Filarial Excretory-Secretory Products Induce Human
Monocytes to Produce Lymphangiogenic Mediators
Tiffany Weinkopff1,2*, Charles Mackenzie3,4, Rob Eversole4, Patrick J. Lammie1
1Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 2Department of Cell Biology, University of Georgia,
Athens, Georgia, United States of America, 3Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, United States of
America, 4Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan, United States of America
The nematodes Wuchereria bancrofti and Brugia spp. infect over 120 million people worldwide, causing lymphedema,
elephantiasis and hydrocele, collectively known as lymphatic filariasis. Most infected individuals appear to be asymptomatic,
but many exhibit sub-clinical manifestations including the lymphangiectasia that likely contributes to the development of
lymphedema and elephantiasis. As adult worm excretory-secretory products (ES) do not directly activate lymphatic
endothelial cells (LEC), we investigated the role of monocyte/macrophage-derived soluble factors in the development of
filarial lymphatic pathology. We analyzed the production of IL-8, IL-6 and VEGF-A by peripheral blood mononuclear cells
(PBMC) from naı ¨ve donors following stimulation with filarial ES products. ES-stimulated PBMCs produced significantly more
IL-8, IL-6 and VEGF-A compared to cells cultured in medium alone; CD14+monocytes appear to be the primary producers of
IL-8 and VEGF-A, but not IL-6. Furthermore, IL-8, IL-6 and VEGF-A induced in vitro tubule formation in LEC Matrigel cultures.
Matrigel plugs supplemented with IL-8, IL-6, VEGF-A, or with supernatants from ES-stimulated PBMCs and implanted in vivo
stimulated lymphangiogenesis. Collectively, these data support the hypothesis that monocytes/macrophages exposed to
filarial ES products may modulate lymphatic function through the secretion of soluble factors that stimulate the vessel
growth associated with the pathogenesis of filarial disease.
Citation: Weinkopff T, Mackenzie C, Eversole R, Lammie PJ (2014) Filarial Excretory-Secretory Products Induce Human Monocytes to Produce Lymphangiogenic
Mediators. PLoS Negl Trop Dis 8(7): e2893. doi:10.1371/journal.pntd.0002893
Editor: Sabine Specht, University Clinic Bonn, Germany
Received June 12, 2013; Accepted April 12, 2014; Published July 10, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by a grant from Glaxo SmithKline to MSU for filarial disease investigation as well as a training grant to the Center for Tropical
and Emerging Infectious Diseases (T32 AI 060546) with additional support from the Centers for Disease Control and Prevention’s Emerging Infectious Disease
Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: I have read the journal’s policy and have the following conflicts: I have received funding from Glaxo SmithKline in the past, but this
funding was not used or related in any way to the present study. This does not alter our adherence to all PLOS NTDs policies on sharing data and materials.
* Email: email@example.com
Lymphatic vessels (LVs) are important components of a system
vital to the body’s maintenance that includes immune surveillance
and fat absorption; the primary function of these vessels is to drain
excess interstitial fluids and to prevent tissue swelling .
Lymphangiectasia is a condition in which LVs are abnormally
dilated and this pathology is often associated with the development
of lymphedema, when lymphatic fluid becomes stagnant and leaks
back into the surrounding interstitium. Lymphatic dilation may
result from a variety of causes including trauma, cancer-related
treatment regimes such as lymphadenectomy, and genetic
mutations in FOXC2 or VEGFR-3. However, the majority of
lymphatic pathology seen worldwide is associated with the filarial
nematode parasites, Wuchereria bancrofti and Brugia malayi which
cause lymphedema in millions of individuals.
An estimated 120 million people worldwide are infected by
filarial parasites . Lymphatic filariasis is an infection with
varying degrees of clinical disease, where infected individuals can
exhibit overt clinical symptoms such as lymphedema and hydrocele
or be asymptomatic yet with microfilaremia. Although these
asymptomatic microfilaremic individuals do not display any overt
clinical manifestations, they do present with hidden sub-clinical
complications [2,3] such as dilated and tortuous lymphatics [4,5],
and scrotal lymphangiectasia in men [6,7]. Ultrasonographic
examination of the scrotal region of 14 asymptomatic Brazilians
revealed that 50% of microfilaremic individuals demonstrated
lymphatic dilation and tortuosity . In microfilaremic indi-
viduals, abnormal lymphatics are present in 69% of limbs by
static lymphoscintigraphy and in 100% of limbs by dynamic
flow lymphoscintigraphy, which are sensitive indicators of
lymphatic dysfunction [4,5,9]. In addition, studies of superfi-
cial skin punch biopsies have revealed that 78% and 68% of
limbs from patients with clinical disease and asymptomatic
microfilaremia, respectively, contained LVs that were abnor-
mally dilated [5,10]. More recently, it was also demonstrated
that children as young as three years of age can present with
lymphangiectasia as measured by lymphoscintigraphy suggest-
ing that sub-clinical pathology can occur at a very early age
The causes for the lymphatic dilation in filarial-infected
individuals remain unknown, but lymphangiectasia is seen in
SCID mice infected with Brugia suggesting that the worm and/or
innate mechanisms, and not the host’s adaptive immune system,
are involved in the development of lymphatic dilation [12,13].
Furthermore, the dilation can be reversed in nude mice by
removing or killing the adult worms [14,15]. An important finding
was made by Shenoy et al. who showed that there is a reduction in
PLOS Neglected Tropical Diseases | www.plosntds.org1 July 2014 | Volume 8 | Issue 7 | e2893
lymphatic dilation following worm death induced by DEC
The molecules involved in the proliferation and maintenance of
endothelial cells (EC) are a family of growth factors known as the
vascular endothelial growth factors (VEGF) as well as cytokines
such as IL-3, IL-6, IL-7 and IL-8 [17–25]. VEGF-A, VEGF-C and
VEGF-D, and their corresponding receptors, have all been shown
to support lymphatic endothelial cell (LEC) proliferation, migra-
tion, survival and tubule formation; thus these molecules are
potent regulators of lymphangiogenesis [17,26]. Several studies
have shown that plasma levels of these lymphangiogenic factors,
including VEGF-A, VEGF-C, VEGF-D and angiopoietins, are
significantly elevated in filarial-infected individuals including those
with filarial lymphedema compared to endemic normal control
subjects [27,28]. Elevated plasma levels of VEGF-A were also seen
in individuals with hydrocele . Furthermore, human infection
with the filarid, Onchocerca volvulus, induces lymphangiogenesis in
parasite-containing nodules  and this neovascularization is
associated with the expression of lymphangiogenic molecules such
as VEGF-C .
Monocytes/macrophages appear to be the predominant pro-
ducers of the VEGFs and the presence of monocytes/macrophag-
es has been correlated with lymphangiogenesis [32–36]. In human
onchocercal nodules, some mononuclear cells expressed both the
macrophage marker, MAC-1, and the lymphatic-specific marker,
LYVE-1, and these double-positive cells were integrated into the
lymphatic endothelium [30,31]. Thus in this present study we have
addressed the role of monocytes/macrophages contributing to the
production of lymphangiogenic mediators in response to filarial ES
and their influence on lymphatic ECs.
Materials and Methods
For human studies, informed consent was obtained from all
human subjects and approved by the Institutional Review Board
at CDC. For animal studies August rats, imported from the MRC
London, UK and bred locally at Western Michigan University,
and maintained in standard animal laboratory housing conditions,
were used. All animals were anesthetized using isoflurane gaseous
equipment (Summit Medical Equipment Company, Foster City,
CA). The animals were housed individually for the course of the
experiment in the Animal Facilities of Western Michigan
University. All the animal procedures were approved by the
Western Michigan University Animal Use and Care Committee
(IACUC) under project 10-01-07 before the project was begun.
The study conformed to the Guide for the Care and Use of
Research Animals published by the National Research Council.
Parasite ES Products
Brugia malayi adult female worms were collected from the
peritoneal cavity of infected jirds, Meriones unguiculatus, obtained
from the NIAID Filariasis Research Reagent Repository at the
University of Georgia (Athens, GA). Worms were isolated 4–12
months post infection from jirds and some of the adult females will
have been gravid at this point. For the collection of ES products,
50 live adult female worms were cultured in vitro for 7 days at 37uC
in 10 mL serum-free RPMI 1640 media (GIBCO) supplemented
with 2 mM L-glutamine and antibiotics (100 U/mL penicillin and
100 mg/mL streptomycin). Supernatants were collected and fresh
medium added daily. Supernatants containing the ES products
were centrifuged at 10006 g for 10 min to remove the
microfilariae and the microfilariae were resuspended in PBS and
counted to ensure worm viability. Supernatants were then
concentrated with a Centricon filter (Millipore, Bedford, MA) to
a volume of ,300 mL. This process resulted in ,670 ng/mL of
worm protein. ES products were stored at 4uC until further use.
Male worms were not used because they do not secrete the same
quantity of protein material as females (unpublished observations).
Prior to cell stimulations with ES products, ES products were
filtered using 0.45 mm Millex-HA syringe filters (Millipore,
Carrigtwohill, Ireland) and used in a dose-dependent (diluted at
1:10, 1:50) manner across various replicates and batches. A batch
is defined as a specimen containing the concentrated ES products
from 50 female worms over one week pooled together. All batches
of ES products were tested for endotoxin activity using the
Limulus Amebocyte Lysate QCL-1000 assay (Lonza, Walkersville,
MD) and ES products were only used for experiments when
endotoxin concentrations were #0.1 EU/mL.
Isolation of Peripheral Blood Mononuclear Cells
Human PBMCs were isolated using lymphocyte separation
media (MP Biomedicals, Solon, OH) as directed by the
manufacturer. In brief, blood was collected from normal healthy
donors by venipuncture in 10 mL EDTA Vacutainer tubes
(Becton Dickinson, Franklin Lakes, NJ). After centrifugation the
buffy coat was removed, resuspended in RPMI 1640 media
supplemented with 10% FBS (Atlas Biologicals, Fort Collins, CO),
2 mM L-glutamine and antibiotics and layered over lymphocyte
separation media. Cells were centrifuged for 30 min at 10006g at
4uC, the buffy coat was removed, washed and cells were counted
using a hemocytometer.
Human CD14+monocytes were enriched by positive selection
from PBMCs using CD14+MACS technology (Miltenyi Biotec,
Auburn, CA) as directed by manufacturer. CD14+monocyte
isolation was confirmed by flow cytometry using mouse anti-
human CD14+PE (BD Pharmingen, San Jose, CA) and CD14+
cells were routinely enriched to a purity of 94–98%.
Culture of LECs
Human dermal lymphatic microvascular endothelial cells
(HMVEC-dLy) were purchased from Clonetics (Lonza) and
maintained in EBM-2 basal media supplemented with EGM-2
Lymphatic filariasis is caused by parasitic worms with
approximately 120 million people infected worldwide and
over 1 billion people at risk. The adult worms reside in host
lymphatic vessels (LV) but most infected individuals do not
present with overt clinical symptoms. Individuals exhibit-
ing lymphedema, a common form of the disease, are often
antigen negative; however, infected individuals, though
often asymptomatic, have dilated LVs suggesting that
early damage to the lymphatic architecture may lead to
lymphedema in these infected individuals. In the LVs, adult
worms release excretory-secretory (ES) products. Filarial ES
products do not directly activate lymphatic endothelial
cells (LEC), so we hypothesized that accessory cells may
activate LECs indirectly and contribute to the development
of disease. Here, we show that adult filarial ES products
induce human blood cells, specifically monocytes, to
produce lymphangiogenic factors such as IL-8 and VEGF-
A and that these factors induce the formation of LVs in
vivo. These results support a role for filarial ES products in
altering the lymphatic architecture in filarial-infected
individuals and this may contribute to LV pathology and
the development of lymphedema.
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MV SingleQuots (Lonza) according to manufacturer’s instruc-
tions. Cells were used from passages 4-8.
Production of Lymphangiogenic Factors by PBMCs and
Cells were plated at 16106PBMCs or 56105CD14+cells in
500 mL RPMI 1640 media supplemented with 10% FBS, 2 mM
L-glutamine and antibiotics and stimulated with or without
100 ng/mL LPS or ES diluted at 1:10 and 1:50 for 72 h. The
final concentration of the ES in the dilutions used to stimulate the
human cells was approximately 10 to 67 ng/mL. Cell culture
supernatants were collected and analyzed for IL-3, IL-6, IL-7, IL-
8 and VEGF-A using the Bio-Plex Pro multiplex suspension array
system (Bio-Rad, Hercules, CA) according to the manufacturer’s
instructions. Data was obtained using low PMT voltage settings
and analyzed by the Bio-Plex Manager software version 4.1.1 and
concentrations were calculated based on a standard curve derived
from a recombinant cytokine standard. If the cytokine level in the
sample was higher than the highest value on the standard curve,
which occurred in many of the LPS stimulations, the highest value
of the standard curve was reported for that data point. All samples
were stimulated in parallel with ES products diluted at 1:10 and
1:50, but only results from the ES concentration which generated
optimal stimulation were reported. VEGF-C and VEGF-D
production were analyzed by the Quantikine Immunoassay kits
(R&D, Minneapolis, MN) as directed by the manufacturer.
In Vitro Matrigel Tubule Formation by LEC
LECs were released from the flask by gentle trypsinization
(Lonza), washed, counted and 16105LECs were stimulated in
200 mL EGM-2 MV SingleQuot media devoid of VEGF and
spiked with 10 ng/mL IL-6 (R&D), 10 ng/mL IL-8 (Sigma, St.
Louis, MO) or 1 ng/mL VEGF-A (R&D) for 10 min at 37uC
before seeding. Cells were plated onto 100 mL Growth Factor-
reduced Matrigel Matrix (BD Biosciences, Bedford, MA) coating a
24 well plate using the thin gel method as per manufacturer’s
instructions. After 24 h, 5 randomized fields per well were
photographed at 56 magnification on a Zeiss AxioVert 200M
microscope (Carl Zeiss, Thornwood, NY). The images were
opened and analyzed in AxioVision release 4.7.2. At a scaling ratio
of 1:1 image analysis was performed; the total number of tubules
was counted and the length of each tubule measured.
In Vivo Matrigel Tubule Formation Assay
These experiments were carried out in August rats as previous
work using this strain of rat demonstrated that the most
appropriate time to sample for dermal vascular growth is 9 days
. Rat carrier-free recombinant proteins including IL-8, IL-6,
VEGF164 were purchased from R&D Systems. Growth Factor-
reduced Matrigel was injected into rats with or without 10 ng/mL
IL-8, 10 ng/mL IL-6 or 10 ng/mL VEGF-A as directed by the
manufacturer. For the injections of the recombinant proteins,
3024 mL of liquid Matrigel was mixed with 576 mL of the
recombinant rat lymphangiogenic proteins yielding a final
concentration of 10 ng/mL for each protein with each animal
receiving a 0.5 mL injection. In addition, we collected superna-
tants from PBMCs stimulated with or without worm ES products
(1:10) as previously mentioned. Supernatants from 5 different
individuals were pooled and 576 mL of the pooled supernatants
was mixed with 3024 mL of liquid Matrigel and 0.5 mL of this mix
was injected into each rat . These supernatants were analyzed
by luminex bead technology using the Bio-Plex 8-plex kit (IL-2,
IL-4, IL-6, IL-8, IL-10, GM-CSF, IFNc, TNFa) as well as IL-5,
IL-13 and VEGF (Bio-Rad) according to the manufacturer’s
instructions. Regardless of Matrigel dilution with either recombi-
nant proteins or PBMC supernatants, the Matrigel concentration
was kept constant across all parameters and animals at 6.64 mg/
Six rats were used per group and the various Matrigel-test agent
samples were placed in the sub-dermal tissue of the flank using an
18G needle; the same position was used on each anesthetized
animal, with only one plug being injected in a single rat. All
injections were made by the one person and care taken with each
injection to maintain a constant injection pressure and to produce
a uniformly distributed plug of the material in the tissues. All rats
were observed at least twice daily during the course of the
experiment; they tolerated the procedures without any difficulty
and did not interfere in any way with the sites where the Matrigel
plugs were located.
Preparation of Histopathology Samples
Animals were sacrificed at day 9 as described above. During
plug excision, the skin and underlying tissues/body wall were
carefully dissected to observe the status of the plug and the
surrounding tissues noting color, presence of scar tissue, vascula-
ture and presence of any abnormal host tissue reaction.
The plugs and adjacent dermal tissues were removed intact
from the animals and cut three times through their longest axis to
provide three relatively equal slices before fixing. The plugs from
each animal were photographed in situ and after removal and
sectioning. Tissue sections were then taken from the cut faces of
these portions to provide three different areas of each plug for
histological preparation and for quantitative assessment. All tissues
were fixed in 3.7% buffered formalin for a maximum of 24 h
whilst maintaining that the fixation solution remained clear.
Tissues were stored for processing in 60% ethanol. Specimens
were then processed, embedded in paraffin, and sectioned on a
rotary microtome at 4–6 mm. Sections were placed on slides
coated with 2% 3-amino-propyl-tri-ethoxysilane and dried at 56uC
overnight. Following de-paraffinizing in xylene and hydrating
through descending concentrations of ethyl alcohol to distilled
water (DW), the slides were placed in Tris-buffered saline (TBS)
pH 7.5 (Scytek Labs, Logan, UT) for 5 min for pH adjustment.
Following TBS, the podoplanin and IgG test slides underwent
heat-induced epitope retrieval utilizing citrate buffer pH 6.0
(Scytek) in a vegetable steamer for 30 min at 100uC, allowed to
cool on the counter at RT for 10 min and rinsed in several
changes of DW. Von Willebrand Factor VIII (vWF) slides
underwent enzyme-induced epitope retrieval utilizing 0.03%
Pronase E in TBS for 10 min at 37uC followed by running tap
and DW rinses. Prior to test antibody (Ab) use, the sections were
subjected to an endogenous peroxidase blocking step (3%
hydrogen peroxide/methanol bath for 30 min followed by
running tap water and DW rinses), a nonspecific protein blocking
step for 30 min (normal horse serum) (Vector Labs, Burlingame,
CA), and finally an avidin/biotin blocking system (avidin, Vector
Labs; biotin, Sigma Chemicals, St. Louis, MO) for 15 min.
Following pretreatment, avidin-biotin complex staining steps were
performed at RT on the Dako Autostainer (Dako North America,
Inc., Carpinteria, CA). All staining steps were followed by two
rinses in TBS+Tween 20 (Scytek). After the sections were rinsed in
TBS/Tween20, they were incubated at various times (usually 40–
60 min) and various concentrations (from 1 in 40 to 1 in 200) with
the various test (primary) Abs. The optimal procedures for each test
Ab were determined following assessment under the microscope.
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Abs were diluted with Normal Antibody Diluent (NAD) (Scytek,
Logan, UT). Primary Ab slides were incubated for 60 min with the
monoclonal mouse anti-rat podoplanin (ReliaTech/Angio-Proteo-
mie, Boston, MA) diluted 1:400, or the biotin-conjugated
polyclonal rabbit anti-rat IgG (Novus Biologicals, Littleton, CO)
diluted 1:100 in NAD. Slides were rinsed in 2 changes of TBS/
Tween20, and then incubated in appropriate biotinylated
secondary Ab for the host species of the primary Ab (biotinylated
anti-rat, anti-goat, and anti-mouse from Vector Labs) at 10–
11.0 mg/mL in NAD incubated for 30 min. The slides were then
again rinsed in TBS/Tween20, and then R.T.U. Vectastain Elite
ABC Reagent (Vector Labs) was applied for 30 min. The slides
were rinsed with TBS/Tween20 and developed using NovaRED
peroxidase substrate kit (Vector Labs) for 15 min. After a rinsing
in DW, they were finally counterstained using Gill 2 (Lerner)
hematoxylin (Thermo Fisher, Kalamazoo, MI), differentiated in
1% aqueous glacial acetic acid, and rinsed in tap water. Slides
were then dehydrated, cleared with xylene, and mounted using
Flotex permanent mounting media (Thermo Shandon, Pittsburg,
During the establishment of optimal immunostaining for
Matrigel sections we compared three antibodies as markers of
lymphatic endothelia: a monoclonal mouse anti-rat podoplanin
(ReliaTech/Angio-Proteomie, Boston, MA), a monoclonal mouse
anti-human D2-40 (DakoCytomation) and a rabbit polyclonal
anti-LYVE-1 Ab (Angiobio, Del Mar, CA). From this pre-study we
selected the first of these reagents as being the most suitable for
quantification. Control tissues used in these studies included rat
lymph nodes and dermal neoplasia, and the staining controls
included omitting the primary antibody. Routine hematoxylin and
eosin staining (H&E) was also employed to examine the tissues and
establish the most suitable area for quantification.
Quantitative Assessment of the Cellular Components of
To avoid any complication from the natural host cellular
response to Matrigel, the areas used for cellular assessment for
vascular invasion were those central areas free of any overt host
cellular response to Matrigel itself; from an examination of all the
samples this free area was seen to cover a minimum of 4.0 mm2
centered around the mid point of the plug. This central area of the
Matrigel plug was quantitatively assessed for the presence of anti-
podoplanin and anti-vWF positivity in serial sections. Photographs
were taken using bright field and Differential Interference
Contrast Microscopy (DIC).
The Chalkley Point Array random sampling technique (The
Graticules Ltd. Chalkley Point Array - Model NG52) was used to
quantify the immuno-positive staining elements present in the
Matrigel plug and thus a relationship to the proportion of the two
vascular components present in different groups; the number of
points lying over a positively stained entity is statistically
proportional to the area occupied by that component . Three
areas in each section taken from the three slices of each individual
plug were quantified and the number of immuno-positive
components was recorded. This provided nine counts for each
sample, and thus 54 counts for each treatment group.
In addition, confirmatory values were obtained using a
commercial image analysis system - Image-Pro (MediaCyber-
netics, Bethesda, MD) and Image J (NIH - rsbweb.nih.gov/ij/)
and examining the central 4.0 mm2test area of each Matrigel
slice. The immunohistochemical staining intensity was standard-
ized for each section by setting the positivity limit for each marker
using the respective cell or tissue component in the dermal tissue
surrounding the plug in each section. Each area was measured
using a pixel color gate (i.e. for marker positive cells), and
subtracting the background using collagen tissue as the negative.
Triplicate runs were made with the pixel number calculations for
each assessment area.
The Signed Rank Test was used in the Statistical Analysis
Software (SAS) version 9.1 to compare median cytokine and
growth factor production by PBMCs in stimulated and control
supernatants. The Signed Rank Test was also used to compare the
production of these factors by CD14+monocytes compared to
non-CD14 cells. GraphPad Prism 5 software (San Diego, CA) was
used to carry out additional statistical analyses to compare the
number of tubules per microscopic field in response to stimuli. It
was determined from a standard power calculation, a minimum
number of animals in each test group to obtain an acceptable
significant result was 5; therefore we used 6 animals in each group.
The animals were injected in random order and the tissues were
assessed blinded to minimize bias. Student’s t test and ANOVA
were used to assess the results.
Filarial ES Products Induce the Production of
Lymphangiogenic Factors in PBMCs
We evaluated the ability of Brugia ES products to induce the
secretion of molecules known to exhibit lymphangiogenic potential
in other in vivo and in vitro settings. Human PBMCs were isolated
from healthy volunteers and cultured with or without filarial ES
products for 72 h. The supernatant fluids were collected and
analyzed for the production of the potentially lymphangiogenic
molecules IL-3, IL-6, IL-7, IL-8 and VEGF-A by luminex
technology. PBMCs cultured in media alone supplemented with
10% FBS served as a negative control. Cells cultured with filarial
ES products secreted significantly higher levels of IL-8, IL-6 and
VEGF-A compared to cells cultured in media alone (Fig. 1). We
did not detect IL-3 or IL-7 in any of our supernatants. We also
attempted to measure VEGF-C and VEGF-D by ELISA, but
these were below the limit of detection. Taken together, these data
suggest that Brugia ES products are capable of inducing the
secretion of lymphangiogenic molecules by circulating PBMCs.
CD14+Monocytes Are the Primary Producers of the
Monocytes/macrophages have been shown to play an impor-
tant role in the production of VEGFs in tumors and inflammation,
so we hypothesized monocytes could be the PBMC in the
periphery contributing to the production of IL-8, IL-6 and VEGF-
A seen in response to worm ES products. We carried out CD14
fractionation experiments using MACS technology to isolate
CD14+monocytes from total PBMCs. As seen in Fig. 2A and 2C,
CD14+monocytes secreted significantly higher amounts of IL-8
and VEGF-A compared to CD14-depleted cells in response to
filarial ES products. However, CD14-enriched and depleted cell
populations produced similar levels of IL-6 (Fig. 2B). CD14+
monocytes produced significantly more IL-8 and VEGF-A
spontaneously compared to CD14-depleted cells (Fig. 2A and
2C). CD14+monocytes stimulated with Brugia ES products also
secreted significantly higher levels of IL-8 and IL-6 compared to
CD14+cells cultured in media alone. LPS was used as a positive
control for the production of IL-8 and IL-6 and robust IL-8 and
IL-6 responses were seen following LPS stimulation. Taken
together, these data suggest that CD14+monocytes are the
primary producers of the lymphangiogenic molecules IL-8 and
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PLOS Neglected Tropical Diseases | www.plosntds.org4 July 2014 | Volume 8 | Issue 7 | e2893
VEGF-A in response to worm ES products, but CD14+monocytes
are not the major cell type contributing to the production of IL-6
in response to worm ES products.
ES-Induced Lymphangiogenic Mediators Stimulate LECs
to Form Tubules In Vitro
Since we were able to demonstrate the production of
lymphangiogenic molecules by PBMCs in response to Brugia ES
products, we examined the ability of these mediators detected
following ES stimulation to alter LEC function as measured by
tubule formation. LECs were layered on Matrigel cultures and
stimulated with concentrations of IL-8, IL-6 and VEGF-A
comparable to the amounts detected in supernatants of ES-
stimulated PBMCs. After 24 h, LECs cultured in the presence of
IL-8, IL-6 and VEGF-A formed a more elaborate tubule network
compared to cells cultured in media alone (Fig. 3A). Using image
analysis software used to quantify tubule formation, cells cultured
in the presence of IL-8, IL-6 or VEGF-A formed a greater number
of tubules per microscopic field compared to LECs cultured
without stimulus (Fig. 3B).
ES-Induced Lymphangiogenic Molecules Result in
Vascularization of Matrigel Plugs In Vivo
Given that mediators produced by PBMCs in response to filarial
ES stimulation such as IL-8, IL-6 and VEGF-A induced LEC
tubule formation in vitro, we hypothesized these molecules could
also promote LV formation in vivo. To determine if the soluble
mediators present in ES-induced supernatants could induce vessel
formation in vivo, we injected rats with Matrigel containing
supernatants from PBMCs (collected from 5 different individuals)
that were stimulated with ES products or cultured in media alone.
Characterization of the pooled PBMC supernatants which
included measurable concentrations of IL-2, IL-6, IL-8 and
VEGF is seen in Table 1. In parallel rats were injected with
Matrigel containing rat recombinant IL-8, IL-6 or VEGF-A in
case the human mediators released by PBMCs in response to
filarial ES did not induce a cross species effect and stimulate vessel
formation in rats. Given that Matrigel contains a variety of
basement membrane proteins including laminin and collagen,
Matrigel alone was used as a non-specific protein negative control.
After 9 days, the plugs were excised and subjected to gross
inspection for vessel infiltration (Fig. 4A and 4B). Surprisingly,
even upon initial gross examination in situ, the Matrigel plugs
displayed an overt difference between treated groups and controls.
Animals given ES-stimulated PBMC supernatants had increased
redness in the plug denoting blood vessel infiltration compared to
supernatants from unstimulated PBMCs. Furthermore, rats
injected with lymphangiogenic cytokines also had an increased
redness compared to Matrigel alone control plugs. The plugs in
situ were generally uniform in size and shape. All except one had
formed a distinct flattened oval shaped plug; one of six samples
from experimental Group 2 was not clearly a round elliptical
entity and was dispersed over a wide and indistinct area in the
dermis; this was discarded. There was quite considerable variation
in color, ranging from yellow-brown to deep pink/red. The
Figure 1. Brugia ES products induce the production of lymphangiogenic molecules by human PBMCs. PBMCs were isolated from a
minimum of 10 healthy human volunteers and 16106cells were stimulated with or without ES for 72 h. Cell supernatants were assessed for the
presence of IL-8, IL-6 and VEGF-A by luminex bead analysis. Brugia ES products induced the production of (A) IL-8 (n=15), (B) IL-6 (n=10) and (C)
VEGF-A (n=15) by PBMCs compared to cells in media alone as assessed by the Signed Rank test. Medians are presented as bars.
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control animals showed the yellow-brown end of the spectrum
while those in groups receiving lymphangiogenic factors were
generally a deeper red color (Fig. 4).
The Matrigel plugs were first examined histologically with H&E
staining to identify and quantify the cellular infiltration into the
central area of the plugs. Different degrees of cellular infiltration
were seen in the specific quantification sites of the plugs in different
test groups (Fig. 5). The principle cellular elements present were
vascular; other cellular elements such as lymphocytes and
monocytes were only seen within these vascular elements and
not independently in the extra-vascular areas. The presentation of
the vascular elements varied from tubular formations (Fig. 5B and
5C) to distinct elongated vessels (Fig. 5D). The number of cells
present in the examined areas of the Matrigel plugs varied
between the groups, although there was consistency in form and
amount within each treatment group. Immunohistochemical
staining for the presence of vWF and podoplanin was carried
out to identify blood and lymphatic vessels, respectively (Fig. 5E
Overall, staining against podoplanin which identifies the
lymphatic endothelium was more prevalent in the Matrigel plugs
from all groups when compared to anti-vWF staining which
Figure 2. Brugia ES products induce the production of IL-8 and VEGF-A by human CD14+monocytes. Human CD14+monocytes were
isolated and compared to CD14-depleted cells for IL-8, IL-6 and VEGF-A production in response to worm ES products or LPS. Cell supernatants were
assessed for the presence of (A) IL-8 (n=12), (B) IL-6 (n=7) and (C) VEGF-A (n=7) after 72 h of stimulation. Data presented represents the mean +SEM
of at least 7 people per factor and comparisons were made using the Signed Rank test. LPS was used as a positive control and stimulated the
production of IL-8 (p,0.003) and IL-6 (p,0.02) compared to cells cultured in media alone.
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Figure 3. Filarial ES-induced lymphangiogenic mediators induce LEC tubule formation in vitro. LECs were grown on Matrigel in the
presence or absence of IL-8, IL-6 or VEGF-A and lymphatic networks were photographed (A). (B) The number of tubules was quantified using image
analysis software. The data represented here are the means +SEM of one experiment representative of 4 independent experiments performed in
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identifies the blood vascular endothelium. When comparing
different treatments for the presence of lymphatic endothelial
elements, Groups 1 (Matrigel alone) and 2 (Unstimulated PBMCs
alone) were not significantly different, whereas Groups 3–6, or
those containing the ES-stimulated supernatants and lymphangio-
genic mediators, had significantly more lymphatic vascular
elements than either Group 1 or 2 (Table 2). Plugs from Groups
3–6 had significantly more blood vascular elements than either the
control Matrigel alone (Group 1) or unstimulated PBMC Matrigel
(Group 2). Assessment of the color intensity by pixel enumeration
with either podoplanin or vWF also showed similar significant
differences between the groups VEGF-A, IL-8 and IL-6 compared
to control samples and a significant difference between the ES-
PBMC group compared to the unstimulated PBMC supernatant
group (Table S1).
Lymphangiectasia, or the dilation of LVs, and lymphangiogen-
esis are subclinical features of filarial infection. LVs containing
adult worms from infected individuals are characterized as
distended, dilated, tortuous and highly indented [40–42]. In
Table 1. Cytokine and growth factor levels (pg/mL) in PBMC supernatantsa.
Group Unstimulated supernatantsES-stimulated supernatantsa
IL-4 Undetectable Undetectable
IL-5 Undetectable Undetectable
IL-10 Undetectable Undetectable
IL-13 Undetectable Undetectable
VEGF 46.04 115.65
a16106PBMCs were stimulated with worm ES or cultured in media alone for 72 h.
Supernatants from 5 different individuals were pooled and cytokines and growth factors were analyzed by luminex bead technology. Matrigel plugs were
supplemented with 80 mL of the pooled supernatants and used for rat in vivo vessel formation experiments.
Figure 4. Matrigel plugs in situ. Matrigel was injected into rats with or without 10 ng/mL IL-8, 10 ng/mL IL-6 or 10 ng/mL VEGF-A in
a total volume of 0.5 mL. Matrigel was supplemented with supernatants from PBMCs stimulated with filarial ES for 72 h and injected into rats.
Matrigel alone and Matrigel containing supernatants from PBMCs cultured in media alone were injected as controls. Matrigel plugs were analyzed at
day 9. Representative in situ observations are presented from a single experiment using 6 rats per group. (A) A subcutaneous Matrigel plug (arrow)
containing VEGF-A showing a red-colored vascular response in the surrounding tissues and infiltrating the plug. (B) A cross section of a plug
containing filarial ES-PBMC products showing discoloration. (C) Distinct outline of the injected plug (arrow) in the sub-cutaneous tissues. (D) A control
Matrigel plug free of coloration. (E and F) Matrigel plugs from IL-6-treated (E) and VEGF-A-treated (F) animals showing a significant vascular response
with a dark red area. The scale bars represent 1 cm.
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dilated lymphatics, flow is impaired leading to improper drainage
of interstitial fluids. The progression of mild lymphangiectasia to
clinical lymphedema may be due to the accumulation of lymphatic
fluid in the tissues over time following damage to the LVs.
Lymphangiectasia is not restricted to the site of the worm nest, but
is found along the length of the infected vessel  arguing that a
soluble factor secreted by the worm, that can travel the length of
the vessel, is responsible for the altered lymphatic pathology.
Additionally, lymphangiectasia is greatest near the worm nest and
the removal or killing of worms can reduce lymphatic dilation [14–
16,40] suggesting living adult worms and their ES products have
the strongest biological effects locally and are associated with
altering lymphatic pathology.
A number of factors may play a role in the development of
lymphangiectasia and our data suggest that parasite products are
central in this process. Since no direct effects of ES products on
LECs were detected, we hypothesized that ES products activate
the lymphatic endothelium indirectly through an accessory cell
. Here, we have demonstrated that Brugia ES products
stimulate host cells to produce lymphangiogenic mediators such
as IL-8, IL-6 and VEGF-A. Autocrine stimulation by these
molecules on the PBMCs themselves may have also amplified the
response in our system. Next, we demonstrated these same
mediators altered LEC phenotypes. Moreover, the mediators
tested in this study not only induced LV formation in vivo using a
Matrigel plug model, but these mediators also induced angiogen-
esis. Therefore, the production of these molecules could contribute
to the development of lymphangiectasia in filarial-infected
Other studies have supported the role of parasite molecules in
lymphangiogenesis and lymphangiectasia. Bennuru et al. showed
microfilariae stimulate LEC proliferation and alter LEC junction
Figure 5. Cellular responses in the central assessment area of Matrigel plugs. Matrigel was injected in the presence or absence of 10 ng/mL
IL-8, IL-6 or VEGF-A in 0.5 mL. Matrigel alone was injected as a control. Matrigel was supplemented with supernatants collected from ES-stimulated
PBMCs or PBMCs cultured in media as a control and injected into rats. After 9 days, Matrigel plugs were excised, sectioned and analyzed.
Representative observations are presented from a single experiment using 6 rats per group. (A) Matrigel alone (control) - H&E stain. (B) Vascular
response in VEGF-A plug - H&E stain. (C) Cellular response in PBMC+ES Matrigel plug – H&E stain. (D) Lymphatic vessels (green arrow) together with
blood vessels (black arrow) in an IL-6-treated Matrigel plug. (E) Example of anti-vWF staining of blood vessels in PBMC+ES Matrigel plug. (F) High
power of the anti-podoplanin staining in an IL-6-containing Matrigel plug at 9 days. The scale bars represent 50 microns in A, B, D and E; 100 microns
in 5C; and 10 microns in 5F.
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adherence pathways which could contribute to lymphatic dilation
. Microfilariae may also contribute to the development of
lymphatic disease as this stage is released simultaneously with adult
ES products and microfilarial ES is found in our adult worm ES.
Others have proposed that parasite endosymbiont Wolbachia is
responsible for elevated lymphangiogenic mediators, but Bennuru
et al. elegantly demonstrated that the levels of VEGF-A, VEGF-C
and VEGF-D pre- and post-DEC treatment did not change
suggesting a minimal role for Wolbachia . Bacterial infection,
including Wolbachia, has been linked with IL-8 production , so
the levels of other lymphangiogenic mediators such as IL-8 and IL-
6 will also need to be examined in this setting. Furthermore,
human ECs exposed to live intact microfilariae either carrying or
free of Wolbachia or not, only induced a limited number of
cytokines and angiogenic mediators suggesting Wolbachia is not a
strong stimuli altering the EC phenotype .
In this present study, we aimed to mimic the relationship
between the living adult worm and the lymphatic endothelium,
and not the changes associated with dead worms, thus we used
Brugia ES products rather than adult worm or microfilariae
extracts. Crude extracts would be more representative of stimuli
associated with worm death, a different scenario. Upon worm
death, there is an immense inflammatory reaction which is distinct
from the lack of inflammation associated with the presence of the
living worm. Responses to living worms differ histopathologically
from the granulomatous responses seen with dead worms
(Mackenzie, unpublished observations). Monocytes/macrophages
appear to be central in both responses, although they may be
acting differently in each situation. Filarial ES products are
generally thought to be immunosuppressive but here ES induced
PBMCs to produce IL-8 and IL-6 which can lead to a massive
recruitment of inflammatory cells. However, the lack of inflam-
mation adjacent to living worms suggests IL-8 and IL-6
production does not lead to a massive inflammatory reaction in
vivo. In contrast, worm death either by drug treatment or natural
attrition may exacerbate the development of lymphatic pathology
if the acute inflammatory reaction provides a stimulus for
downstream processes leading to lymphatic insufficiencies. Future
studies will be needed to compare the production of lymphangio-
genic mediators and the induction of LVs in vivo in response to ES
products versus crude extracts.
Even though the expression of lymphangiogenic mediators is
generally perceived to be beneficial for the formation of new LVs
and to reverse malfunctioning LVs [47–49], the over-expression of
lymphangiogenic molecules over an extended period of time has
been shown to be detrimental and to impair lymphatic function. A
massive expansion of the lymphatic network can lead to defective
LVs and thus decreased drainage and lymphedema. For example,
VEGF-A and VEGF-C over-expression results in structurally and
functionally abnormal and dilated lymphatics [50–52]. ES-
stimulated host cells may compromise lymphatic function by
secreting lymphangiogenic factors over many years throughout the
duration of worm infection. It is important to note that a worm
infection can last five years or more so the kinetics and molecular
mechanisms associated with altering lymphatic pathology may
differ from those involved in acute infection and may be
cumulative over time. The cumulative amounts/effects of these
soluble mediators may parallel those observed in over-expression
model systems leading to defective lymphatics. For instance,
elevated plasma levels of VEGF-C have been found in micro-
filaremic individuals compared to endemic normal individuals 
suggesting the same VEGF and cytokine molecules involved in
lymphangiogenesis and lymphangiectasia in other models are also
present in filarial infection. These lymphangiogenic cytokines and
growth factors may be binding their receptors which are expressed
on LECs lining the vessel [20,53,54]. Besides the chronicity of
filarial infections, worm infections, and specifically worm ES
products, are also associated with a down regulation of the
immune response so future experiments will also need to address
how a chronic infection alters the formation of LVs in the presence
of a dampened proinflammatory response.
Even though we did see the production of VEGF-A by PBMCs
in response to worm ES, we did not see the production of VEGF-
C that was previously shown to be elevated in filarial-infected
individuals [28,31]. We also did not detect elevated levels of
VEGF-D or lymphangiogenic cytokines IL-3 or IL-7. The lack of
detection of VEGF-C, VEGF-D, IL-3 or IL-7 may be because we
were examining the production of these molecules by PBMCs
which may not be the cellular source; these molecules may be
produced by a cell found focally at the infection site. VEGF-C and
VEGF-D signaling through VEGFR-3 is the primary and most
well-characterized mechanism contributing to lymphangiogenesis,
but there is also an emerging role for VEGF-A in lymphangiogen-
esis [52,55–57], so it is possible that this molecule may be playing
an important role in filarial-induced lymphatic pathologies. In
addition to potential systemic versus local differences in lymphan-
giogenic mediators, differences between individual responses were
also noted. The variability in lymphangiogenic mediators,
Table 2. Quantitative assessment of the presence of podoplanin positive areas (lymphatic endothelial elements) and vWF positive
areas (blood endothelial elements) in treated Matrigel plugs recovered from rats 9 days after sub-cutaneous implantation.
GROUP PODOPLANIN POSITIVE AREAS (CPA+ +/2 2 SE)vWF POSITIVE AREAS (CPA+ +/2 2 SE)
1 MATRIGEL ALONE0.21 (0.1)0.18 (0.1)
2 UNSTIMULATED PBMCs2.71 (0.8)0.08 (0)
3 ES-STIMULATED PBMCs10.50 (2.3)* 3.10 (1.0)***
4 IL-6 11.57 (3.9)* 5.10 (1.3)***
5IL-8 5.29 (1.7)** 1.23 (0.3)****
6 VEGF-A 12.21 (3.1)*4.64 (1.2)***
A total of 9 areas were examined in each sample (54 areas per treatment) and assessed using a Chalkley Point Array count (CPA).
* Significantly different (p,0.005) from anti-podoplanin Groups 1 and 2.
** Significantly different (p,0.05) from anti-podoplanin Groups 1 and 2.
*** Significantly different (p,0.005) from anti-vWF Groups 1 and 2.
**** Significantly different (p,0.05 from anti-vWF Groups 1 and 2.
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especially for IL-8, produced by PBMCs basally and after ES
stimulation made control experiments injecting supernatants from
unstimulated PBMCs of paramount importance. Regardless,
supernatants from ES-stimulated PBMCs induced significantly
more podoplanin and vWF staining compared to supernatants
from unstimulated PBMCs. Furthermore, supernatants from
unstimulated PBMCs induced more vessel formation than
Matrigel alone confirming the basal production of these mediators
and providing an important baseline control beyond Matrigel
Monocytes and macrophages play a major role in supporting
lymphangiogenesis. They can produce lymphangiogenic factors
such as VEGFs and cytokines which induce LEC proliferation,
survival, migration and tubule formation [33,34]. In this present
study monocytes were primarily responsible for the production of
IL-8 and VEGF-A in response to ES products; however we did not
identify the cell type responsible for the production of IL-6, so
future experiments need to identify the source of IL-6.
Monocytes and macrophages may play a role in the lymphatic
pathology associated with filarial infection. Typically, LVs from
infected individuals are thought to be devoid of an inflammatory
response ; however, some have noted small lymph thrombi
composed of mononuclear cells and multinucleated giant cells
within the lumen . Here, we defined CD14+cells as the
primary producer of IL-8 and VEGF-A in response to Brugia ES
products and others have also reported the presence of mono-
cytes/macrophages in regions of lymphangiectasia and lymphan-
giogenesis in O. volvulus infection [30,31]. In nodules isolated from
humans infected with O. volvulus, the predominant cell type
associated with the worms was the macrophage and many
macrophages stained positive for the lymphatic-specific marker
LYVE-1 . Additionally, some LYVE-1+macrophages were
integrating into the lymphatic endothelium . Taken together,
these data suggest that monocytes/macrophages are important in
lymphangiectasia and lymphangiogenesis in filarial infections and
future research is needed to define the role of these cells in
One could speculate that the worm induces lymphangiogenesis
and lymphangiectasia for many reasons. The worm may increase
vessel diameter to provide a larger space for habitation; increasing
the vessel diameter also slows lymphatic flow and increases the
availability of nutrients and resources. The worm may stimulate
expansion of the lymphatic network by inducing host production
of VEGFs and cytokines to increase LEC proliferation and
differentiation as a mechanism of LV dilation. We also demon-
strated tubule formation in response to ES-stimulated mediators.
Filarial worms may induce the formation of new LVs to expand
their biological niche, to maintain flow through a collateral
network, or to increase the likelihood that their microfilariae reach
the periphery for transmission.
In this study we have begun to dissect the molecular
mechanisms involved in the development of lymphangiectasia
and lymphangiogenesis; however, similar studies must be carried
out in cells isolated from endemic populations to confirm that the
same molecules and cell types occur in filarial-infected individuals.
Given that parasite products induce the production of lymphan-
giogenic molecules and that infected persons exhibit lymphangi-
ectasia, we hypothesize that these molecules are elevated in
infected individuals. We are currently examining the production of
VEGFs and cytokines by microfilaremic individuals, endemic
normals and those with lymphedema in response to ES products.
Since many infected individuals exhibit lymphangiectasia, which
may progress to a lymphedema, we need to define the initial
molecular mechanisms responsible for the development of disease.
Given many of the lymphangiogenic mediators identified in this
study are expressed in a variety of inflammatory settings, we
hypothesize that lymphangiogenesis is a hallmark of inflammation.
Therefore, understanding the pathogenesis of lymphatic filariasis
may identify potential molecular targets for preventing disease
initiation and progression as well as a greater understanding of the
molecular mechanisms associated with lymphatic pathologies from
cancer and inflammation.
vWF or podoplanin positive elements on vascular structures in the
SA by measuring the number of positive pixels in a standard area
(4 sq.mm) and the difference is related to the Matrigel alone
control. A total of 3 areas (and 3–4 fields) per Matrigel plug were
quantified, and there were 6 animals tested per treatment, except
there were only 5 animals in Group 2. * p,0.005 statistically
significant differences between Group 2 and Group 3 for both
Quantitative assessment: Assessment of the presence of
We would like to thank the staff of the Investigative Histopathology
Laboratory (Amy Porter, Kathy Jacobs and Rick Rosebury) at Michigan
State University for their skill in providing high quality immunocytochem-
ical material for this study. We would also like to thank Delynn Moss and
Dr. Mike Arrowood (CDC, Atlanta, GA) for their skillful technical
assistance as well as Drs. Evan Secor and Diana Martin (CDC, Atlanta,
GA) for their support and input during the study. Also we are very
appreciative to Dr. Mike Dzimianski and the NIAID/NIH Filariasis
Research Repository Center (FR3, UGA, Athens, GA) for supplying
Conceived and designed the experiments: TW PJL CM. Performed the
experiments: TW CM RE. Analyzed the data: TW CM RE PJL.
Contributed reagents/materials/analysis tools: CM RE PJL. Wrote the
paper: TW CM PJL.
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Lymphangiectasia in Lymphatic Filariasis
PLOS Neglected Tropical Diseases | www.plosntds.org12 July 2014 | Volume 8 | Issue 7 | e2893