1164 • JID 2006:193 (15 April) • Ramirez et al.
M A J O R A R T I C L E
Brugia malayi Asparaginyl–Transfer RNA Synthetase
Induces Chemotaxis of Human Leukocytes
and Activates G-Protein–Coupled Receptors CXCR1
Bernadette L. Ramirez,1O. M. Zack Howard,2Hui Fang Dong,3Takeo Edamatsu,2Ping Gao,2Michael Hartlein,4
and Michael Kron5
1Department of Biochemistry and Molecular Biology, College of Medicine, University of the Philippines, Manila;
for Cancer Research, Laboratory of Molecular Immunoregulation, and
Program, National Cancer Institute-Frederick, Frederick, Maryland;
Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee
2National Cancer Institute, Center
3Science Applications International Corporation–Frederick Basic Research
4Institute Max von Laue, Paul Langevin, and
5Department of Medicine,
acute and chronic inflammatory reactions in the host bloodstream and lymphatics. Excretory-secretory products
derived from filariae are believed to play an important role in the development of associated immunologic
conditions; however, the specific mechanisms involved in these changes are not well understood. Recently, human
were shown to activate chemokine receptors on T lymphocytes, monocytes, and immature dendritic cells by
recruiting immune cells that could induce innate and adaptive immune responses. Filarial (Brugia malayi) aspar-
aginyl-tRNA synthetase (AsnRS) is known to be an immunodominant antigen that induces strong human im-
munoglobulin G3 responses.
Recombinant B. malayi AsnRS was used to performcellularfunctionassays—forexample,chemotaxis
and kinase activation assays.
Unlike human AsnRS, parasite AsnRS is chemotactic for neutrophils and eosinophils. Recombinant
B. malayi AsnRS but not recombinant human AsnRS induced chemotaxis of CXCR1 and CXCR2 single-receptor–
transfected HEK-293 cell lines, blocked CXCL1-induced calcium flux, and induced mitogen-activated protein
Our findings suggest that a filarial parasite chemoattractant protein may contribute to the de-
velopment of chronic inflammatory disease and that chemokine receptors may be therapeutic targets to ameliorate
Lymphatic filariasis is a chronic human parasitic disease in which the parasites repeatedlyprovoke
Lymphatic filariasis caused by Wuchereria bancrofti,Bru-
gia malayi, and Brugia timori is a complex human nem-
atode disease that affects 1200 million peopleworldwide
Received 18 January 2005; accepted 9 November 2005; electronically published
6 March 2006.
Potential conflicts of interest: none reported.
The content of this publication does not necessarily reflect the views or policies
of the Department of Health and Human Services, nor does mention of trade
names, commercial products, or organization imply endorsement by the US
Financial support: National Institutes of Health (contract CO-12400; grants TW-
06625 and AI-53877).
Reprints or correspondence: Dr. O. M. Zack Howard, PO Box B, 1050 Boyles
St., Frederick, MD 21702 (email@example.com).
The Journal of Infectious Diseases
? 2006 by the Infectious Diseases Society of America. All rights reserved.
. B. malayi is found only in Asia, from India in the
west to Korea in the northeast and Indonesia in the
south, and infects ∼13 million people [1, 2]. The course
of infection with filarial parasites may last for decades
in the human host.
Filarial disease develops with the parasite repeated-
ly provoking host-derived acute, as well as long-term
chronic, inflammatory reactions within the lymphatic
channels and nodes, where the worms eventuallyreside
and reach sexual maturity . In Brugian filariasis, ep-
isodes of prolonged fever, adenolymphangitis,abscesses
of affected lymph nodes, and local residual scarring
occur frequently in infected persons . Withrecurrent
episodes of filarial lymphadenitis and complication of
secondary bacterial infection, progression to elephan-
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BmAsnRS Activates CXCR1 and CXCR2 • JID 2006:193 (15 April) • 1165
aginyl-tRNA synthetase (BmAsnRS) and human (Hu) AsnRS placed in the
lower wells of a microBoyden chemotaxis chamber. The cell type being
tested is shown above each graph (iDC, immature dendritic cells); 0
indicates background. B, Chemoattraction of primary eosinophils to
BmAsnRS at 10 ng/mL chemoattractant. Binding medium indicates back-
ground. Eotaxin was used as a positive control. The concentrations of
BmAsnRS are shown on the X-axes, whereas the mean (?SD) numbers
of cells/high-power field (hpf; ?200) are shown on the Y-axes. Each
condition was tested in triplicate with each assay performed a minimum
of 3 times. Unpaired Student’s t tests were used to compare the number
of migrating cells in the binding medium versus the number of migrating
cells in individual chemoattractant concentrations. *
A, Migration of primary leukocytes to Brugia malayi aspar-
.P ? .001
tiasis occurs in a small population of cases. The swelling is
often restricted to distal extremities beyond the knee or elbow.
Sclerotic cordlike lymphatics and enlarged firm nodes of the
arms and legs are usual. Further proliferation of endothelial
cells and connective tissue cells may lead to obliterative lym-
phangitis with scarred vessels.
induce histologically obvious lesions, the most severe reactions
occur around dead or dying worms that produce infiltrates of
polymorphonuclear cells and histiocytes . This may later pro-
gress to local necrosis and marked edema of the surrounding
tissue. The gross pathological sequelae of repeated inflammatory
reactions result in obstruction of normal lymphatic flow, dila-
tation of lymphatic vessels, valvular incompetence of theafferent
lymphatic obstruction. The skin can become greatly thickened
in elephantiasis, which is characterized by markedly increased
hyperplasia of the connective tissues, edema, and diffuse infil-
tration of plasma cells, eosinophils, and monocytes.
It had long been believed that excretory-secretory worm
products might significantly participate in the endothelial ac-
tivation that triggers massive host inflammatory processes.
One potentially important excretory-secretory worm product
may well be a 63-kDa immunodominant B. malayi antigen,
which, in earlier community-based studies of lymphatic fil-
ariasis [4–6], was reported to induce a strong IgG3 response
among infected patients. Subsequent characterization of this
antigen led to the identification and cloning of the corre-
sponding gene that encodes a biologically activehomodimeric
cytoplasmic B. malayi asparaginyl–transfer(t)RNAsynthetase
(BmAsnRS) [7, 8].
Our group previously reported the chemotactic effects of hu-
man asparaginyl-tRNA synthetase and histidyl-tRNAsynthetase,
which are known autoantigens in myositis, a debilitating muscle
disease associated with an inflammatory cellular infiltrate .
We observed that these autoantigenic human aminoacyl-tRNA
synthetases (HuAsnRS) specifically activate chemokinereceptors
may perpetuate the development of myositis by recruiting and
activating immune cells, thereby promoting innate and adaptive
immune responses. The possibility that similar chemotactic ef-
fects of BmAsnRS may contribute to the induction of acute and/
or chronic host inflammatory responses against the filarial par-
asite is the subject of the current investigation.
In the present study, we determined that parasite BmAsnRS,
like HuAsnRS, served as a chemoattractant for primary human
lymphocytes and immature dendritic cells; however, BmAsnRS
also chemoattracted neutrophils and eosinophils, whereas
HuAsnRS did not. BmAsnRS, unlike HuAsnRS, did notactivate
the chemokine receptor CCR3. Instead, BmAsnRS interacted
with both human CXCR1 and CXCR2 chemokine receptors.
In addition, we have shown that BmAsnRS desensitizedgrowth
regulating oncogene (Gro)–a–induced calcium mobilizationin
neutrophils and activated the mitogen-activatedproteinkinases
(MAPKs) Erk1 and Erk2 in the cell migration–signal trans-
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1166 • JID 2006:193 (15 April) • Ramirez et al.
aginyl-tRNA synthetase (BmAsnRS).
Checkerboard analysis of neutrophil migration in response to Brugia malayi aspar-
in lower compartment, ng/mL
BmAsnRS concentration in upper compartment, ng/mL
0 0.1 1.0 10.0
201.7 ? 10.4
262.3 ? 18.3
378.0 ? 33.7
360.0 ? 14.4
185.3 ? 30.0
208.5 ? 23.0
323.3 ? 33.0
304.2 ? 13.7
193.0 ? 18.0
188.6 ? 36.0
279.7 ? 22.2
300.0 ? 14.1
206.7 ? 37.0
251.2 ? 25.9
225.0 ? 14.3
241.8 ? 9.70
Data are the mean ? SD number of cells migrated per high-power field.
MATERIALS AND METHODS
Peprotech. All monoclonal antibodies used to phenotype den-
dritic cells were purchased from BD Biosciences, unless oth-
erwise indicated. All chemicals were obtained from Sigma-
Aldrich, unless otherwise indicated. Human recombinant
HuAsnRS and parasite recombinant BmAsnRS were prepared
as described elsewhere [8, 9].
Primary human leukocytes were isolated from fresh
normal donor leukapheresis packs under an approved human
subjects protocol, as described elsewhere . In some studies,
Percoll-purified lymphocytes or monocytes were cultured at a
concentration of 106cells/mL in RPMI 1640 containing 10%
fetal bovine serum (HyClone), 2 mmol/L glutamine, and 100
U/mL each penicillin and streptomycin (Quality Biologicals),
with 100 U/mL recombinant human interleukin (IL)-2 for 16
h or 7 days in a 5% CO2humidified tissue culture incubator.
HEK-293 cells were cultured in Dulbecco’s modified Eagle me-
dium (Bio Whittaker) containing 10% fetal bovine serum (Hy-
clone), 2 mmol/L glutamine, and 100 U/mL penicillin and
streptomycin (Quality Biologicals).
Human immature dendritic cells were generated from pu-
rified human peripheral blood monocytes (195%), as described
elsewhere , and were confirmed by flow cytometryanalysis.
cDNA for human CXCR2 (P25025), human CXCR3 (P49682),
or chimera of CXCR2 and CXCR3 were cloned into pcDNA3.1
(Invitrogen) containing immunoglobulin k light-chain signal
sequence and a hemagglutinin epitope. The chimera 2333 was
composed of 1–48 aa of CXCR2 and 54–368 aa of CXCR3. The
chimera 3233 was composed of amino acids 106–120 of CXCR2,
which replaced amino acids 111–125 of CXCR3; the remaining
amino acids corresponded to CXCR3. The chimera 3323 was
composed of amino acids 184–208 of CXCR2, which replaced
amino acids 190–212 of CXCR3; the remaining amino acids
of amino acids 276–294 of CXCR2, which replaced amino acids
280–298 of CXCR3; the remaining amino acids correspondedto
CXCR3. The sequence of each expression vector was confirmed
All chemokines and cytokines were obtained from
Expression vectors containing
by high-throughput sequencing performed by the Laboratory of
Molecular Technology, Science Applications International Cor-
poration–Frederick (Frederick, MD). Stable HEK-293 transfec-
tants expressing individual receptors or receptor chimera were
produced using Lipofectamine, as described bythemanufacturer
(Invitrogen). Surface expression was confirmed by antihemag-
glutinin staining (data not shown).
Cells were resuspended in chemotaxis media
L HEPES [pH 8.0]) at a concentration of 1–
Chemokines diluted in chemotaxis medium were placed in the
lower wells of a microBoyden chemotaxis chamber (Neu-
roprobe). When circulating leukocytes were analyzed, 5-mm
polycarbonate membranes were placed over the chemokines.
Detection of chemotaxis by lymphocytes required that the
membranes be precoated with 50 mg/mLfibronectin.WhenHEK
transfectants were analyzed, 10-mm polycarbonate membranes
precoated with 50 mg/mL rat tail collagen type 1 (Collaborative
Biomedical Products) at 37?C for 2 h were placed over the che-
moattractants. After the microchemotaxis chamber was assem-
bled, 50 mL of cells was placed in the upper wells. The filled
chemotaxis chambers were incubated in a humidified CO2in-
cubator for 60 min for neutrophils and eosinophils, for 90 min
for monocytes and immature human dendritic cells, for 3 h for
lymphocytes, or for 5 h for HEK transfectants. After incubation,
the membranes were removed from the chemotaxis chamber
assembly and then gently removed from the upper side of the
membrane. The cells on the lower side of the membrane were
stained using Rapid Stain (Richard Allen). The number of mi-
grated cells in 3 high-powered fields(?200)wascountedbylight
microscopy after samples were coded. Quantization of human
cell migrations was performed with computer assistance, using
the BIOQUANT program (R & M Biometrics). Paired Student’s
t tests were performed between the binding medium controland
given chemokine concentrations. All experiments were repeated
at least 3 times.
Primary human neutrophils were
loaded with Fura-2 in the following manner:
loading medium (Dulbecco’s modified Eagle medium with 10%
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BmAsnRS Activates CXCR1 and CXCR2 • JID 2006:193 (15 April) • 1167
synthetase (BmAsnRS)–induced neutrophil migration. The concentrations
of pertussis toxin used to pretreat human neutrophils for 1 h are shown
on the X-axis, whereas the mean (?SD) numbers of cells/high-power
field (hpf; ?200) are shown on the Y-axis. Media control indicates back-
ground; human CXCL8 (interleukin-8) was used as a positive control. Each
condition was tested in triplicate, with each assay being performed a
minimum of 3 times. Unpaired Student’s t tests were used to compare
the number of migrating cells without pertussis toxin treatment versus the
number of migrating cells with pertussis toxin treatment.
significant reduction in migrating cells was observedat200ng/mLpertussis
toxin for both CXCL8 and BmAsnRS.
Pertussis toxin sensitivity of Brugia malayi asparaginyl-tRNA
. AP ? .0001
and CXCR2/HEK-293 (B)–transfected cell lines by Brugia malayi as-
paraginyl-tRNA synthetase (BmAsnRS) (100 ng/mL). Human (Hu) AsnRS,
failed to induce chemotaxis of these cells. Human CXCL8 (interleukin-8)
was used as positive control. The mean (?SD) numbers of cells/high-
power field (hpf; ?200) are shown on the Y-axis. Unpaired Student’s t
tests were used to compare the number of migrating cells in the binding
medium versus thenumber ofmigratingcellsinindividualchemoattractant
concentrations. *.P ? .0001
Induction of migration of CXCR1/HEK-293–transfected (A)
fetal calf serum) was incubated with 5 mmol/L Fura-2 AM (Mo-
lecular Probes) for 30 min at room temperature in the dark. The
dye-loaded cells were washed 3 times and resuspended in saline
buffer (138 mmol/L NaCl, 6 mmol/L KCl, 1 mmol/L CaCl2, 19
mmol/L HEPES [pH 7.4], 5 mmol/L glucose, and 0.1% bovine
serum albumin). The cells were then transferred into contin-
uously stirred quartz cuvettes (
placed in a 37?C luminescence spectrophotometer (LS-50B;
Perkin-Elmer). Stimulants at different concentrations were
added in 20 mL volume to each cuvette at the indicated time
points. The relative ratio of fluorescence excited at 340- and
380-nm wavelengths was recorded and calculated every second
using FL WinLab program (Perkin Elmer).
Activation of MAPKs.
The activation of MAPKs p44
(Erk1) and p42 (Erk2) was tested by Western blotting.CXCR2-
transfected HEK-293 cells were grown to a high confluence in
culture and starved in 0.5% fetal bovine serum–containingme-
dium. The transfected cells (CXCR2/HEK-293 cells) were then
treated with 1 mg/mL BmAsnRS in fresh 0.5% fetal bovineserum
medium. IL-8 (CXCL8) was used as positive control. Cells were
lysed by adding 1? SDS sample buffer, followed by sonication
to shear DNA and reduce sample viscosity. Samples were then
cells in 2 mL) that were
onto a mini SDS-PAGE gel. Electrophoresed bands were elec-
trotransferred to a polyvinylidene fluoride membraneforWest-
ern immunoblotting using phospho-p44/p42 MAPK mouse
monoclonal antibodies (Cell Signaling Technology). After ex-
posure to antibody, the membrane was washed 3 times with
Tris-buffered saline with Tween 20, followed by incubationwith
a working solution of ECLplus DetectionReagents(Amersham)
for 5 min and exposure to radiographic film, which was then
developed using an automatic processor (Kodak X-OMAT
(62.5 mmol/L Tris-HCl, pH 6.7, 2% SDS, 100 mmol/L 2-mer-
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1168 • JID 2006:193 (15 April) • Ramirez et al.
synthetase (BmAsnRS). Primary human neutrophils were loaded with Fura-2 and stimulated with increasing concentrations of CXCL1. B, Lack of change
in bound versus free calcium ratio by BmAsnRS but blockade of CXCL1-induced ratio changes. Fura-2–loaded neutrophils were exposed to 2 mg/mL
BmAsnRS 200 s before exposure to CXCL1. C, CXCL1 and formylmethionylleucylphenylalanine (fMLP) do not desensitize each other. Fura-2–loaded
neutrophils were exposed to 3.5 ng/mL CXCL1 followed by 1 nmol/L (darker line) or 1 nmol/L (lighter line) fMLP followed by 3.5 ng/mL CXCL1. D,
Lack of induction of calcium flux by BmAsnRS, even over 3 logs of concentration, can suppress CXCL1-induced calcium flux without limiting fMLP-
induced calcium flux by neutrophils. Lines are offset for ease of visualization. All lines show Fura-2–loaded neutrophils, which were exposed in the
upper line (lightest color) to only 3.5 ng/mL CXCL1. The middle line shows calcium flux by neutrophils exposed to 0.2 mg/mL BmAsnRS followed at
200 s by 3.5 ng/mL CXCL1 and at 300 s by 1 nmol/L fMLP. The lower line (darkest color) shows calcium flux by neutrophils exposed to 20 mg/mL
BmAsnRS followed at 200 s by 3.5 ng/mL CXCL1 and at 300 s by 1 nmol/L fMLP.
A, Blockade of CXCL1 (growth regulating oncogene [Gro]–a)–induced calcium flux in neutrophils by Brugia malayi asparaginyl-tRNA
captoethanol) at 50?C for 30 min and then reprobed as before
for total Erk.
Chemotactic effects of BmAsnRS.
mary cells to determine whether BmAsnRS, like HuAsnRS,che-
moattracted the same populations of leukocytes. As can be seen
in figure 1, BmAsnRS and HuAsnRS chemoattractedseveralsub-
sets of leukocytes, including primary human lymphocytes and
immature dendritic cells. In contrast, BmAsnRS also specifically
chemoattracted neutrophils. To further evaluate BmAsnRS with
respect to cells observed in filarial disease, we determined that
eosinophils were also chemoattracted by BmAsnRS.
We next determined that the migratory activity of BmAsnRS
was likely due to chemotaxis rather than chemokinesis by per-
forming a desensitization or checkerboard assay. In this assay,
increasing amounts of BmAsnRS are added to the upper wells
We initially evaluated pri-
of a microBoyden chamber. As can be seen in table1,increasing
amounts of BmAsnRS in the upper chamber reduced the num-
ber of migrating cells, indicating the BmAsnRS induced a che-
motactic rather than a chemokinetic response. To determine
whether the chemotactic response induced by BmAsnRS was
due to a Gaiprotein–coupled receptor, neutrophils were pre-
treated for 1 h with pertussis toxin at the indicated concen-
trations and then weresubjectedtoastandardchemotaxisassay.
As can be seen in figure 2, both CXCL8- andBmAsnRS-induced
chemotaxis were inhibited by pertussis toxin treatment.
We next investigated whether parasite BmAsnRS interacted
with the same chemokine receptor as did HuAsnRS, which we
know from previous studies induces CCR3 receptor–expressing
cells to migrate [8, 9]. Using single chemokine receptor–trans-
fected HEK-293 cells in a microBoyden chemotaxis chamber,we
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BmAsnRS Activates CXCR1 and CXCR2 • JID 2006:193 (15 April) • 1169
and Erk2. Western blot analysis of extracts of Brugia malayi asparaginyl-
tRNA synthetase (BmAsnRS)–treated cells (CXCR2-transfected HEK-293
cells) using phospho-p44/42 MAPK mouse monoclonal antibody (Cell Sig-
naling Technology). Interleukin-8 (IL-8) was used as a positive control.
Activation of mitogen-activatedproteinkinases(MAPKs)Erk1
asparaginyl-tRNA synthetase (BmAsnRS) chemotactic signal.
Domains responsible for transmission of Brugia malayi
Receptor, chemoattractant ConcentrationChemotaxis
?, no induced cell migration; +, 11.5-fold increase over spontaneousmigration
IL, interleukin; ITAC, interferon-inducible T cell a-chemoattractant;
that BmAsnRS does not use the same receptor as does HuAsnRS
to induce cell migration.
Knowing that BmAsnRS chemoattracted neutrophils and
eosinophils, we proceeded to useCXCR1-andCXCR2-transfect-
ed cell lines to determine which of these chemokine receptors
might be activated by the parasite ligand. Figure 3 shows that
BmAsnRS (100 ng/mL) induced the migration of both human
CXCR1- and CXCR1/HEK-293–transfected cell lines.HuAsnRS,
however, failed to induce chemotaxis of these cells. Both CXCR1
and CXCR2 chemokine receptors were therefore activatedbythe
parasite ligand but not by the human homologue.
Effect of BmAsnRS on calcium flux.
consequence of BmAsnRS interacting with chemokinereceptors,
Gro-a (CXCL1) or BmAsnRS, using real-time observation of
Fura 2–loaded cells. Calcium mobilization is a proximal event
in chemokine receptor signaling. Gro-a (CXCL1) activated che-
mokine receptors on human neutrophils, as evidenced by in-
flux by itself, but it desensitized Gro-a–induced calcium flux
while not affecting the calcium flux induced by formylmethio-
nylleucylphenylalanine, which uses a different chemoattractant
receptor. The human autoantigens similarly inhibited cognate
chemokine ligand–induced calcium fluxes but did not induce
a calcium flux of their own .
Induction of signal cascades by BmAsnRS.
treated CXCR2-transfected HEK-293 cells using phospho-p44/42
MAPK mouse monoclonal antibody (Cell Signaling Technolo-
gy). For both IL-8– and BmAsnRS-treated cells, phospho-p44/
42 MAPK mouse monoclonal antibody showed endogenous
levels of p44 and p42 MAPK (Erk 1 and Erk2) to be dually
phosphorylated at threonine 202 and tyrosine 204. This pro-
vides evidence that BmAsnRS activates the protein kinase cas-
cade that is downstream of chemokine receptor activation.
Chimeric receptors were tested to determinewhichdomain(s)
transmit BmAsnRS chemotactic signal. The results are reported
in table 2. As was previously reported for human histidyl-tRNA
synthetase, BmAsnRS requires the third extracellulardomain(al-
To test the functional
Figure 5 shows
ternatively known asthesecond extracellularloop)ofitsreceptor
to transduce a chemotactic signal.
This work showed that BmAsnRS is a potent (?0.5 nmol/L)
chemoattractant for human lymphocytes, immature dendritic
cells, neutrophils, and eosinophils by activating human CXCR1
and CXCR2 receptors. The recruitment of eosinophilsinBrugia
infections has been proposed as essential for the elimination
of larvae [12–14] in murine models. Very early work showed
a requirement for complement-mediated cellular adhesion for
both eosinophils and monocytes to localize with the filarial
larvae of Dipetalonema viteae . Recent work has shown that
CXCR2 is essential for neutrophils to firmly attach to endo-
thelial cells . In light of these earlier studies, our study
suggests that parasite BmAsnRS, by activating either CXCR1 or
CXCR2, may both act as a chemoattractant for selectleukocytes
and induce the adhesion of those leukocytes. Furthermore, by
chemoattracting immature dendritic cells, the parasite has the
means to promote the host immune response . Semnani et
al. [18, 19] showed that B. malayi placed in human skin blisters
recruited skin dendritic cells or Langerhans’ cells with reduced
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1170 • JID 2006:193 (15 April) • Ramirez et al.
surface expression of major histocompatibility complex I and II
and reduced ability to activate CD4 T cells.
CXCR1 and CXCR2 are expressed by several additional leu-
kocyte subsets that may participateinthepathologicalprocesses
of filarial parasites, including mast cells [20–22], CD1d NK T
cells , and CD8 T cells [24, 25]. Mast cells have long been
associated with innate immune responses to cutaneous inflam-
mation and granuloma formation. Recent work has shown that
limiting mast cell degranulation blocks mast cell–mediated an-
giogenesis , which is associated with such chronic inflam-
matory diseases as rheumatoid arthritis and possibly contrib-
utes to increasing granulomatous disease. CD1d NK T cells are
very interesting innate immune cells, because their activating
ligand is a lipid and they express CXCR1 and CXCR2 receptors
. B. malayi nematode egg and embryo express an unusual
parasite-specific lipid-binding protein, but whether the lipid-
binding protein expressed by the parasite inhibits NK T func-
tion has not been evaluated . The recruitment of effector
CD8 T cells to participate in human host defense against viral
infections has recently been shown to require CXCR1 [24, 25].
The human effector CD8 cells with the greatest expression of
CXCR1 also showed the greatest expression of cytolytic factors,
perforin, and granzyme B , and interferon-g. However, the
role of interferon-g in murine models of filariasis is contro-
versial [28, 29]. The immunocompetent mouse is not permis-
sive for human filarial (B. malayi) infection and does not ex-
press CXCR1 . In contrast, rats and guinea pigs express
both CXCR1 and CXCL8 homologues  and are permissive
for human filarial infection . Further evaluation of CD8
cells in human filarial infection is needed to clarify the role of
cytolytic T cells and interferon-g in filarial disease.
The work of Wakasugi and Schimmel [33, 34] first showed
that a human aminoacyl-tRNA synthetase could act as both a
chemoattractant and activating cytokine for human myeloid
cells. Human tyrosyl-tRNA synthetase (TyrRS) binds to human
CXCR1, inducing neutrophil migration, but the Saccharomyces
cerevisiae and Escherichia coli homologues of TyrRS do not [33,
34]. The exact domains of human CXCR1 involved in human
TyrRS binding have not been reported; however, the domains
needed for HisRS-induction of CCR5-mediated migrationwere
shown to include the third and fourth extracellular domains.
In this study, we have mapped association of BmAsnRS to only
ies of chemokines and their receptors have shown this domain
to be a low-affinity chemokine-binding domain that is essential
for chemokine-mediated migratory signal transduction.Be-
cause we have observed Erk activation by BmAsnRS, it would
seem likely that the third extracellular domain of CXCR2 is also
essential for the transmission of BmAsnRS-mediated signal. Al-
though there is no obvious primary sequencehomologybetween
BmAsnRS and human TyrRS–BmAsnRS even lacks the 3-aa ELR
(glutamate-leucine-arginine) chemokine homologydomainsug-
gested by Wakasugi and Schimmel  as a possible TyrRS/
CXCR1–binding domain–both proteins have clusters of negative
charged amino acids, which might interact with the 2 clusters
of positively charged amino acids found in the CXCR2 third
extracellular domain. When they are available, analysis of the
tertiary structures should suggesthowBmAsnRSisabletomimic
a human chemoattractant.
In summary, lymphatic filariasis is a chronic human parasitic
disease whose excretory-secretory products are likely to play a
role in development of associated pathological processes. Filarial
(B. malayi) AsnRS is an immunodominant antigen that induces
a strong human IgG3 response in persons with lymphatic fila-
riasis. We have determined that BmAsnRS, like HuAsnRS, che-
moattracts populations of leukocytes, including circulating hu-
man lymphocytes, immature dendritic cells, eosinophils, and
neutrophils. Recombinant BmAsnRS, but not recombinant
HuAsnRS, induced chemotaxis of CXCR1 and CXCR2 single-
receptor–transfected HEK-293 cell lines, blocked CXCL1-in-
duced calcium flux, and induced MAPK activation, as expected
for chemokine-induced activation of signal transduction. The
present findings suggest that a filarial parasite chemoattractant
protein possesses the potential to contribute to development of
chronic inflammatory disease. These observations also suggest
be therapeutic against parasite-induced pathology.
We thank Kip Wigmore and Nancy Dunlop for expert technical assis-
tance in the laboratory and Drs. J. J. Oppenheim and J. A. Turpin for
critical manuscript review.
1. Duerr HP, Dietz K, Eichner M. Determinants of the eradicability of
filarial infections: a conceptual approach. Trends Parasitol 2005;21:
2. Kron M, Walker E, Hernandez L, Torres E, Libranda-Ramirez B. Lym-
phatic filariasis in the Philippines. Parasitol Today 2000;16:329–33.
3. King CL, Freedman DO. Filariasis. In: Strickland GT, ed. Hunter’s
tropical medicine and emerging infectious diseases. 8th ed. Phil-
adelphia: WB Sauders, 1999:740–55.
4. Kazura JW, Hazlett FE Jr, Pearlman E, et al. Antigenicity of a protective
recombinant filarial protein in human bancroftian filariasis. J Infect
5. Nilsen TW, Maroney PA, Goodwin RG, et al. Cloning and character-
ization of a potentially protective antigen in lymphatic filariasis. Proc
Natl Acad Sci USA 1988;85:3604–7.
6. Perrine KG, Denker JA, Nilsen TW. A multi-copy gene encodes a
potentially protective antigen in Brugia malayi. Mol Biochem Parasitol
7. Kron M, Marquard K, Hartlein M, Price S, Leberman R. An immu-
nodominant antigen of Brugia malayi is an asparaginyl-tRNA synthe-
tase. FEBS Lett 1995;374:122–4.
8. Kron M, Petridis M, Milev Y, Leykam J, Hartlein M. Expression, lo-
by guest on November 15, 2015
BmAsnRS Activates CXCR1 and CXCR2 • JID 2006:193 (15 April) • 1171
calization, and alternative function of cytoplasmic asparaginyl-tRNA
synthetase in Brugia malayi. Mol Biochem Parasitol 2003;129:33–9.
9. Howard OM, Dong HF, Yang D, et al. Histidyl-tRNA synthetase and
asparaginyl-tRNA synthetase, autoantigens in myositis, activate che-
mokine receptors on T lymphocytes and immature dendritic cells. J
Exp Med 2002;196:781–91.
10. Grimm MC, Ben-Baruch A, Taub DD, Howard OM, Wang JM, Op-
penheim JJ. Opiate inhibition of chemokine-induced chemotaxis. Ann
N Y Acad Sci 1998;840:9–20.
11. Yang D, Howard OM, Chen Q, Oppenheim JJ. Cutting edge: immature
dendritic cells generated from monocytes in the presence of TGF-beta
1 express functional C-C chemokine receptor 6. J Immunol 1999;163:
12. Ramalingam T, Ganley-Leal L, Porte P, Rajan TV. Impaired clearance
of primary but not secondary Brugia infections in IL-5 deficient mice.
Exp Parasitol 2003;105:131–9.
13. Rajan TV, Ganley L, Paciorkowski N, Spencer L, Klei TR, Shultz LD.
Brugian infections in the peritoneal cavities of laboratorymice:kinetics
of infection and cellular responses. Exp Parasitol 2002;100:235–47.
14. Simons JE, Rothenberg ME, Lawrence RA. Eotaxin-1–regulated eosin-
ophils have a critical role in innate immunity against experimental
Brugia malayi infection. Eur J Immunol 2005;35:189–97.
15. Haque A, Ouaissi A, Santoro F, des Moutis I, Capron A. Complement-
mediated leukocyte adherence to infective larvaeofDipetalonemaviteae
(Filarioidea): requirement for eosinophils or eosinophil products in
effecting macrophage adherence. J Immunol 1982;129:2219–25.
16. Smith ML, Olson TS, Ley K. CXCR2- and E-selectin–induced neutro-
phil arrest during inflammation in vivo. J Exp Med 2004;200:935–9.
17. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic
cells. Annu Rev Immunol 2003;21:685–711.
18. Semnani RT, Law M, Kubofcik J, Nutman TB. Filaria-inducedimmune
evasion: suppression by the infective stage of Brugia malayi at the
earliest host-parasite interface. J Immunol 2004;172:6229–38.
19. Semnani RT, Nutman TB. Toward an understanding of the interaction
between filarial parasites and host antigen-presenting cells. Immunol
20. Lippert U, Zachmann K, Henz BM, Neumann C. Human T lympho-
cytes and mast cells differentially express and regulate extra- and in-
tracellular CXCR1 and CXCR2. Exp Dermatol 2004;13:520–5.
21. Inamura H, Kurosawa M, Okano A, Kayaba H, Majima M. Expression
cultured human mast cells. Int Arch Allergy Immunol 2002;128:142–50.
22. Nilsson G, Mikovits JA, Metcalfe DD, Taub DD. Mast cell migratory
response to interleukin-8 is mediated through interaction with che-
mokine receptor CXCR2/interleukin-8RB. Blood 1999;93:2791–7.
23. Thomas SY, Hou R, Boyson JE, et al. CD1d-restrictedNKTcellsexpress
a chemokine receptor profile indicative of Th1-type inflammatory
homing cells. J Immunol 2003;171:2571–80.
24. Takata H, Tomiyama H, Fujiwara M, Kobayashi N, Takiguchi M. Cut-
ting edge: expression of chemokine receptor CXCR1onhumaneffector
CD8+T cells. J Immunol 2004;173:2231–5.
25. Hess C, Means TK, Autissier P, et al. IL-8 responsiveness defines a
subset of CD8 T cells poised to kill. Blood 2004;104:3463–71.
26. Russo A, Russo G, Peticca M, Pietropaolo C, Di Rosa M, Iuvone T.
Inhibition of granuloma-associated angiogenesis by controlling mast cell
mediator release: role of mast cell protease-5. Br J Pharmacol 2005;145:
27. Michalski ML, Monsey JD, Cistola DP, Weil GJ. An embryo-associated
fatty acid–binding protein in the filarial nematode Brugia malayi. Mol
Biochem Parasitol 2002;124:1–10.
28. Gray CA, Lawrence RA. Interferon-gamma and nitricoxideproduction
are not required for the immune-mediated clearance of Brugia malayi
microfilariae in mice. Parasite Immunol 2002;24:329–36.
29. SaeftelM, VolkmannL, KortenS,etal.Lackofinterferon-gammaconfers
impaired neutrophil granulocyte function and impartsprolongedsurviv-
al of adult filarial worms in murine filariasis. Microbes Infect 2001;3:
30. Mestas J, Hughes CC. Of mice and not men:differencesbetweenmouse
and human immunology. J Immunol 2004;172:2731–8.
31. Malgorzata Goczalik I, Raap M, Weick M, et al. The activation of IL-
8 receptors in cultured guinea pig Muller glial cells is modified by
signals from retinal pigment epithelium. J Neuroimmunol 2005;161:
32. Bell RG, Adams L, Coleman S, Negrao-Correa D, Klei T. Brugia pa-
hangi: quantitative analysis of infection in several inbred rat strains.
Exp Parasitol 1999;92:120–30.
33. Wakasugi K, Schimmel P. Two distinctcytokinesreleasedfromahuman
aminoacyl-tRNA synthetase. Science 1999;284:147–51.
34. Wakasugi K, Schimmel P. Highly differentiated motifs responsible for
two cytokine activities of a split human tRNA synthetase. J Biol Chem
35. Oppermann M. Chemokine receptor CCR5: insights into structure,
function, and regulation. Cell Signal 2004;16:1201–10.
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