negative controls for all experiments were produced
and analyzed via the same instrumental and image-processing
procedures. Average intensity projections representative of the
data are shown. All experiments were repeated at minimum in
triplicates.
Immunohistochemistry of liver and lymph node sections. Liver and
mesenteric lymph node cross sections were cut using a cryostat at
10 ␮m at ⫺20°C. Sections were air-dried for 30 min at room
temperature and then fixed overnight in Bouin’s solution at room
temperature. Masson Trichrome staining to detect fibrosis was then
carried out as described earlier (67). For immunohistochemical
analysis of liver and lymph node, the sections were fixed in cooled
acetone for 60 min at ⫺20°C. Samples were blocked with 10%
serum from the host of the secondary antibody in TBS (100 ␮l) for
30 min. The appropriate primary antibodies, [NE, SP, myeloperoxidase (MPO)] were then applied in blocking buffer, and sections
were incubated for 1 h at room temperature. After three washes
with PBS, the secondary antibodies Alexa Fluor 488 or Alexa
Fluor 594 (Invitrogen) were placed on the sections (1:200 dilution
in PBS), which were incubated for 30 min. A cover slip was placed
over the sections and fixed with ProLong DAPI mounting medium
(Life Technologies). Images were acquired by Zeiss 510 META
confocal microscope at ⫻40 magnification and quantified using the
NIH ImageJ analysis program.
Immunofluorescence analysis of lymphatic muscle cells and isolated mesenteric vessels. Immunofluorescence experiments were
carried out using cultured LMCs and stained with NF-␬B antibody,
as described earlier (19). Briefly, the LMCs were grown to about
70% confluence and plated onto coverslips. They were treated with
LPS (20 ng/ml) for 6 or 24 h. Cells were then fixed with 2%
paraformaldehyde, permeabilized with ice-cold methanol and incubated with NF-␬B primary antibody for 1 h. Goat anti-mouse
IgG-Alexa Fluor was used as a secondary. Images were scanned
using the Leica AOBS SP2 confocal microscope (Wetzlar, Germany). For isolated, paraformaldehyde-fixed rat mesenteric collecting lymphatics, immunofluorescence was also performed as
previously described (78). Briefly, fixed lymphatic vessels were
incubated in blocking solution (1% BSA, 5% normal goat serum in
PBS) for 1 h at RT and then divided into two pieces. The two
sections were incubated in blocking solution overnight at 4°C in
the presence of intralumenal and extralumenal primary NF-␬B or
the corresponding normal mouse Ig (negative control), respectively. Both segments were then incubated with the secondary goat
anti-mouse IgG Alexa Fluor 488. The vessels were then cannulated
and tied onto two glass pipettes, pressurized at 2 cmH2O and
INFLAMMATION AND IMMUNE CELLS IMPACT LYMPHATIC FUNCTION
the most abundant cell population within the tissue under
normal conditions and bear similarity to adipose tissue macrophages (Fig. 3, A–E). These CD163⫹CD206⫹ cells reside
both on the lymphatic collecting vessels and within the peripheral adipose (Fig. 3B). In response to 72-h LPS, the number of
these CD163⫹CD206⫹ macrophages increases twofold (Fig. 3,
F–J) and demonstrate a tendency toward residing on or within
the lymphatic vessel wall (Fig. 3H). Additionally, LPS injections resulted in the recruitment of CD163⫺CD206⫹ macrophages with ⬃19 per FOV that were preferentially found in
tight association with lymphatic vessels, as opposed to the
surrounding mesentery/adipose tissue (Fig. 3I). Macrophage
polarization is described as a dynamic spectrum, and the
single-positive (CD163⫺CD206⫹) cells represent a different
M2 phenotype than the double-positive (CD163⫹CD206⫹)
cells. The summary of quantification of CD163⫹CD206⫹ and
CD163⫺CD206⫹ cells on the lymphatic vessel, periphery, and
total counts for normal and LPS groups are shown in Fig. 3K.
Under control conditions, the MHCII⫹ macrophages demonstrate a significant spatial tropism to reside along or within the
mesenteric lymphatic vessels or other microvasculature structures (Fig. 3, L–Q). In contrast to the CD163⫹CD206⫹ macrophages, the number of MHCII⫹ cells per FOV does not
dramatically increase in response to LPS (Fig. 3, L–Q and
summary of quantification in Fig. 3R).
Impairment of lymphatic contractility in response to LPS
induced inflammation. Mesenteric lymphatic vessels were isolated from rats at various times after vehicle or LPS injection
and studied. Exposure to LPS resulted in significant impairment of mesenteric lymphatic vessel contractile activity in
regard to both tonic and phasic contractile parameters.
Mesenteric lymphatic vessels isolated from LPS injected
rats after 72 h had significantly reduced vessel tone (Fig. 4A)
at transmural pressures of 3 and 5 cmH2O compared with
their control counterparts [values for LPS vessels (n ⫽ 7)
were 4.8 ⫾ 0.86 and 4.2 ⫾ 0.66 vs. control vessels (n ⫽ 13)
13.2 ⫾ 2.6 and 11.2 ⫾ 2.6 at 3 and 5 cmH2O, respectively].
(Here, n represents the number of animals used). Four out of
seven rats injected with LPS examined at 6 and 72 h
completely lacked spontaneous mesenteric lymphatic contractions at all tested transmural pressures. In addition to the
disruption in vessel tone, LPS injections resulted in reduction in the phasic contraction frequency, which was significant at 6 h after injection (1.50 ⫾ 0.98 in LPS compared
with 6.25 ⫾ 1.05 in control at pressure 5 cmH2O), as well
as at 72 h (0.57 ⫾ 0.29, 0.59 ⫾ 0.30, 0.59 ⫾ 0.31 at
pressures 1, 3, and 5 cmH2O, respectively) compared with
the control vessels (10.913 ⫾ 1.14, 14.499 ⫾ 1.17, and
16.74 ⫾ 1.27 at 1, 3, and 5 cmH2O, respectively) (Fig. 4B).
Lymph pump flow (Fig. 4C) was also significantly reduced
in the 72-h LPS-injected rats at transmural pressure of 3 and
5 cmH2O (54.1 ⫾ 2.75 and 3.7 ⫾ 1.98 nl/min LPS; 26.1 ⫾
4.1 nl and 96.3 ⫾ 11 nl/min controls, respectively). As
shown in Fig. 4D, fractional pump flow was significantly
reduced in the 72-h LPS-injected animals at all pressures
examined (0.69 ⫾ 0.33, 1.86 ⫾ 0.94, and 2.8 ⫾ 1.46 at 1,
3, and 5 cmH2O, respectively) compared with its controls
(3.6 ⫾ 0.39, 5.77 ⫾ 0.58, and 6.38 ⫾ 0.61 at 1, 3, and 5
cmH2O, respectively). Despite changes in both vessel tone
and phasic contraction frequency, there were no significant
differences in the stroke volume, ejection fraction, and
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the clear mesenteric beds, which is indicative of an inflammatory response. As shown in the representative images in
Fig. 1C, by 6 h, there is an increase in CD11b⫹ cells after
LPS treatment. By 24 h and 72 h, the mesenteric bed and the
lymphatic vessels show a very robust infiltration of CD11b⫹
cells into the mesenteric plexus and neurovascular bundles.
LPS decreases the number of neutrophils on and in the
vicinity of the lymphatics. Mesenteric whole mounts from normal and LPS-injected animals were stained for a neutrophilspecific marker, NE. In control animals, a large number of
NE-positive cells were found associated with the lymphatic vessel, in the close vicinity of the vessels, as well as in the surrounding clear mesentery (Fig. 2). The number of neutrophils near the
lymphatic vessels did not show a significant change after 6 h or 24
h of LPS treatment (Fig. 2A). However, a significant decrease in
the numbers of neutrophils was found after 3 days in the LPSinjected group compared with the control mesentery (49.67 ⫾
5.24% decrease in LPS-injected group compared with control
mesentery) (Fig. 2A). To ascertain whether the neutrophils were
on the walls of lymphatic vessels, we costained some of these
vessels with the lymphatic muscle marker, ␣-smooth muscle
actin. As shown in control lymphatic vessels Fig. 2, B and C, a
number of the NE-positive cells directly adhered to the vessel wall
of the collecting lymphatics; three-dimensional orthogonal confocal projections revealed that some of the neutrophils were also
present beneath the muscle cell fibrils. In addition, a significantly
increased number of NE-positive cells were found in the mesenteric lymph nodes from the LPS-treated animals (Fig. 2D). The
NE-positive cells did not costain with CD11b, indicating that
these are two distinct populations of cells (data not shown). The
morphology of the neutrophils identified by NE staining was
found to be similar to those reported previously in other studies
(38, 79).
Neutrophils in the vicinity of the lymphatics and in the lymph
node stain positively for substance P. Substance P (SP) is a
known modulator of lymphatic contractility, and it has been
shown that during inflammatory conditions, various immune
cells, including neutrophils secrete SP (4, 26, 49, 75).
Hence, we costained the lymphatic mesenteric tissue arcades with NE and SP to determine whether the neutrophils
associated with the lymphatics also expressed SP. As seen in
Fig. 2E, most of the NE-positive cells express SP. We also
found that a number of cells that do not stain for NE, but
positively stain for SP, are present on the lymphatics.
Further, we costained SP with MHCII to show whether the
SP-expressing cells were dendritic cells or macrophages.
SP-MHCII coexpressing cells were not found on the wall of
lymphatics and/or in the mesentery (Fig. 2F).
Characterization of macrophages associated with lymphatic
collecting vessels. As shown in Fig. 1, there was a significant
population of CD11b⫹ cells that were recruited in abundance
to the mesenteric lymphatic collecting vessels in response to
LPS in a time-dependent manner (Fig. 1C). To further characterize the polarization of these cells, we performed immunofluorescence for the M1 marker MHCII and the common M2
markers CD163 and CD206 in 72-h LPS-treated animals and
their corresponding controls. Under control physiological conditions, we observed at a minimum two distinct and prominent
macrophage populations invested within the mesenteric neurovascular bundles that were identified as CD163⫹CD206⫹ or
MHCII⫹ (Fig. 3). The CD163⫹CD206⫹M2 macrophages were
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Fig. 1. Induction of inflammation in response to LPS. Lymph nodes and liver cryostat sections were made from both control and LPS-injected animals. A: Mason
trichrome staining was carried out on the liver and lymph node sections. Blue color indicates fibrotic areas, and red indicates normal cellular area. B: lymph node
and liver sections were also stained with myeloperoxidase to ascertain the degree of inflammation. The percentage of fluorescent intensity was calculated as
detailed in the METHODS section and plotted. A minimum of four to six sections was quantified per animal. Data are represented as means ⫾ SE. aP ⬍ 0.05;
n ⫽ 3. C: whole-mount mesenteric arcades harvested at 6 h, 24 h, and 72 h LPS postinjection were stained for the CD11b along with the control. Top: recruitment
of CD11b⫹ cells in response to LPS on the mesenteric lymphatic vessel. Confocal images were acquired at ⫻40. Bottom: time-dependent increases in infiltration
of CD11b⫹ cells at 6 h, 24 h, and 72 h in the clear field mesentery. Confocal images were acquired at ⫻20. A common scale bar for all figure panels is indicated.
Scale bar: 150 ␮m.
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Fig. 2. LPS causes significant decreases in the neutrophil population in the vicinity of the lymphatics. A: whole-mount mesenteric arcades from control and
LPS-injected animals were stained for the neutrophil-specific serine protease NE at 6 h, 24 h, and 72 h postinjection. Confocal images were acquired at ⫻20.
Scale bar: 150 ␮m. A single scale bar for entire panel is indicated. Left: neutrophil elastase (NE) staining near the lymphatics and on the lymphatic vessel wall.
Right: quantification of the neutrophils on or near the lymphatic vessel in response to LPS at 6 h, 24 h, and 72 h compared with the untreated control. A minimum
of five ⫻20 fields were counted and averaged per animal (n ⫽ 6). Data are presented as means ⫾ SE. *Significant difference, P ⬍ 0.05. B: confocal images
of whole-mount mesenteric preparations from LPS-treated rats showing that neutrophils (red) adhere to the lymphatic vessel muscle layer stained by ␣-smooth
muscle actin (SMA; green). Magnification: ⫻63; scale bar: 50 ␮m. C: orthogonal projection of a lymphatic vessel, i.e., cross sections through the vessel wall
in the x–z and y–z directions on the zoomed rotated and cropped part of the “Merge” image. The yellow lines depicting locations of the cross sections that show
the neutrophils between and under lymphatic muscle cells (LMC) fibrils. Scale bar: 50 ␮m. D: significant increase in numbers of neutrophils were observed in
lymph node sections from control and LPS-treated animals stained for NE. The percentage of fluorescent intensity was calculated and plotted. Minimum of four
to six sections were quantified per animal. Data are represented as means ⫾ SE; aP ⬍ 0.05; n ⫽ 3. A common scale bar for all figure panels is indicated. Scale
bar: 50 ␮m. E: confocal images of whole mount mesentery showing that neutrophils also express substance P (SP). Many other immune cells on the mesenteric
lymphatics apart from neutrophils also express SP. Magnification: ⫻20; scale bar: 150 ␮m. F: SP and MHCII costain was also carried out. MHCII and SP stain
separate populations of cells. Magnification: ⫻20; scale bar: 75 ␮m.
diastolic diameter in the 72-h LPS-treated animals (data not
shown). However, both the diastolic diameter and stroke
volume were significantly reduced in the 6-h LPS animals
(diameter: 82.40 ⫾ 20.74 in LPS vs. 144.33 ⫾ 12.01 in
controls; and stroke volume: 1.88 ⫾ 0.86 in LPS vs. 7.98 ⫾
2.55 in controls).
To further determine whether LPS would directly influence
the lymphatic contractile activity, the isolated mesenteric lym-
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Fig. 3. Macrophage accumulation and M2 polarization in the LPS-treated rat mesentery. Top: mesenteric arcades from control (A–E) and intraperitoneal LPS-injected
(F–J) were stained for CD163 (green) and CD206 (red). Arrows indicate the single-positive CD163-CD206⫹ cells. Mesenteric lymphatic collecting vessels are outlined
with dashed white lines. K: quantification of the M2 macrophage phenotypes in control and LPS-injected rats; *P ⬍ 0.05. DP denotes double-positive (CD163⫹206⫹)
cells; SP denotes single-positive cells (CD163⫺206⫹). A minimum of five ⫻40 fields were quantified and averaged per animal (n ⫽ 4). Within the FOV for every image
stack analyzed, cells that were directly in contact with the vessel were counted and represented as “vessel”, and the remaining cells were considered to be in the
“periphery”. Magnification in A, B, F, G: ⫻20 magnification; scale bar ⫽ 150 ␮m. A common scale bar for all figure panels is indicated. Magnification in C, D, E, H,
I, J: ⫻40; scale bar ⫽ 75 ␮m. A common scale bar for all figure panels is indicated . Data are represented as means ⫾ SE. *Significant difference, P ⬍ 0.05. L–R: LPS
treatment does not promote M1 but M2 macrophage phenotype on and around the mesenteric lymphatics. Control (L–N) and LPS (O–Q)-treated animals were stained
for MHCII (blue) and CD206 (red). Magnification in L and O: ⫻40; scale bar: 75 ␮m. Magnification in M and P: ⫻20 magnification; scale bar: 150 ␮m. A common
scale bar for all figure panels is indicated. A minimum of five ⫻40 fields were quantified and averaged per animal (n ⫽ 4).
phatic vessels were treated with LPS as described in MATERIALS
As shown in Fig. 4E, the vessels that were
treated directly with LPS showed a significant decrease in
phasic contraction frequency at both 5 and 7 cmH2O compared
with the sham controls.
AND METHODS.
Differential expression of key adhesion molecules on LMCs
in response to LPS. As we found distinct shifts in the recruitment of immune cells in the vicinity of the lymphatics in
response to LPS, we analyzed the expression of key cell
adhesion molecules that are known to regulate this process in
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cultured LMCs. LPS significantly increased the expression of
VCAM1 and ICAM1 at 24 h and 72 h compared with the
untreated controls. Expression of CXCR2, a key receptor for
the neutrophil-associated cytokine IL-8 showed a slight decrease at 24 h but was significantly reduced in LPS-treated
LMCs at 72 h. Galectin 9, another neutrophil chemoattractant
was significantly reduced at both 24 h and 72 h in LPS-treated
cells (Fig. 5A). Our real-time PCR data further corroborated
these findings in LPS-treated LMCs (Fig. 5B).
LPS activates parallel inflammatory and contractile pathways in LMCs in a p-AKT and p-ERK dependent manner. To
further investigate the detailed molecular mechanisms underlying LPS-induced lymphatic contractile impairment, we analyzed the effects of LPS treatment on LMCs at varying concentrations and times. LPS treatment (20 –100 ng/ml) significantly inhibited MLC20 phosphorylation (Fig. 6A). Further, to
ascertain that LPS acts through Toll-like receptor (TLR), we
used the TLR4 inhibitor (LPS-RS) and evaluated its effects on
pMLC20, both in the presence and absence of LPS (Fig. 6B). In
the presence of TLR4 inhibitor, MLC20 in 24-h LPS-treated
LMCs, reverted to normal levels, but MLC20 phosphorylation
in 72-h LPS-treated LMCs was significantly increased. Further,
LPS was found to significantly increase p-AKT, p-ERK, and
pI-␬B␣ levels in LMCs at both 24 h and 72 h (Fig. 6C).
Further, to elucidate the roles of p-AKT and p-ERK in modulating downstream signaling by LPS, we used p-AKT inhibitor (LY294002) or p-ERK inhibitor (PD98059) in conjunction
with LPS on LMCs. Inhibition of p-ERK was found to significantly reduce pERK1/2 and I␬B␣ levels, both in the presence
or absence of LPS (Fig. 6D). Similarly, LY294002 caused a
significant reduction of p-AKT levels. Interestingly pMLC20
was found to increase when p-AKT was suppressed both in the
presence or absence of LPS (Fig. 6E). These results suggest
that two parallel pathways may be activated in LMCs by LPS
signaling through TLR4, one that depresses MLC20 phosphorylation levels in LMCs via activation of AKT phosphorylation
and the second that increases the downstream inflammatory
pathways regulated by pI-␬B␣ by its effects on ERK phosphorylation. Because cytokines released by immune cells have
been shown to affect muscle tone and function, we used LMCs
to evaluate the effects of IL-8, one of the key cytokines
released by neutrophils, as well as a prominent neutrophil
chemoattractant. As shown in Fig. 6F, IL-8 treatment increased
pMLC20 significantly in LMCs at different time points, and
IL-8 was shown to induce levels of pMLC20, even in the
presence of LPS.
LPS activates NF-␬B in LMCs. LMCs treated for 6 h and 24
h with LPS showed a robust increase in NF-␬B nuclear
translocation in a time-dependent manner (Fig. 7A). This was
also associated with increases in the levels of p-I␬B in LMCs
on treatment with LPS (Fig. 7B). Similar response was also
seen in mesenteric lymphatic vessels isolated from control and
6 h LPS-treated animals with a rapid increase in nuclear NF-␬B
levels in lymphatic vessels from LPS animals (Fig. 7C).
Inflammatory cytokines and chemokines are induced in the
mesenteric lymphatic bed in response to LPS. We sought to
determine the expression of key immunomodulatory and inflammatory cytokines within the specific tissue space in the
mesentery in response to LPS-induced inflammation. Mesenteric arcades from untreated and 24-h LPS-treated rats were
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Fig. 4. LPS causes significant impairment of
collecting lymphatic vessels. Pressurized isolated lymphatic vessels from control, and 72-h
LPS-treated animals were subjected to transmural pressures of 1, 3, and 5 cmH2O. Lymphatic tonic index (A), frequency (B), lymph
pump flow (C), and fractional pump flow (D)
were measured as described in the MATERIALS
AND METHODS. E: contraction frequency measured in vessels mounted on a pressure myograph after a 24-h incubation in media without
(Sham) and with 10 ␮g/ml LPS. Data are
presented as means ⫾ SE. Two-way ANOVA
was carried out. * and ** denote significance
at P ⬍ 0.05 and P ⬍ 0.01 vs. sham, respectively. Dark bars denote control, and striped
bars denote LPS-treated.
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INFLAMMATION AND IMMUNE CELLS IMPACT LYMPHATIC FUNCTION
subjected to protein cytokine array analysis. Among the 29
inflammatory cytokines analyzed, LPS stimulation significantly upregulated the expression of 10 cytokines by more than
twofold compared with the untreated controls (Fig. 7D), indicating a complex cytokine signaling in the mesenteric milieu in
response to an inflammatory signal. As expected for acute
inflammation, six of those were found to be chemoattractants
for neutrophils, macrophages, and monocytes. These included
cytokine-induced neutrophil chemoattractant 1 (CINC1), cytokine-induced neutrophil chemoattractant 2, or macrophage inflammatory protein 2␣ (CINC2␣ also known as MIP2␣),
macrophage inflammatory protein 1␣ (MIP1␣ also known as
CCL3), macrophage inflammatory protein 3␣ (MIP3␣ also
known as CCL20), LPS-inducible CXC chemokine or chemokine (C-X-C motif) ligand 5 (LIX also known as CXCL5) and
chemokine (C-X3-C motif) ligand 1 (CX3CL1 also known as
fractalkine). In addition, proinflammatory interleukins such as
IL-1␣, IL-1␤, and anti-inflammatory IL-1ra were also induced
in the mesenteric beds. Further, elevated levels of, tissue
inhibitor of metalloproteases (TIMP1) and monokine induced
by IFN-␥ (MIG) were also observed (Fig. 7D).
DISCUSSION
The data presented in this study are the first to identify the
effects of LPS on temporal and spatial dynamics of immune
cells (specifically neutrophils and macrophages) associated
with lymphatics and their impact on lymphatic contractility
and function. Using a rat model of LPS-induced peritonitis,
we focus on the lymphatic mesenteric plexus, which is
subject to the most dynamic physiological changes in fluid
flow (associated with feeding patterns) and alterations in the
surrounding microenvironment due to interactions between
inflammatory mediators, cytokines, chemokines, and immune cells. In this study, we provide clear evidence that
LPS causes significant impairment of lymphatic contractility in both tonic and phasic contractile parameters and, as a
result, inhibits lymphatic pump flow. Further, we demonstrate that exposure to exogenous LPS causes significant
changes in the recruitment of neutrophils and macrophages,
and favors a shift to a M2 macrophage polarized state that
acts in parallel with activation of a number of inflammatory
cytokines and chemokines in the mesenteric beds (Fig. 8).
Fig. 6. LPS activates contractile and inflammatory pathways in a pAKT and p-ERK-dependent manner. A: representative Western blot showing the expression of
pMLC20 in LMCs in the absence or presence of LPS in a dose-dependent manner. B: representative Western blot showing that LPS-mediated inhibition of pMLC20 is
TLR4 dependent. LMCs were treated with LPS and polymixin B (TLR4 inhibitor) for 72 h, and the effects on pMLC20 was determined. Bottom: relative expression
of pMLC20/MLC20 was quantified and plotted. Data are represented as means ⫾ SE. *P ⬍ 0.05. C, top: representative Western blots showing LPS-mediated activation
of pAKT, pERK, p-I-␬B, and inhibition of pMLC20 at 24 h and 72 h after treatment. Bottom: relative expression of pAKT/AKT, pERK/ERK, p-I-␬B/I-␬B, and
pMLC20/MLC20 was quantified and plotted. Data are represented as means ⫾ SE. *P ⬍ 0.05. D, left: representative Western blots showing the expression of pERK
and p-I-␬B in the presence of ERK inhibitor, PD98059. Right: quantification from triplicate experiments. *P ⬍ 0.05 vs. control; #P ⬍ 0.05 vs. LPS. E, left: representative
Western blots showing that the expression of pAKT and MLC20 in the presence of AKT inhibitor, LY294002. Right: quantification from triplicate experiments. *P ⬍
0.05 vs. control; #P ⬍ 0.05 vs. LPS. F, top: representative Western blots showing effects of IL-8 on pMLC20 in LMCs in the absence or presence of LPS. Bottom: relative
expression of pMLC20/MLC20 and pAKT/AKT was quantified and plotted. Data are represented as means ⫾ SE. *P ⬍ 0.05; n ⫽ 3.
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Fig. 5. LPS modulates the levels of key
adhesion molecules and chemoattractants on
LMCs. A, left: representative Western blot
showing the expression of VCAM1 ICAM1,
CXCR2, and Gal-9 in LMCs in the absence
and presence of LPS in a time-dependent
manner. Right: quantitative values from
three independent experiments are plotted as
a ratio vs. housekeeping control ␤-actin.
Data are represented as means ⫾ SE; *P ⬍
0.05; n ⫽ 3. B: quantitative real-time PCR
showing the expression of VCAM1 ICAM1,
CXCR2, and Gal-9. Real-time quantification
was carried out, and the relative fold change
over the untreated control was calculated.
RPL19 was used as the housekeeping control. Experiments were done in triplicate,
and the values are presented as means ⫾ SE.
*P ⬍ 0.05.
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The rat model of LPS used in this study does cause a
systemic inflammation, as shown in the immunohistochemical
staining of the liver and lymph node sections (Fig. 1) and, in
fact, very closely resembles a peritonitis model, as demonstrated by Kim et al. (47). In addition, this model has pronounced global effects immediately after administration of
LPS, as rats exhibited raised body temperature, huddled posture, slow gait, and symptoms of uveitis. Further, molecular
data suggest that systemic responses are created in the mesenteric bed in response to LPS as a number of inflammatory
cytokines and chemokines, known mediators of inflammatory
responses, appeared dysregulated in the mesenteric tissue arcades (Fig. 7D).
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Fig. 7. LPS causes activation of the NF-␬B
and proinflammatory cytokines and chemokines in the lymphatics. A: LPS activates
NF-␬B in cultured mesenteric LMCs. LMCs
were treated with LPS (20 ng/ml) for 6 h and
24 h, and nuclear translocation of NF-␬B
was assessed using immunofluorescence.
Magnification is ⫻20. B: LPS activates I-␬B
in cultured mesenteric LMCs. LMCs were
treated with LPS (20 ng/ml) for 6, 24, and 72
h, and Western blot analysis was carried out.
Magnification is ⫻20. C: LPS activates
NF-␬B in mesenteric lymphatic vessels.
Mesenteric lymphatic vessels isolated from a
6-h LPS-treated rat were assessed for nuclear translocation of NF-␬B using immunofluorescence. Magnification is ⫻20. Scale
bar: 150 ␮m. D: LPS induces the expression
of several cytokines and chemokines in lymphatic mesenteric tissue arcades. Proteins
isolated from mesenteric tissue arcades from
untreated and LPS-treated animals were analyzed by inflammatory cytokine array.
Mean pixel density of each analyte was
quantified and plotted and represented as
fold change over control. Data are represented as means ⫾ SE. B and D: *P ⬍ 0.05;
n ⫽ 3.
LPS causes endotoxin tolerance in the mesentery. Our data
clearly indicate that there are many neutrophils adhered to
and/or present beneath the muscle cells of the afferent collecting lymphatic vessels (Fig. 2, B and C), and 72 h after initiation
of the LPS challenge, there is a dramatic decrease in the
number of neutrophils in the vicinity of the lymphatics (Fig.
2A). Furthermore, immunohistochemical analyses of the mesenteric lymph nodes from the same animals indicate a significant upregulation in the numbers of neutrophils in the lymph
node (Fig. 2D). Hence, we speculate that in response to LPS,
neutrophils migrate through the mesenteric lymphatic vessels
to reach the lymph nodes, where they may be important players
in eliciting an immune response. Indeed, the ability of neutro-
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INFLAMMATION AND IMMUNE CELLS IMPACT LYMPHATIC FUNCTION
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phils to migrate to lymph nodes has been previously documented. Abadie et al. (1) provided in vivo evidence that
neutrophils migrate via afferent lymphatics to lymphoid tissues
and can shuttle live microorganisms there. It has also been
shown that neutrophils are recruited to sites of inflammation
and can then coordinate lymph node lymphangiogenesis and
modulate immune responses (22, 71). The confocal imaging
data, together with the finding that mesenteric lymphatic arcades from LPS-injected animals show a significant induction
of several neutrophil chemoattractants such as CINC1, CINC2,
MIP1␣ (Fig. 7D), support our speculation of inflammationmediated neutrophil migration through collecting lymphatics.
However, additional experiments using fluorescence-tagged
neutrophils studying their migration in the mesenteric region
are warranted.
As noted earlier, we observed significant populations of
CD11b⫹ cells in and around the lymphatic vessels and surrounding mesenteric tissue beds, which based solely on
CD11b⫹ staining could be monocytes, macrophages, or even
activated neutrophils (47). However, the CD11b⫹ cells identified in this population of immune cells in our studies did not
costain with the neutrophil-specific marker, NE (data not
shown), indicating that these cells were either macrophages or
monocytes, as previously described by Kim et al. (47). Previous microarray data collected on the mesenteric collecting
vessels demonstrated a large immune fingerprint, particularly
that of antigen-presenting cells (13). The data presented in this
study corroborate those findings, as a strong contingent of
MHCII⫹ macrophages were seen not only in the adipose tissue
surrounding the mesenteric lymphatics but also invested within
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Fig. 8. A: schematic representation of LPS
mediated immune cell modulation and impairment of lymphatic function. Neutrophils
and macrophages are present in the lymphatic mesenteric bed in normal physiological conditions. After 3-day LPS challenge, a
significant decrease in neutrophils and increase in M2 polarized macrophages is noted
in the vicinity of the lymphatics. LPS significantly reduces key neutrophil-associated
cell adhesion molecules as CXCR2 and
Gal-9 on the LMCs, while activating ICAM1
and VCAM1 that attract monocytes and
macrophages. LPS further causes a significant impairment of lymphatic contractility
by directly activating NO-mediated inhibitory signaling by activation of NF-␬B. The
alterations in the immune cell population
on/near the mesenteric lymphatics also cause
changes in the complex cytokine-mediated
signaling that affect lymphatic vessel function. B: schematic representation of LPSTLR4 pathways in LMCs. In the LMCs, LPS
signals through the TLR4 receptor and activates parallel contractile and inflammatory
pathways by activation of pAKT and pERK,
respectively. Inhibition of ERK inhibits
I-␬B-mediated induction of inflammatory
cytokines, NO and NF-␬B activation. On the
other hand, induction of pAKT represses
pMLC20. IL-8 may also directly activate
pMLC20.
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INFLAMMATION AND IMMUNE CELLS IMPACT LYMPHATIC FUNCTION
neutrophils and to increase macrophage recruitment by reduction of some of its key receptors and adhesion molecules. In
addition, the neutrophils that we identified as adhering to the
mesenteric lymphatics also strongly expressed SP. We have
previously shown that SP is a strong modulator of lymphatic
contractility and activates contractile pathways in the lymphatics (4, 18, 26). Although SP is released by C-sensory fibers in
the vicinity of the lymphatics, changes in SP levels from
surrounding immune cells, such as the neutrophils, may also
affect lymphatic contractile functions. Thus, our data suggest
that cytokines directly expressed by immune cells in the
vicinity of the lymphatics could be potentially involved in
modulating the lymphatic contractile function in response to
various immune and inflammatory stimuli.
Corroborating these findings, our cytokine array analysis
provides novel insights into the complex crosstalk of cytokines
and chemokines in the mesenteric bed in response to LPS that
could play a critical role in determining the balance between
inflammation progression and resolution. Several key neutrophil and macrophage/monocyte chemoattractants (CINC1,
CINC2␣, MIP1␣, MIP3␣, CXCL5, and CX3CL1) were induced in mesenteric lymphatic beds in response to LPS (Fig.
7D). This is interesting as CINC1 is a member of the IL-8
family and is a major neutrophil chemoattractant released in
response to tissue injury (15). Similarly, members of the MIP1
family of proteins play a critical role in leukocyte chemotaxis,
as well as the production of proinflammatory cytokines, and
they are fundamental components of the acute phase response
to sepsis (59). Proinflammatory interleukins such as IL-1␣,
IL-1␤, as well as anti-inflammatory IL-1ra were also induced
in the lymphatic mesenteric beds, indicating that the lymphatics may actively participate both in the progression and resolution of inflammation. Furthermore, TIMP1 that is known to
be associated with tissue degradation and remodeling is elevated in inflamed lymphatic tissue beds. CX3CL1, or fractalkine, is directly involved in trafficking of antigen-loaded
dendritic cells from the periphery via afferent lymphatics to
draining lymph node during tissue inflammation and has been
significantly correlated with the severity of inflammatory diseases (41, 73). As it was significantly induced in mesenteric
lymphatic arcades from the LPS-treated group, this could be
setting the stage for a more widespread systemic inflammatory
response due to LPS. It is not clear whether the production of
cytokines and chemokines is a cause or consequence of the
immune cell infiltration or, more likely, a result of the systemic
inflammatory mechanisms activated by LPS on the mesenteric
tissue bed.
LPS-TLR4 pathway activates both contractile and inflammatory pathways in LMCs. We have previously demonstrated
that the lymphatic pump is regulated by intrinsic forces, pressure/stretch, and flow/shear, as well as extrinsic mechanisms
such as humoral/neural actions (reviewed in Refs. 17, 30, 81).
One of the most common mechanisms mediating lymphatic
contractile function is the regulation of the phosphorylation
status of the contractile regulatory protein MLC20 (57, 78). We
conjectured that LPS would have a direct role on the lymphatic
contractile machinery by regulating levels of pMLC20, as well
as the cytokines potentially released by the immune cells, or
lack of their availability by changes in immune cell dynamics
would affect the LMC contractile apparatus. Our data show
that LPS directly represses phosphorylation of MLC20 and
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and along the walls of the mesenteric collecting vessels. These
MHCII⫹ macrophages appear to represent M1 skewed cells,
and their tropism for the vessel wall suggests that specific
chemoattractants would be mediating this effect. The chemokine CX3CL1, or fractalkine, that we found to be significantly
induced in the lymphatic beds (Fig. 7) is actively involved in
the recruitment of monocytes/macrophages to inflamed tissue
(73). Although LPS has been associated with the activation of
M1 inflammation, the CD163⫹CD206⫹ M2 phenotype expanded significantly in the LPS-injected animals and was
closely associated with lymphatic tissues. In our study, we
found an increase in the number of CD163⫺CD206⫹ singlepositive cells near the lymphatic vessels as well. These
CD163⫺CD206⫹ cells may be similar to the M2b phenotype
that are also induced by TLR stimulation and are involved in
inflammation resolution. These M2 macrophages are found
within the adipose tissue and along the mesenteric vessels
under physiological conditions, and they are likely of the
fibrotic/tissue repair phenotype. In response to TLR stimulation M2b macrophages that often lack CD163 express CD206
in addition to producing high levels of TNF␣, IL-10, and nitric
oxide (NO) (8). Macrophages, particularly the M2 subpopulation, can mediate tissue repair and growth by the release of a
number of growth-promoting factors and cytokines, including
the anti-inflammatory IL-10.
Our finding that neutrophils are significantly decreased in
the vicinity of the lymphatics coupled with the propagation of
an M2 anti-inflammatory macrophage phenotype after repeated
injections of LPS may be indicative of endotoxin tolerance, a
phenomenon where cells show reduced responsiveness toward
repeated endotoxin or TLR4 stimulation. Recent evidence
suggests that both proinflammatory and anti-inflammatory processes occur early and simultaneously in endotoxin-mediated
sepsis and that immunosuppression is the predominant driving
force for mortality and morbidity in sepsis (39). This is also in
keeping with the recently discovered immunosuppressive roles
of neutrophils in inflammation progression (10). Because of
this characteristic desensitization response, endotoxin tolerance is considered a regulatory mechanism to balance inflammation (7). Pena et al. (61) have shown that substantial similarities exist between M2 polarization and endotoxin tolerance
and suggest that this phenomenon can be considered another
form of alternative activation triggered by bacterial signatures
such as LPS. We surmise that the alterations in the lymphatic
vessel-associated neutrophils and predominant M2 phenotype
that we observe as an acute response to LPS set the stage for
a more profound systemic inflammatory response of the host.
LPS causes conducive environment in LMCs and mesentery
for regulating neutrophils and macrophages. IL-8 acts as a
chemoattractant through CXCR1 and CXCR2 G protein-coupled receptors. During septicemia, neutrophil responsiveness to
IL-8 and other CXC chemokines is reduced via TNF-␣ mediated downregulation of CXCRl and CXCR2 (68, 72). Supporting the existence of a similar mechanism in lymphatics, we
also find that LPS significantly represses CXCR2 in LMCs
(Fig. 5). Further LPS significantly reduced the expression of
Gal-9, another neutrophil chemoattractant, in the LMCs. On
the other hand, LPS significantly increased the expression of
ICAM1 and VCAM1, which are known monocyte and macrophage adhesion molecules (21, 46). Hence, LMCs seemed to
be primed by LPS challenge to reduce the recruitment of
INFLAMMATION AND IMMUNE CELLS IMPACT LYMPHATIC FUNCTION
phatic vessel function in TNBS-treated guinea pigs (80). Corroborating these findings, we have recently shown that lymph
transport significantly decreased after TNBS treatment, and
these changes were preceded by increased numbers of MHCII⫹ cells surrounding mesenteric lymphatics leading to an
altered lymphatic environment favoring dysfunction (23).
These recent findings coupled with the results of this study
underscore the direct link between inflammation-induced activation of immune cell dynamics and alterations in the lymphatic microenvironment with impairment of lymphatic function. While some cytokines have been individually reported to
have an effect on lymphatic contractility (2, 3), as shown in
Fig. 8, our study is the first to demonstrate how inflammation
alters the surrounding immune cells near the lymphatics in vivo
and also activates global inflammatory pathways within the
lymphatic cells. A combination of these events influences the
immune cells’ recruitments and trafficking through the lymphatics. We can speculate that the alterations in cytokines and
chemokines that we observed in response to LPS is an effect of
these dynamic shifts in the immune populations in response to
an inflammatory insult and that these changes, in turn, affect
lymphatic function. It is also possible that these changes are
key in determining whether there will be a progression or
resolution of inflammation by the initial triggering of a systemic response to a pathogenic insult. We know that lymph
flow moves not only fluid and macromolecules but also many
immune cells and, thus, impairment of pump function could
well relate to temporal changes in immune cell numbers.
Indeed, recent work by our lab group and our collaborators has
shown that these cells then traffic to the lymph nodes via the
afferent lymph flow (48, 62). So although we did not directly
measure this in this study, we can infer that the impaired lymph
pump flow will impede the removal of fluid, macromolecules,
and immune cells from the tissues, thereby precipitating further
inflammatory insult, as we observed recently (23). Further
studies are also warranted to carefully evaluate the role of the
specific chemokines and cytokines and cell adhesion molecules
that were identified to be dysregulated in the mesenteric lymphatics on lymphatic contractility. However, our study sets the
stage for further investigation in this area aimed at identifying
other immune cell-mediated alterations in inflammatory pathways that directly affect lymphatic contractility and pump
function and are key determinants of progression or resolution
of inflammation.
GRANTS
This work was supported by National Institutes of Health RO1-DK-99221
to M. Muthuchamy and D. Zawieja.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: S.C., S.D.Z., P.-Y.v.d.W., D.C.Z., and M.M. conception and design of research; S.C., S.D.Z., W.W., Y.L., and Y.J.W. performed
experiments; S.C., S.D.Z., W.W., Y.L., Y.J.W., P.-Y.v.d.W., D.C.Z., and
M.M. analyzed data; S.C., S.D.Z., W.W., Y.L., P.-Y.v.d.W., D.C.Z., and M.M.
interpreted results of experiments; S.C., S.D.Z., and M.M. prepared figures;
S.C., S.D.Z., and M.M. drafted manuscript; S.C., S.D.Z., P.-Y.v.d.W., D.C.Z.,
and M.M. edited and revised manuscript; S.C., S.D.Z., W.W., Y.L., Y.J.W.,
P.-Y.v.d.W., D.C.Z., and M.M. approved final version of manuscript.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00467.2015 • www.ajpheart.org
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activates parallel pathways mediated by the phosphorylation of
AKT and ERK to modulate lymphatic contraction and inflammation, respectively (Fig. 6C). Interestingly, LPS increased
p-AKT levels while decreasing pMLC20. It has been shown
that p-AKT inhibits pMLC20 by inhibiting pMYPT1 (51). We
have previously shown that increased p-MYPT1 directly correlates with increased pMLC20 in the LMCs in response to
inflammatory agonist (18). Hence, our data provide the basis
for a novel mechanism of pMLC20 inhibition in LMCs by
activation of p-AKT (Fig. 8). This was further corroborated, as
inhibition of p-AKT increased pMLC20 levels (Fig. 6E). Previously, we have also demonstrated that p-ERK is required for
MLC20 phosphorylation (18). Hence, we concluded that pERK was not playing a direct role in MLC20 phosphorylation
in response to LPS, as effects of LPS elicited opposite responses (Fig. 6C). However, p-ERK inhibition by pharmacological inhibitors in the presence of LPS directly affected
pI-␬B and, hence, was mediating the inflammatory signaling
downstream of LPS (Fig. 6D).
In addition to activation of various immune cells, LPS
directly stimulates inflammatory cellular responses in LMCs
through TLR4, which subsequently activates the downstream
signaling mechanisms and activation of NF-␬B (83). Previous
studies in sheep show that although endotoxin impaired lymphatic pumping, it did not have a direct effect on the lymphatic
vessels; the LPS-mediated effects were believed to have occurred indirectly through interactions with cellular and humoral factors in blood and lymph (27, 28). A number of recent
studies have demonstrated that inflammatory cytokines significantly affect lymphatic contractile functions, as well as endothelial permeability (3, 24, 43). However, except for one study
in bovine mesenteric lymphatic vessels, the direct effects of
LPS on LMCs have not been previously investigated (53). Our
data are, however, the first to clearly delineate different molecular pathways activated by LPS in LMCs and show that it
initiates proinflammatory pathways in LMCs by causing nuclear translocation of NF-␬B and upregulation of pI-␬B levels.
NF-␬B induction can be directly correlated with induction of
iNOS and NO, which have been documented as a crucial
regulator of lymphatic contractile activity, and high NO levels
leads to strong pumping inhibition (20). Liao et al. (52) have
shown that bone marrow-derived Cd11b⫹ myeloid cells expressing iNOS infiltrate the tissue surrounding the contractile
lymphatics during inflammation and inhibit autonomous lymphatic contraction. This is significant, as we have demonstrated
the recruitment of large numbers of Cd11b⫹ cells on the
collecting lymphatics in response to LPS, and our data support
a direct induction of NF-␬B pathways that may mediate downstream NO signaling. It must also be noted that in addition to
the initiation of inflammatory cascades, the activation of
NF-␬B is also involved in resolution of inflammation and M2
polarization of tumor-associated macrophages (36, 50, 63).
Taken together, our data suggest that the mesenteric lymphatic vessels are highly involved in regulating the inflammatory process, and their contractility is subject to control by
immune activation in the vicinity of the vessel, as well as
inflammatory signaling cascades activated within the tissue.
Immune cell trafficking and its relation with lymphatic dysfunction constitute a relatively underexplored area of lymphatic research. We have previously demonstrated that during
inflammatory conditions, there is a significant loss of lym-
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INFLAMMATION AND IMMUNE CELLS IMPACT LYMPHATIC FUNCTION
REFERENCES
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00467.2015 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 15, 2017
1. Abadie V, Badell E, Douillard P, Ensergueix D, Leenen PJ, Tanguy M,
Fiette L, Saeland S, Gicquel B, Winter N. Neutrophils rapidly migrate
via lymphatics after Mycobacterium bovis BCG intradermal vaccination
and shuttle live bacilli to the draining lymph nodes. Blood 106: 1843–
1850, 2005.
2. Al-Kofahi M, Becker F, Gavins FN, Woolard MD, Tsunoda I, Wang
Y, Ostanin D, Zawieja DC, Muthuchamy M, von der Weid PY,
Alexander JS. IL-1␤ reduces tonic contraction of mesenteric lymphatic
muscle cells, with the involvement of cycloxygenase-2 and prostaglandin
E2. Br J Pharmacol 172: 4038 –4051, 2015.
3. Aldrich MB, Sevick-Muraca EM. Cytokines are systemic effectors of
lymphatic function in acute inflammation. Cytokine 64: 362–369, 2013.
4. Amerini S, Ziche M, Greiner ST, Zawieja DC. Effects of substance P on
mesenteric lymphatic contractility in the rat. Lymph Res Biol 2: 2–10,
2004.
5. Angeli V, Llodra J, Rong JX, Satoh K, Ishii S, Shimizu T, Fisher EA,
Randolph GJ. Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobilization. Immunity 21: 561–574, 2004.
6. Azuma K, Akiyama M, Ebata T, Totsuka M, Hayasaka H. Endogenous
endotoxin absorption and the role of intestinal lymphatics. Jpn J Surg 13:
535–539, 1983.
7. Bain CC, Mowat AM. The monocyte-macrophage axis in the intestine.
Cell Immunol 291: 41–48, 2014.
8. Bain CC, Scott CL, Uronen-Hansson H, Gudjonsson S, Jansson O,
Grip O, Guilliams M, Malissen B, Agace WW, Mowat AM. Resident
and pro-inflammatory macrophages in the colon represent alternative
context-dependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunol 6: 498 –510, 2013.
9. Benoit JN, Zawieja DC, Goodman AH, Granger HJ. Characterization
of intact mesenteric lymphatic pump and its responsiveness to acute
edemagenic stress. Am J Physiol Heart Circ Physiol 257: H2059 –H2069,
1989.
10. Beyrau M, Bodkin JV, Nourshargh S. Neutrophil heterogeneity in
health and disease: a revitalized avenue in inflammation and immunity.
Open Biol 2: 120134, 2012.
11. Bohlen HG, Gasheva OY, Zawieja DC. Nitric oxide formation by
lymphatic bulb and valves is a major regulatory component of lymphatic
pumping. Am J Physiol Heart Circ Physiol 301: H1897–H1906, 2011.
12. Bohlen HG, Wang W, Gashev A, Gasheva O, Zawieja D. Phasic
contractions of rat mesenteric lymphatics increase basal and phasic nitric
oxide generation in vivo. Am J Physiol Heart Circ Physiol 297: H1319 –
H1328, 2009.
13. Bridenbaugh EA, Wang W, Srimushnam M, Cromer WE, Zawieja
SD, Schmidt SE, Jupiter DC, Huang HC, Van Buren V, Zawieja DC.
An immunological fingerprint differentiates muscular lymphatics from
arteries and veins. Lymph Res Biol 11: 155–171, 2013.
14. Butterfield TA, Best TM, Merrick MA. The dual roles of neutrophils
and macrophages in inflammation: a critical balance between tissue
damage and repair. J Athl Train 41: 457–465, 2006.
15. Campbell SJ, Hughes PM, Iredale JP, Wilcockson DC, Waters S,
Docagne F, Perry VH, Anthony DC. CINC-1 is an acute-phase protein
induced by focal brain injury causing leukocyte mobilization and liver
injury. FASEB J 17: 1168 –1170, 2003.
16. Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D,
Neyrinck AM, Fava F, Tuohy KM, Chabo C, Waget A, Delmee E,
Cousin B, Sulpice T, Chamontin B, Ferrieres J, Tanti JF, Gibson GR,
Casteilla L, Delzenne NM, Alessi MC, Burcelin R. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 56: 1761–1772,
2007.
17. Chakraborty S, Davis MJ, Muthuchamy M. Emerging trends in the
pathophysiology of lymphatic contractile function. Sem Cell Dev Biol 38:
55–66, 2015.
18. Chakraborty S, Nepiyushchikh Z, Davis MJ, Zawieja DC, Muthuchamy M. Substance P activates both contractile and inflammatory
pathways in lymphatics through the neurokinin receptors NK1R and
NK3R. Microcirculation 18: 24 –35, 2011.
19. Chakraborty S, Zawieja DC, Davis MJ, Muthuchamy M. MicroRNA
signature of inflamed lymphatic endothelium and role of miR-9 in lymphangiogenesis and inflammation. Am J Physiol Cell Physiol 309: C680 –
C692, 2015.
20. Chatterjee V, Gashev AA. Aging-associated shifts in functional status of
mast cells located by adult and aged mesenteric lymphatic vessels. Am J
Physiol Heart Circ Physiol 303: H693–H702, 2012.
21. Chen Q, Zhang XH, Massague J. Macrophage binding to receptor
VCAM-1 transmits survival signals in breast cancer cells that invade the
lungs. Cancer Cell 20: 538 –549, 2011.
22. Chtanova T, Schaeffer M, Han SJ, van Dooren GG, Nollmann M,
Herzmark P, Chan SW, Satija H, Camfield K, Aaron H, Striepen B,
Robey EA. Dynamics of neutrophil migration in lymph nodes during
infection. Immunity 29: 487–496, 2008.
23. Cromer W, Wang W, Zawieja SD, von der Weid PY, Newell-Rogers
M.K, Zawieja DC. Colonic insult impairs lymph flow, increases cellular
content of the lymph, alters local lymphatic microenvironment, and leads
to sustained inflammation in the rat ileum. Inflamm Bowel Dis 21:
1553–1563, 2015.
24. Cromer WE, Zawieja SD, Tharakan B, Childs EW, Newell MK,
Zawieja DC. The effects of inflammatory cytokines on lymphatic endothelial barrier function. Angiogenesis 17: 395–406, 2014.
25. Davis MJ. An improved, computer-based method to automatically track
internal and external diameter of isolated microvessels. Microcirculation
12: 361–372, 2005.
26. Davis MJ, Lane MM, Davis AM, Durtschi D, Zawieja DC, Muthuchamy M, Gashev AA. Modulation of lymphatic muscle contractility by
the neuropeptide substance P. Am J Physiol Heart Circ Physiol 295:
H587–H597, 2008.
27. Elias RM, Johnston MG. Modulation of lymphatic pumping by lymphborne factors after endotoxin administration in sheep. J Appl Physiol 68:
199 –208, 1990.
28. Elias RM, Johnston MG, Hayashi A, Nelson W. Decreased lymphatic
pumping after intravenous endotoxin administration in sheep. Am J
Physiol Heart Circ Physiol 253: H1349 –H1357, 1987.
29. Gashev AA, Davis MJ, Zawieja DC. Inhibition of the active lymph pump
by flow in rat mesenteric lymphatics and thoracic duct. J Physiol 540:
1023–1037, 2002.
30. Gashev AA, Zawieja DC. Hydrodynamic regulation of lymphatic transport and the impact of aging. Pathophysiology 17: 277–287, 2010.
31. Gasheva OY, Gashev AA, Zawieja DC. Cyclic guanosine monophosphate and the dependent protein kinase regulate lymphatic contractility in
rat thoracic duct. J Physiol 591: 4549 –4565, 2013.
32. Ghoshal S, Witta J, Zhong J, de Villiers W, Eckhardt E. Chylomicrons
promote intestinal absorption of lipopolysaccharides. J Lipid Res 50:
90 –97, 2009.
33. Gordon S. Alternative activation of macrophages. Nat Rev Immunol 3:
23–35, 2003.
34. Gordon S, Pluddemann A, Martinez Estrada F. Macrophage heterogeneity in tissues: phenotypic diversity and functions. Immunol Rev 262:
36 –55, 2014.
35. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev
Immunol 5: 953–964, 2005.
36. Hagemann T, Lawrence T, McNeish I, Charles KA, Kulbe H, Thompson RG, Robinson SC, Balkwill FR. “Re-educating” tumor-associated
macrophages by targeting NF-␬B. J Exp Med 205: 1261–1268, 2008.
37. Haidari M, Leung N, Mahbub F, Uffelman KD, Kohen-Avramoglu R,
Lewis GF, Adeli K. Fasting and postprandial overproduction of intestinally derived lipoproteins in an animal model of insulin resistance.
Evidence that chronic fructose feeding in the hamster is accompanied by
enhanced intestinal de novo lipogenesis and ApoB48-containing lipoprotein overproduction. J Biol Chem 277: 31,646-31,655, 2002.
38. Hickey MJ, Kubes P. Intravascular immunity: the host-pathogen encounter in blood vessels. Nat Rev Immunol 9: 364 –375, 2009.
39. Hotchkiss RS, Monneret G, Payen D. Immunosuppression in sepsis: a
novel understanding of the disorder and a new therapeutic approach.
Lancet Infect Dis 13: 260 –268, 2013.
40. Huggenberger R, Siddiqui SS, Brander D, Ullmann S, Zimmermann
K, Antsiferova M, Werner S, Alitalo K, Detmar M. An important role
of lymphatic vessel activation in limiting acute inflammation. Blood 117:
4667–4678, 2011.
41. Johnson LA, Jackson DG. The chemokine CX3CL1 promotes trafficking
of dendritic cells through inflamed lymphatics. J Cell Sci 126: 5259 –5270,
2013.
42. Juskewitch JE, Knudsen BE, Platt JL, Nath KA, Knutson KL, Brunn
GJ, Grande JP. LPS-induced murine systemic inflammation is driven by
parenchymal cell activation and exclusively predicted by early MCP-1
plasma levels. Am J Pathol 180: 32–40, 2012.
INFLAMMATION AND IMMUNE CELLS IMPACT LYMPHATIC FUNCTION
63. Porta C, Larghi P, Rimoldi M, Totaro MG, Allavena P, Mantovani A,
Sica A. Cellular and molecular pathways linking inflammation and cancer.
Immunobiology 214: 761–777, 2009.
64. Porta C, Rimoldi M, Raes G, Brys L, Ghezzi P, Di Liberto D, Dieli F,
Ghisletti S, Natoli G, De Baetselier P, Mantovani A, Sica A. Tolerance
and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor ␬B. Proc Natl Acad Sci USA 106:
14,978 –14,983, 2009.
65. Randolph GJ, Angeli V, Swartz MA. Dendritic-cell trafficking to lymph
nodes through lymphatic vessels. Nat Rev Immunol 5: 617–628, 2005.
66. Randolph GJ, Sanchez-Schmitz G, Angeli V. Factors and signals that
govern the migration of dendritic cells via lymphatics: recent advances.
Springer Sem Immunopathol 26: 273–287, 2005.
67. Rizwan A, Bulte C, Kalaichelvan A, Cheng M, Krishnamachary B,
Bhujwalla ZM, Jiang L, Glunde K. Metastatic breast cancer cells in
lymph nodes increase nodal collagen density. Sci Rep 5: 10002, 2015.
68. Schraufstatter IU, Chung J, Burger M. IL-8 activates endothelial cell
CXCR1 and CXCR2 through Rho and Rac signaling pathways. Am J
Physiol Lung Cell Mol Physiol 280: L1094 –L1103, 2001.
69. Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo
veritas. J Clin Invest 122: 787–795, 2012.
70. Steinmuller M, Srivastava M, Kuziel WA, Christman JW, Seeger W,
Welte T, Lohmeyer J, Maus UA. Endotoxin induced peritonitis elicits
monocyte immigration into the lung: implications on alveolar space
inflammatory responsiveness. Respir Res 7: 30, 2006.
71. Tan KW, Chong SZ, Wong FH, Evrard M, Tan SM, Keeble J,
Kemeny DM, Ng LG, Abastado JP, Angeli V. Neutrophils contribute to
inflammatory lymphangiogenesis by increasing VEGF-A bioavailability
and secreting VEGF-D. Blood 122: 3666 –3677, 2013.
72. Tikhonov I, Doroshenko T, Chaly Y, Smolnikova V, Pauza CD,
Voitenok N. Down-regulation of CXCR1 and CXCR2 expression on
human neutrophils upon activation of whole blood by S. aureus is
mediated by TNF-␣. Clin Exp Immunol 125: 414 –422, 2001.
73. Truman LA, Ford CA, Pasikowska M, Pound JD, Wilkinson SJ,
Dumitriu IE, Melville L, Melrose LA, Ogden CA, Nibbs R, Graham
G, Combadiere C, Gregory CD. CX3CL1/fractalkine is released from
apoptotic lymphocytes to stimulate macrophage chemotaxis. Blood 112:
5026 –5036, 2008.
74. Tso P, Balint JA. Formation and transport of chylomicrons by enterocytes
to the lymphatics. Am J Physiol Gastrointest Liver Physiol 250: G715–
G726, 1986.
75. Tuncer LI, Alacam T, Oral B. Substance P expression is elevated in
inflamed human periradicular tissue. J Endod 30: 329 –332, 2004.
76. von der Weid PY, Muthuchamy M. Regulatory mechanisms in lymphatic vessel contraction under normal and inflammatory conditions.
Pathophysiology 17: 263–276, 2010.
77. Wang H. Activated macrophage-mediated endogenous prostaglandin and
nitric oxide-dependent relaxation of lymphatic smooth muscles. Jpn J
Physiol 47: 93–100, 1997.
78. Wang W, Nepiyushchikh Z, Zawieja DC, Chakraborty S, Zawieja SD,
Gashev AA, Davis MJ, Muthuchamy M. Inhibition of myosin light
chain phosphorylation decreases rat mesenteric lymphatic contractile activity. Am J Physiol Heart Circ Physiol 297: H726 –H734, 2009.
79. Witko-Sarsat V, Mocek J, Bouayad D, Tamassia N, Ribeil JA, Candalh C, Davezac N, Reuter N, Mouthon L, Hermine O, PederzoliRibeil M, Cassatella MA. Proliferating cell nuclear antigen acts as a
cytoplasmic platform controlling human neutrophil survival. J Exp Med
207: 2631–2645, 2010.
80. Wu TF, Carati CJ, Macnaughton WK, von der Weid PY. Contractile
activity of lymphatic vessels is altered in the TNBS model of guinea pig
ileitis. Am J Physiol Gastrointest Liver Physiol 291: G566 –G574, 2006.
81. Zawieja DC. Contractile physiology of lymphatics. Lymph Res Biol 7:
87–96, 2009.
82. Zawieja SD, Wang W, Wu X, Nepiyushchikh ZV, Zawieja DC,
Muthuchamy M. Impairments in the intrinsic contractility of mesenteric
collecting lymphatics in a rat model of metabolic syndrome. Am J Physiol
Heart Circ Physiol 302: H643–H653, 2012.
83. Zhang G, Ghosh S. Toll-like receptor-mediated NF-␬B activation: a
phylogenetically conserved paradigm in innate immunity. J Clin Invest
107: 13–19, 2001.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00467.2015 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 15, 2017
43. Kakei Y, Akashi M, Shigeta T, Hasegawa T, Komori T. Alteration of
cell-cell junctions in cultured human lymphatic endothelial cells with
inflammatory cytokine stimulation. Lymph Res Biol 12: 136 –143, 2014.
44. Kavanagh K, Wylie AT, Tucker KL, Hamp TJ, Gharaibeh RZ, Fodor
AA, Cullen JM. Dietary fructose induces endotoxemia and hepatic injury
in calorically controlled primates. Am J Clin Nutr 98: 349 –357, 2013.
45. Kelly JL, O’Sullivan C, O’Riordain M, O’Riordain D, Lyons A,
Doherty J, Mannick JA, Rodrick ML. Is circulating endotoxin the
trigger for the systemic inflammatory response syndrome seen after
injury? Ann Surg 225: 530 –541; discussion 541-533, 1997.
46. Kevil CG, Patel RP, Bullard DC. Essential role of ICAM-1 in mediating
monocyte adhesion to aortic endothelial cells. Am J Physiol Cell Physiol
281: C1442–C1447, 2001.
47. Kim KE, Koh YJ, Jeon BH, Jang C, Han J, Kataru RP, Schwendener
RA, Kim JM, Koh GY. Role of CD11b⫹ macrophages in intraperitoneal
lipopolysaccharide-induced aberrant lymphangiogenesis and lymphatic
function in the diaphragm. Am J Pathol 175: 1733–1745, 2009.
48. Kuan EL, Ivanov S, Bridenbaugh EA, Victora G, Wang W, Childs
EW, Platt AM, Jakubzick CV, Mason RJ, Gashev AA, Nussenzweig
M, Swartz MA, Dustin ML, Zawieja DC, Randolph GJ. Collecting
lymphatic vessel permeability facilitates adipose tissue inflammation and
distribution of antigen to lymph node-homing adipose tissue dendritic
cells. J Immunol 194: 5200 –5210, 2015.
49. Lai JP, Douglas SD, Shaheen F, Pleasure DE, Ho WZ. Quantification
of substance P mRNA in human immune cells by real-time reverse
transcriptase PCR assay. Clin Diagn Lab Immunol 9: 138 –143, 2002.
50. Lawrence T. The nuclear factor NF-␬B pathway in inflammation. Cold
Spring Harbor Perspect Biol 1: a001651, 2009.
51. Lee JH, Palaia T, Ragolia L. Impaired insulin-mediated vasorelaxation
in diabetic Goto-Kakizaki rats is caused by impaired Akt phosphorylation.
Am J Physiol Cell Physiol 296: C327–C338, 2009.
52. Liao S, Cheng G, Conner DA, Huang Y, Kucherlapati RS, Munn LL,
Ruddle NH, Jain RK, Fukumura D, Padera TP. Impaired lymphatic
contraction associated with immunosuppression. Proc Natl Acad Sci USA
108: : 18,784 –18,789, 2011.
53. Lobov GI, Kubyshkina NA. Mechanisms underlying the effect of E. coli
endotoxin on contractile function of lymphatic vessels. Bull Exp Biol Med
137: 114 –116, 2004.
54. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage
polarization: tumor-associated macrophages as a paradigm for polarized
M2 mononuclear phagocytes. Trends Immunol 23: 549 –555, 2002.
55. Nagai T, Bridenbaugh EA, Gashev AA. Aging-associated alterations in
contractility of rat mesenteric lymphatic vessels. Microcirculation 18:
463–473, 2011.
56. Nemoto K, Sato H, Tanuma K, Okamura T. Mesenteric lymph flow in
endotoxemic guinea pigs. Lymph Res Biol 9: 129 –134, 2011.
57. Nepiyushchikh ZV, Chakraborty S, Wang W, Davis MJ, Zawieja DC,
Muthuchamy M. Differential effects of myosin light chain kinase inhibition on contractility, force development and myosin light chain 20
phosphorylation of rat cervical and thoracic duct lymphatics. J Physiol
589: 5415–5429, 2011.
58. Niu CY, Hou YL, Zhao ZG, Zhang YF, Ji JJ, Qiao HX, Zhang J, Yao
YM. [Role of intestinal lymphatic pathway in pathogenesis of intestinederived bacteria/endotoxin translocation in rats in shock]. Zhongguo Wei
Zhong Bing Ji Jiu Yi Xue 19: 266 –269, 2007.
59. O’Grady NP, Tropea M, Preas HL, 2nd Reda D, Vandivier RW,
Banks SM, Suffredini AF. Detection of macrophage inflammatory protein (MIP)-1␣ and MIP-1␤ during experimental endotoxemia and human
sepsis. J Infect Dis 179: 136 –141, 1999.
60. Olofsson P, Nylander G, Olsson P. Endotoxin: routes of transport in
experimental peritonitis. Am J Surg 151: 443–446, 1986.
61. Pena OM, Pistolic J, Raj D, Fjell CD, Hancock RE. Endotoxin tolerance represents a distinctive state of alternative polarization (M2) in
human mononuclear cells. J Immunol 186: 7243–7254, 2011.
62. Podgrabinska S, Kamalu O, Mayer L, Shimaoka M, Snoeck H,
Randolph GJ, Skobe M. Inflamed lymphatic endothelium suppresses
dendritic cell maturation and function via Mac-1/ICAM-1-dependent
mechanism. J Immunol 183: 1767–1779, 2009.
H2057