Research Article Warifteine, an Alkaloid Purified from Cissampelos sympodialis, Inhibits Neutrophil Migration In Vitro and In Vivo

Abstract

Cissampelos sympodialis Eichl is a plant from the Northeast and Southeast of Brazil. Its root infusion is popularly used for treatment of inflammatory and allergic diseases. We investigated whether warifteine, its main alkaloid, would have anti-inflammatory effect due to a blockage of neutrophil function. In vivo warifteine treatment inhibited casein-induced neutrophil migration to the peritoneal cavity but did not inhibit neutrophil mobilization from the bone marrow. Analysis of the direct effect of warifteine upon neutrophil adherence and migration in vitro demonstrated that the alkaloid decreased cell adhesion to P and E-selectin-transfected cells. In addition, fLMP-induced neutrophil migration in a transwell system was blocked by warifteine; this effect was mimicked by cAMP mimetic/inducing substances, and warifteine increased intracellular cAMP levels in neutrophils. The production of DNA extracellular traps (NETs) was also blocked by warifteine but there was no alteration on PMA-induced oxidative burst or LPS-stimulated TNFíµí»¼ secretion. Taken together, our data indicate that the alkaloid warifteine is a potent anti-inflammatory substance and that it has an effect on neutrophil migration through a decrease in both cell adhesion and migration.

Full text

Research Article
Warifteine, an Alkaloid Purified from Cissampelos sympodialis,
Inhibits Neutrophil Migration In Vitro and In Vivo
Thaline F. A. Lima,1Juliana D. B. Rocha,1Anderson B. Guimarães-Costa,1
José M. Barbosa-Filho,2Débora Decoté-Ricardo,3Elvira M. Saraiva,1
Luciana B. Arruda,4Marcia R. Piuvezam,2and Ligia M. T. Peçanha1
1Departamento de Imunologia, Instituto de Microbiologia Prof. Paulo de G´
oes, Universidade Federal do Rio de Janeiro,
CCS, Bloco I, Sala I2-062, Ilha do Fund˜
ao, 21944-570 Rio de Janeiro, RJ, Brazil
2Departamento de Fisiologia e Patologia, Universidade Federal da Para´
ıba, 58051-970 Jo˜
ao Pessoa, PB, Brazil
3Instituto de Veterin ´
aria, Universidade Federal Rural do Rio de Janeiro, 23890-000 Serop´
edica, RJ, Brazil
4Departamento de Virologia, Instituto de Microbiologia Prof. Paulo de G´
oes, Universidade Federal do Rio de Janeiro,
21944-570 Rio de Janeiro, RJ, Brazil
Correspondence should be addressed to Ligia M. T. Pec¸anha; lpecanha@micro.ufrj.br
Received  December ; Revised  April ; Accepted  April ; Published  June 
Academic Editor: Mario Clerici
Copyright ©  aline F. A. Lima et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Cissampelos sympodialis Eichl is a plant from the Northeast and Southeast of Brazil. Its root infusion is popularly used for treatment
of inammatory and allergic diseases. We investigated whether warieine, its main alkaloid, would have anti-inammatory eect
due to a blockage of neutrophil function. In vivo warieine treatment inhibited casein-induced neutrophil migration to the
peritoneal cavity but did not inhibit neutrophil mobilization from the bone marrow. Analysis of the direct eect of warieine upon
neutrophil adherence and migration in vitro demonstrated that the alkaloid decreased cell adhesion to P and E-selectin-transfected
cells. In addition, fLMP-induced neutrophil migration in a transwell system was blocked by warieine; this eect was mimicked
by cAMP mimetic/inducing substances, and warieine increased intracellular cAMP levels in neutrophils. e production of DNA
extracellular traps (NETs) was also blocked by warieine but there was no alteration on PMA-induced oxidative burst or LPS-
stimulated TNF𝛼secretion. Taken together, our data indicate that the alkaloid warieine is a potent anti-inammatory substance
and that it has an eect on neutrophil migration through a decrease in both cell adhesion and migration.
1. Introduction
Cissampelos sympodialis is a plant found in the Northeast and
Southeast regions of Brazil [] and its extract is popularly used
in the treatment of both inammatory and allergic diseases
[]. Studies using the aqueous fraction of the ethanolic
extract obtained from its leaves (AFL) have conrmed its
immunomodulatory eects. AFL was shown to decrease
T cell, B cell, and macrophage function in vitro [–].
In vivo studies showed that AFL treatment decreased IgE
levels in ovalbumin sensitized animals [,]andinhibited
carrageenan-induced edema []. ese ndings suggest that
components present in the AFL may be important for the
anti-inammatory and antiallergic eects of C. sympodialis.
Warieine, a bisbenzylisoquinoline alkaloid, is the major
alkaloid puried from both root and leaves of C. sympodialis
[,]. Warieine was described to induce spasmolytic activity
on tracheal and cardiac smooth muscle and to inhibit calcium
channels and to modify the intracellular Ca2+ stores [,].
Also, this alkaloid had immunomodulatory eects since it
reduced T cell response and IgE production in an ovalbumin-
induced allergy model, increased the levels of regulatory
T cells in a food allergy model [,], and decreased B
cell proliferation and immunoglobulin production []. is
late eect was mediated through an increase in intracellular
cAMP levels [].
Neutrophils are key cells in the inammatory response
which migrate to inamed tissue towards the higher
Hindawi Publishing Corporation
Journal of Immunology Research
Volume 2014, Article ID 752923, 12 pages
http://dx.doi.org/10.1155/2014/752923

Journal of Immunology Research
OCH3
CH3
N
N
OH
O
O
1
3
45
6
7
8
9
10
11
12
13
14
𝛼
8a
4aH2C
HO
H3CO
1󳰀
3󳰀
4a󳰀
8a󳰀
5󳰀
6󳰀
7󳰀8󳰀
9󳰀
10󳰀
11󳰀
12󳰀
13󳰀14󳰀
𝛼󳰀
F : Structure of warieine. e structure is based on data previously published [,].
concentration of locally produced chemotactic substances
[].emigratoryresponseisamultistepprocessthat
is initiated by a selectin-dependent neutrophil rolling on
endothelial cells []. Subsequently, an LFA--mediated arrest
occurs and this is followed by lateral motility of leukocytes on
the surface of endothelial cells which is mediated by the Mac-
 integrin and this is followed by neutrophil transendothelial
migration []. Blockage of any of the steps in the described
processes will decrease the magnitude of the inammatory
responseandinducesevereinfection-associateddisease[,
]. Neutrophils will participate in the local response through
the production of several antimicrobial substances like the
induction of oxidative burst []. Also, neutrophils were
shown to kill microorganisms through the production of
neutrophil extracellular traps (NETs) which are formed by the
chromatin associated to granular and cytoplasm components
[,]. NETs can be induced by IL-, LPS, fMLP, PMA,
bacteria, fungi, and the protozoan Leishmania as well as by
HIV [–]. Moreover, NETs were recently identied in
human atopic asthmatic airways in vivo [,]. Despite
NETs’ reported microbicidal function, exaggerated NET for-
mation may be also associated with pathological conditions
like autoimmune and inammatory disorders [,].
Severalmodelshavebeenemployedformeasuringneu-
trophil mobilization in vivo.Caseininjectionwasshown
to induce a massive increase in neutrophil numbers in
the peritoneal cavity [,]. e neutrophil levels reach
an average content of %. ose cells are only partially
activated, since they still respond to inammatory stimuli
[,].
Given that warieine has been described as a potential
anti-inammatory compound, in the present study we inves-
tigated whether the anti-inammatory eect of warieine
wouldbemediatedthroughaninhibitionofneutrophil
mobilization and response in vitro and in vivo.Weemployed
here the model of neutrophil migration to the peritoneal
cavity aer injection of casein.
2. Materials and Methods
2.1. Reagents. DMEN tissue culture medium, fetal calf serum,
andgentamicin(usedat𝜇g/mL) were obtained from
GIBCO (Invitrogen Corporation, Carlsbad, CA, USA). is
medium was supplemented with HEPES, sodium bicar-
bonate, l-glutamine, and 𝛽-mercaptoethanol (obtained from
Sigma-Aldrich, St. Louis, MO, USA). Warieine was puried
from roots and leaves of plants grown in the botanical
garden of Departamento Fisiologia e Patologia from Federal
University of Paraiba. A voucher specimen (Agra-) was
deposited at Herbarium Lauro Pires Xavier from the Federal
University of Paraiba. Hydroalcoholic material from dried
roots and leaves was extracted with % ethanol. is step
was followed by several extractions with chloroform and a
nal purication by TLC [,]. Detailed information on
the purication and structure of the warieine was previously
published [,]anditsstructureisshowninFigure.e
warieine used throughout was in its crystal form and had a
purity of %. Warieine was endotoxin free, as determined
by NMR and mass spectroscopy. e warieine solutions
were prepared by adding  𝜇L of HCl N to mg of the
crystalfollowedbytheadditionof𝜇Lofdistilledwater.
epHwasadjustedto.withNaOHN.edilutionswere
made in PBS []. Under these conditions the alkaloid showed
% solubility. Lipopolysaccharide (LPS) (extracted from E.
coli :B), casein, phorbol myristate acetate (PMA), and N-
formyl-methionine-leucine-phenylalanine (fMLP) were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Animals and Treatments. MaleandfemaleBALB/cmice
withagerangingfromtoweekswereobtainedfrom
the animal facility of the Instituto de Microbiologia from
Federal University of Rio de Janeiro. e animals were bred
and housed according to institutional policies for animal care
and usage. All experimental protocols were approved by the
EthicalCommitteefromtheCenterofHealthSciencesfrom
Federal University of Rio de Janeiro (protocol no. IMIPPG
) and adhered strictly to the guidelines of the Brazilian
Law no. . from  which established the rules for use
and care of laboratory animals.
For in vivo studies, mice were injected (i.p.) with a
% solution of casein diluted in PBS. Warieine-treated
animals were injected i.p. with the alkaloid ( 𝜇g/mice)  h
before casein inoculation. e doses used were established
in previous dose-response studies and were in accordance
with the doses described in previous reports [,]. For bone
marrowanalysis,micewerekeptuntreatedorwereinjected

Journal of Immunology Research 
i.p. with casein, warieine, or both substances. Cells were
obtained  h later from tibia and femur. Samples containing
2×10
5cells were put into microscopy slides. A stained
cytospin preparation was set up. Dierential cell counting
was performed by analyzing  cells per slide. For blood
cell analysis, samples were obtained from mice treated as
described above. ree hours aer treatment with casein, a
drop of blood was obtained from the lateral tail vein and a
blood smear was prepared. e samples were stained with
Giemsa dye. Dierential cell counting was performed aer
analyzing a total of  cells per slide. Neutrophil percentage
in peritoneal exudates was determined by optical microscopy.
Peritoneal exudates were obtained aer warieine treatment
followed by a h treatment with casein. Samples (2×10
5
cells) were applied to microscopy slides and they were
submitted to cytospin. e slides were then stained with May-
Grunwald Giemsa. Percentage of neutrophils in the samples
was obtained by dierential cell count. Around  cells
were counted in each slide. e PMN cell population in
peritoneal exudates was also identied by ow cytometry
using a FACSCalibur (BD Biosciences, San Jose, CA, USA).
Forward scatter (FSC) by side scatter (SSC) prole was
determined and the number of neutrophils was counted in
thePMNgate.Gr-expressioninthegatedpopulationwas
also employed to conrm these cells were neutrophils. A total
of , cells were analyzed by ow cytometry. Data were
analyzed using the Cell Quest Soware.
2.3. Neutrophil Enrichment from Peritoneal Exudates for
In Vitro Studies. In vitro studies were performed using
neutrophil-rich exudates obtained from casein-treated mice
as described above. e cells were obtained  h aer casein
injection and the material was analyzed for neutrophil per-
centage in stained cytospin preparations before use. Treat-
ment was performed using the protocol described by Luo and
Dorf []. is protocol provides a neutrophil enriched cell
preparation with up to % neutrophils as determined by ow
cytometry. is neutrophil-rich cell preparation will be called
neutrophils throughout the paper for simplicity.
2.4. Cell Viability Studies. Propidium iodide labeling []:
neutrophils were obtained from the peritoneal cavity of mice
 h aer i.p. injection of casein. ese cells were cultured
for  min in either the presence or absence of PMA ( nM)
and warieine (added at various concentrations  min before
PMA addition). is was followed by staining with a FITC-
labeled anti-Gr antibody (clone RB-C, BD Pharmingen).
Aerwards, propidium iodide ( 𝜇Lat.𝜇g/mL in saline)
was added to the samples that were incubated for  min
and subsequently analyzed. e samples were analyzed for
propidium iodide labeling by ow cytometry.
XTT cell viability assay []: neutrophils were obtained
from the peritoneal cavity of casein-treated mice. ese cells
were cultured for min in either the presence or absence of
the following reagents: LPS ( ng/mL), PMA ( nM), and
warieine (added at a range of concentrations min before
addition of either PMA or LPS). Aer incubation, the cells
weretreatedfortohwith𝜇L of XTT solution (used
at  𝜇g/mL) at ∘C under a % CO2atmosphere. Triton
X--treatedcultureswereusedaspositive(deadcells)
controls. Cell viability was visualized by the development of
a colored orange derivative by viable cells. Plates were then
read using a microplate reader for determining A490.
2.5. Reactive Oxygen Species (ROS) Measurement. Asuspen-
sion of neutrophils obtained from the peritoneal cavity of
casein-injectedmicewastreatedwithPMAin vitro ( nM).
Some cultures also received dierent doses of warieine,
which was added  min before PMA treatment. e samples
were incubated for  h at ∘Candwerethenincubated
with the CM-H2DCFDA probe (. 𝜇M, Molecular Probes,
Invitrogen Corporation, Carlsbad, CA, USA) for  min in
the dark at ∘Cinanatmospherecontaining%CO
2.e
sampleswerethenanalyzedbyowcytometryformeasuring
ROS production.
2.6. Cell Binding Assay. CHO cells and CHO cells transfected
with the genes for E-selectin (CHOE) or P-selectin (CHOP)
[] were added to -well plates containing a coverslip
and the cultures were incubated overnight for establishing
a cell monolayer. Cell suspensions were prepared in DMEM
culture medium supplemented with % FCS. e following
day a neutrophil-rich cell suspension was obtained from
the peritoneal cavity of mice pretreated with casein. Some
cultures were incubated with dierent doses of warieine for
 min. Aer incubation, the neutrophil samples were added
to cultures containing a monolayer of CHO, CHOE, or CHOP
cells. e coculture was carried out for  min. Aerwards,
the plates were washed with PBS to remove nonadherent
cells and the coverslips were stained with Giemsa, mounted
on slides, and counted by light microscopy. Around 
CHOcellswerecountedandthenumberofneutrophilsthat
adhered per cell was determined.
2.7. In Vitro Transwell Cell Migration Assay. Cell chemotaxis
was determined using a transwell system (Corning, NY,
USA). Transwell inserts with a  𝜇mpolycarbonatemem-
branewereused.Neutrophils(1.5×106) were le untreated or
were incubated with dierent doses of warieine for  min at
∘Cina%CO
2atmosphere. e cell samples were added
into transwell inserts placed in a -well plate containing
fMLP ( nM) in the outside compartment. e migration
system was incubated for  h at ∘Cina%CO
2atmo-
sphere. e number of cells in the fMLP compartment was
determined by direct cell counting using a hemocytometer.
e chemotaxis index for each sample was calculated by
the ratio of the cell number in the outside compartment in
cultures with chemotactic reagents and the cell number in
medium only cultures (random spontaneous cell migration).
DbAMPc or forskolin treatment was performed as follows:
cell samples of 1.5 × 106neutrophils were incubated for
 min with warieine ( 𝜇M), DbAMPc ( 𝜇M), or forskolin
( 𝜇M). Cultures were set up as described above. Pretreated
cell samples were added to transwell inserts and fMLP was
added in the outside compartment as described above.

Journal of Immunology Research
2.8. Measurement of Neutrophil Extracellular DNA Traps
(NETs). ExtracellularDNAwasmeasuredaspreviously
described []. Neutrophils (6)wereincubatedwithseveral
doses of warieine for  min. e cells were then stimulated
with fMLP ( nM). e cultures supernatants were obtained
 h later and treated with the restriction enzymes ECOR
and HINDIII ( units/mL) and incubated for an additional
 h period. e supernatants were harvested and the DNA
content was quantied using the dsDNA Picogreen kit (Invit-
rogen Corporation, Carlsbad, CA, USA). NET evaluation by
microscopy analysis was performed as follows: neutrophils
(2×10
5) were transferred to round  mm coverslips and
incubated for  min with dierent doses of warieine.
Aerwards the cells were stimulated with fMLP as shown
above for  min. e coverslips were then xed with %
paraformaldehyde and stained with DAPI. e samples were
analyzed by phase contrast and epiuorescence microscopy
using a Zeiss Axioplan microscope.
2.9. Measurement of Intracellular cAMP. Cyclic AMP was
quantied using Gilman’s competitive binding assay []
modied as previously described []. Briey, neutrophils
were incubated for  min at ∘C in RPMI medium (pH .)
containing . mM isobutylmethylxanthine and  M ascor-
bic acid and incubated for  h with either warieine ( 𝜇M) or
forskolin ( 𝜇M—that raises levels of cAMP and was used as
a positive control). e reaction was stopped by the addition
of trichloroacetic acid. e samples were centrifugated and
the supernatant was individually passed through an ion-
exchange resin column (Dowex ) to remove trichloroacetic
acid and other nucleotides. e samples obtained were then
used in a competition assay with the regulatory subunit of
PKA with the addition of a xed, trace amount of [3H]cAMP.
2.10. Statistical Analysis. e results are shown as arithmetic
means ±SD. Student’s 𝑡-test for independent samples was
performed using the PrismGraphPad  soware. Level of
signicance was set at 𝑃 ≤ 0.05.
3. Results
3.1. Treatment with Warieine Decreased Cell Migration In
Vivo. e initial studies aimed to identify whether warieine
wouldhaveaninhibitoryeectonneutrophilmigrationin
vivo.Micewereinjectedwithcaseinandwarieineandthe
eect of the alkaloid in neutrophil migration was investi-
gated. Intraperitoneal casein injection induced, as previously
described, an increase in the percentage of neutrophils in
theperitonealuid(Figure(a)). Peritoneal exudates were
obtained between  and  h aer casein injection since kinetic
studies have shown that neutrophil migration would peak in
thistimeinterval(datanotshown).edosesofwarieine
used in vivo were the ones that were previously characterized
as able to signicantly inhibit allergic inammation [,]
and to induce maximal inhibition of neutrophil migration in
preliminary studies (data not shown). In order to thoroughly
investigate the eect of warieine in neutrophil migration
in vivo, the inhibitory eect of warieine in casein-induced
in vivo neutrophil mobilization was analyzed and determined
by three approaches: determination of the number of cells in
the PMN gate by ow cytometry analysis in plots of forward
scatter (FSC) by side scatter (SSC) (Figures (a) and (b)),
calculation of the percentage of Gr-+ cells in the PMN
gate measured by ow cytometry (Figures (c) and (d)),
and, nally, determination of the percentage of neutrophils
in cytospin smears (Figure (e)). In vivo pretreatment with
warieineinhibitedthenumberofneutrophilsinthePMN
gate of casein-treated animals in around % (Figures (a)
and (b)). Warieine treatment in vivo decreased the per-
centage of GR+ cells among the cells in the PMN leukocytes
gate (from . to .%, Figure (c)). e total number
of Gr+ cells was diminished in % (Figure (d)). Finally,
the percentage of neutrophils in cytospin smears was also
decreased in around % (Figure (e)).
To evaluate whether the decreased cell migration would
be due to a direct toxic eect of warieine, which would
decrease total in vivo neutrophil numbers, we cultured
fresh and PMA-activated neutrophils with the alkaloid and
analyzedcellviabilitybybothPIincorporationandXTT
metabolization. Warieine did not aect PI staining of resting
or PMA-activated cells (Figure (a)). e same pattern of
responsewasobservedinLPS-stimulatedcultures(datanot
shown). Cell viability, as measured by the metabolization of
the XTT dye, was not modied by warieine treatment (Fig-
ure (b)). Warieine would not induce an overall blockage
in neutrophil function since it did not induce a decrease
in oxidative burst in neutrophils stimulated by either PMA
(Figure )orLPSin vitro (data not shown).e decrease
in neutrophil recruitment to the peritoneal cavity could not
also be explained by a decrease in neutrophil mobilization
from bone marrow since there was a decline in neutrophil
numbersinbonemarrowdespitetheprevioustreatmentwith
warieine before casein-induced neutrophil mobilization
(Figure (a)). Interestingly, unlike what was observed in
casein-onlytreatedanimals,micetreatedwithbothcasein
and warieine showed an accumulation of neutrophils in
peripheral blood aer warieine treatment (Figure (b)).
3.2. Warieine Reduced Neutrophil Migration through a
Decrease in Both Cell Adhesion and Chemotactic Response
and Induced an Increase in Intracellular cAMP Levels. We
next investigated the mechanism by which warieine would
decrease neutrophil migration by analyzing its eect on cell
adhesion and chemotaxis in vitro. We observed that adhesion
of unstimulated casein-mobilized neutrophils to CHO cells
expressing either P or E selection was blocked by dierent
doses of warieine, indicating that the compound strongly
inhibited the selectin-mediated adhesion of neutrophils (Fig-
ure ).
Also, the addition of warieine to transwell cham-
bers blocked the migration of neutrophils induced by the
chemoattractants fMLP (Figure (a)) and LPS (data not
shown).eeectofwarieineoncellchemotaxiswasmim-
icked by the addition of DbcAMP and forskolin (Figure (b))
and warieine treatment induced an increase in intracellular
cAMP levels (Figure (c)).

Journal of Immunology Research 
R1 R1
1000
800
600
400
200
0
1000
8006004002000
Side scatter
Forward scatter
1000
800
600
400
200
0
1000
8006004002000
Side scatter
Forward scatter
Data.001 Data.003
(a)
Number of PMN in R1
0
2000
4000
6000
Control
War i  e i ne
Casein
Warieine + casein
∗
∗∗
(b)
M1
91.2%
M1
76.5%
Casein Warieine and casein
Gr1 FITC
Cell counts
40
30
20
10
0
100101102103104
Gr1 FITC
Cell counts
40
30
20
10
0
100101102103104
(c)
0
500
1000
1500
2000
2500
Control
War i  e i ne
Casein
Warieine + casein
∗
∗∗
Number of cells Gr1+ in R1
(d)
0
20
40
60
80
100
Neutrophils (%)
Control
War i  e i ne
Casein
Warieine + casein
∗
∗∗
(e)
F : Neutrophil number in the peritoneal cavity of mice treated with casein and warieine. (a, b) Cell number evaluation by ow
cytometry. Peritoneal cells were obtained from mice aer treatment with warieine and casein. e cells were prepared for analysis by ow
cytometry. (a) Side scatter and forward scatter proles of normal (le) and casein-treated (right) animals to indicate the R gate. (b) Total PMN
cell number in the R gate (as dened in (a)). e FSC versus SSC plots are representative of three independent experiments. e mean value of
neutrophil numbers in R gates from three independent experiments is shown (𝑛=3animals per group/experiment). ∗𝑃≤ 0.05as compared
to untreated controls and ∗∗𝑃≤ 0.05 as compared to casein-treated animals. (c) Analysis of the percentage of Gr+ cells in the peritoneal
cavity uid from animals treated with casein and warieine. Peritoneal uid was obtained from warieine and casein-treated animals. e
samples were incubated with an anti-Gr-FITC labeled antibody. Histograms show the Gr-expressing cells in the R gate. e value shown
in M indicates the percentage of Gr+ cells. Data shown in the histogram plots are representative of two independent experiments. (d)
Quantication of the number of Gr+ cells in the R gate. Data shown indicate the total number of GR+ cells from experiments shown in
(a) and are the mean value from two experiments (𝑛=4animals per group/experiment): ∗𝑃≤ 0.05 in comparison with control animals
and ∗∗𝑃≤ 0.05 in relation to animals treated with casein alone. (e) Percentage of neutrophils in peritoneal exudate obtained from mice
treated with casein and warieine. Peritoneal exudates were obtained aer a treatment with warieine and casein and were submitted to
cytospin followed by May-Grunwald Giemsa staining. Data in (e) show the percentage of neutrophils in the samples obtained by dierential
cell count. Data shown are mean value from six independent experiments (𝑛=3animals per group/experiment). ∗𝑃≤ 0.05 when comparing
with untreated controls and ∗∗𝑃≤ 0.05 when comparing data with those from the group treated with casein only.

Journal of Immunology Research
Medium
PMA
Untreated
Warieine Warieine
42.2%
4.8%
56.2%
2.2%
48.3%
2.8%
43.3%
3.2%
43.9%
7.7% 4.2%
6.7%
31.8%
FL2-PI
FL1-Gr-1
100101102103104
100
101
102
103
104
FL2-PI
FL1-Gr-1
100101102103104
100
101
102
103
104
FL2-PI
FL1-Gr-1
100101102103104
100
101
102
103
104
FL2-PI
FL1-Gr-1
100101102103104
100
101
102
103
104
FL2-PI
FL1-Gr-1
100101102103104
100
101
102
103
104
FL2-PI
FL1-Gr-1
100101102103104
100
101
102
103
104
8.1% 5.3% 7.4%
0.2 𝜇M2𝜇M
8.8%
5.0%
(a)
LPSMedium
Triton
X-100
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Control
PMA
Warieine 0.02 𝜇M
Warieine 0.2 𝜇M
Warieine 2𝜇M
A490
(b)
F : Evaluation of the toxic eect of warieine on neutrophils. (a) Isolated neutrophil suspensions were incubated with the indicated
doses of warieine and subsequently stimulated with PMA. Cultures were then incubated with FITC-labeled anti-Gr- antibody, which was
followed by incubation with a solution of propidium iodide. e samples were analyzed for propidium iodide labeling by ow cytometry. e
plots indicate the labeling of cells in the PMN gate (as dened in Figure ) and are representative of two independent experiments. (b) Isolated
neutrophil suspensions were treated for  min with PMA. Some cultures also received the indicated doses of warieine. e cells were then
incubated with the XTT dye and XTT reduction by viable cells was determined. Triton X--treated cultures were used as positive (dead
cells) controls. Statistical analysis showed no signicant dierences between warieine-treated and untreated groups. Data are representative
of three independent experiments.
3.3. Eect of Warieine on Neutrophil Function. We next
investigate whether other parameters associated with neu-
trophil activation would be modied by warieine. NETs
were not induced by the alkaloid, which inhibited NET
release induced by fMLP (Figure ). Warieine, as shown
above, did not decrease induction of oxidative burst in
neutrophils stimulated by PMA (Figure ). Warieine neither
modied the secretion of TNF-𝛼in vitro aer LPS-induced
stimulation (data not shown) nor decreased PMA-induced
neutrophil degranulation as measured by ow cytometry,
SSC scatter (data not shown).
4. Discussion
Our previous study has shown that the aqueous fraction
obtained from the ethanolic extract of roots and leaves
of C. sympodialis inhibited carrageenan-induced neutrophil

Journal of Immunology Research 
War i  e in e
Warieine War i  e in e
M1
1.5%
M1
88.4%
M1
78.5%
M1
83.5%
M1
81.8%
Untreated
M1
0.9%
M1
1.1%
M1
0.5%
Medium
PMA
Cell counts
60
50
40
30
20
10
0
100101102103104
0.2 𝜇M
0.02 𝜇M2𝜇M
CM-H2DCFDA
Cell counts
60
50
40
30
20
10
0
100101102103104
CM-H2DCFDA
Cell counts
60
50
40
30
20
10
0
100101102103104
CM-H2DCFDA
Cell counts
60
50
40
30
20
10
0
100101102103104
CM-H2DCFDA
Cell counts
60
50
40
30
20
10
0
100101102103104
CM-H2DCFDA
Cell counts
60
50
40
30
20
10
0
100101102103104
CM-H2DCFDA CM-H2DCFDA
Cell counts
60
50
40
30
20
10
0
100101102103104
Cell counts
60
50
40
30
20
10
0
100101102103104
CM-H2DCFDA
F : Measurement of reactive oxygen species in cultures stimulated with PMA and treated with warieine. Neutrophils were incubated
in either the presence or absence of PMA. Some cultures were also treated with the indicated doses of warieine. e samples were then
incubated with the probe CM-H2DCFDA. e production of ROS was analyzed by ow cytometry. e M marker shows the percentage of
cells that metabolized the probe. e data are representative of three independent experiments.
migration []. e alkaloid warieine, the main alkaloid
isolated from this plant extract, was previously shown to
inhibit leukotriene production, which would impart an anti-
inammatory eect to this alkaloid [,]. In the present
study we further characterized the anti-inammatory mech-
anism of warieine and described the eect of the alkaloid in
neutrophil migration.
A previous study has shown that the IC50for warieine
ranged from  to  𝜇M when the alkaloid is added to
cultures of either broblast or hepatic cell lines []. ese
doses are quite higher than the ones we used in the present
study. Also, warieine did not induce an overall blockage in
neutrophil function since it did not show any toxic eect on
thiscelltypeandweobservedthatthealkaloiddidnotblock
PMA-induced oxidative burst.
Several factors have been shown to induce a rapid
blood neutrophilia, like the chemokine MIP-, which induces
both an increase in circulating levels of neutrophils and
its accumulation in the peritoneal cavity aer thioglycollate
injection []. e casein-induced neutrophilia was main-
tained and was even increased aer warieine injection,
which indicated that warieine did not modify neutrophil
mobilization from the bone marrow in vivo.However,the
next step of the process (the migration to the peritoneal
cavity), which will depend on cell adhesion, was blocked by
warieine. Our in vitro measurement of neutrophil adhesion
to selectin-transfected CHO cells directly indicated that
selectin-mediated adhesion was blocked by warieine and
this eect can explain the decrease in neutrophil migration
to the peritoneal cavity we observed in vivo.
Previous studies have characterized alkaloids with anti-
inammatory eect []. Some bisbenzylisoquinoline alka-
loids (tetrandrine and berbamine) inhibited neutrophil adhe-
sion and locomotion [,]. e alkaloid tetrandrine had
also immunosuppressive eects and blocked T cell signaling
[,] and the isoquinoline alkaloid berberine was shown
to block an inammatory T cell response and the expression
of costimulatory molecules by dendritic cells []. One of the
mechanisms described to corroborate the inhibitory eect of
such alkaloids on neutrophils adhesion was the antioxidant
eect of substances like tetrandrine, which inhibited ROS
formation []. is does not seem to be the case for
warieine, since this substance had no eect on the oxidative
burst stimulated in vitro by PMA. Another mechanism that
imparts anti-inammatory eect to bis-benzylisoquinoline
alkaloids is the inhibition of secretion of cytokines like IL-
, TNF-𝛼, IL-, and IL- [,]. We did not test the
production of all these cytokines in our system, but inhibition
of cytokine production may not be the mechanisms through
which warieine would exert an anti-inammatory response,
since we did not observe a decrease in the secretion of TNF-𝛼
by neutrophils stimulated in the presence of warieine (data
not shown).
Neutrophil transmigration is the last step in the process
of neutrophil response to an inammatory stimulus in vivo
[] and this response was also inhibited by warieine, as
observed in transwell migration assays. Previous studies
analyzing the eect of warieine in B cell response have
shown that this alkaloid decreased the activation-induced
intracellular calcium increase []. Also, this alkaloid was

Journal of Immunology Research
Number of neutrophils in 300 cells
0
10
20
30
40
50 Bone marrow
Control
War i f e i ne
Casein
Warifeine + casein
∗
∗
(a)
Number of neutrophils in 300 cells
0
20
40
60
80 Blood
∗
∗∗
Control
War i  e i ne
Casein
Warieine + casein
(b)
F : Number of polymorphonuclear leukocytes in the bone marrow and peripheral blood of mice treated with casein and warieine.
Mice were treated with warieine, casein, or both substances. Aer a three-hour period past casein treatment, bone marrow (a) and blood
samples (b) were obtained. Blood smears and bone marrow cytospins were analyzed by optical microscopy aer staining. e graph shown
in (a) indicates the mean value from data obtained in two experiments (𝑛=3animals per group). ∗𝑃≤ 0.05 when comparing the group of
animals treated with casein with untreated animals (controls). ere was no statistical dierence between the group treated with casein and
the one treated with warieine plus casein. Data shown in (b) indicate the percentage of blood neutrophils in dierent groups and are the
mean value from three independent experiments (𝑛=3animals per group/experiment). ∗𝑃≤ 0.05 when comparing animals treated with
casein with the untreated ones and ∗∗𝑃≤ 0.05 in the comparison of the groups of animals treated with warieine and casein with the group
treated with casein only.
0
20
40
60
CHO CHOE CHOP
Number of adhered
neutrophils
∗∗ ∗∗ ∗∗
∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗
Control
Warieine 0.02 𝜇M
Warieine 0.2 𝜇M
Warieine 2𝜇M
F : Adhesion of neutrophils to CHO cells and to CHO cells
expressing P and E selectin. CHO cells transfected with either P
(CHOP) or E selectin (CHOE) genes were used. A cell monolayer
was obtained by overnight incubation of normal or transfected CHO
cells in coverslips. Peritoneal exudate neutrophils were obtained and
treatedwithwarieinein vitro. ose cells were then transferred
to the monolayers of CHO cells and incubated. Coverslips were
removed, washed with PBS, and Giemsa-stained. ey were ana-
lyzed by optical microscopy. e number of neutrophils adhered
to CHO cells was scored. e graphs shown indicate the mean
values from four independent experiments performed in triplicate.
∗∗𝑃≤ 0.05 in the comparison of cultures treated with warieine
(in dierent concentrations) and cultures treated with medium only
without warieine.
shown to inhibit calcium channels and modied the intracel-
lular Ca2+ stores in smooth muscle cells []. erefore, the
eect of warieine in cell migration could be due to its eect
in intracellular calcium levels in neutrophils. Even though we
did not measure calcium levels in warieine-treated cells, the
alkaloid did not alter cytoskeleton movement in either PMA
or fMLP-treated cells (data not shown). Other studies have
characterized alkaloids that inhibit calcium signaling [,]
and have suggested that the addition of rolipram or dibutyryl
cyclic AMP to neutrophil cultures would decrease calcium
levels in fMLP-stimulated cells due to an enhancement of
cyclic AMP-dependent calcium sequestration []. is may
be occurring in warieine-treated cultures, since we observed
that warieine indeed increased the levels of intracellular
cAMP and that the blocking eect of warieine in cell
migration was in fact mimicked by in vitro treatment with a
cAMP analog (DbcAMP) and by incubation with the adenyl
cyclase activator forskolin.
During an infection, aer migration to inamed tissue,
neutrophils would destroy local infecting microorganisms. A
recently described microbicidal mechanism is the production
of NETs []. We observed that warieine inhibited NET
formation in neutrophils. NET formation was shown to be
dependent on ROS produced by the NADPH oxidase, elas-
tase, hydrogen peroxide, and peptidyl arginine deiminase-
(PAD) through a mechanism poorly understood [–].
Our nding that fMLP-induced NET release was inhibited
by warieine despite no eect on the stimulation of oxidative
burst by PMA suggested that additional steps exist between
these two biological phenomena and this unidentied bio-
chemical step was subjected to inhibition by warieine.
Although the physiological or pathological roles of NETs
were unknown, these structures were identied in human
atopic subjects []. In this milieu, NETs could protect

Journal of Immunology Research 
Medium fMLP
Chemotactic index
0
2
4
6
8
10
12
∗∗ ∗∗
∗∗
∗
Control
Warieine 0.02 𝜇M
Warieine 0.2 𝜇M
Warieine 2𝜇M
(a)
fMLPMedium
Chemotactic index
0
1
2
3
4
5
6
DbAMPc
Forskolin
∗∗
∗
∗∗ ∗∗
Control
Warieine
(b)
Control Warieine
Forskolin
0
2
4
6
8
10
cAMP (fold increase)
(c)
F : Inhibition of neutrophil migration in the presence of warieine and induction of increased intracellular cAMP levels. (a) Solutions
of chemoattractant (fMLP) were added to a -well plate. A neutrophil suspension was obtained and either le untreated or incubated with
the indicated doses of warieine. eses samples were added to transwell inserts which were placed into the fMLP lled wells. Cell migration
was determined by counting cell number in the outside (fMLP) compartment using a hemocytometer. e chemotactic index was calculated
for each sample. Data shown indicate the mean value obtained from two independent experiments. ∗𝑃≤ 0.05 when comparing untreated
and fMLP-stimulated cultures; ∗∗𝑃≤ 0.05 when comparing data from warieine-treated and untreated cultures. (b) Samples obtained as
described in (a) were incubated with warieine, DbAMPc, or forskolin. Cultures were set up as in (a). Pretreated cell samples were added
into a transwell insert placed in fMLP containing wells (outside compartment). e chemotactic index is shown. (c) Neutrophil cultures
were incubated with either forskolin or warieine. Control cultures received no pretreatment. Intracellular cAMP levels were measured by a
competitive binding assay. Data indicate the fold increase of cAMP levels compared to untreated controls (set as ).
airways from infections through their microbicidal proper-
ties, contribute to the airways damage and/or remodeling,
and also modulate the immune response involved in asthma
development [,].
5. Conclusion
A hallmark feature of an inammatory response is the inl-
tration of activated neutrophils and this response is altered
by several anti-inammatory drugs. Inhibition of neutrophil
migration is a rational target for treating inammation and
drugs that inhibit this event would be an important option
forthetreatmentofthisdiseasealoneorinassociation
with other drugs. Our ndings described here indicated
that the alkaloid warieine was a potent anti-inammatory
substancethathadaneectonneutrophilmigrationthrough
both a decrease in cell adhesion and migration. Warieine
also inhibited antimicrobial events like NET production.
ese characteristics impart to warieine a potential use
in the treatment of inammatory diseases and pathological
conditions associated with NETs production.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
is research was supported by CNPq, PRONEX—MCT,
PUC/PETROBRAS, and FAPERJ. aline F. A. Lima and
JulianaD.B.RochawererecipientsoftheCNPqand
CAPES/MEC fellowships, respectively. e authors are
indebted to Mr. Sidney Gomes da Costa for the technical

 Journal of Immunology Research
0
500
1000
1500
2000
2500
3000
Medium fMLP
Extra cellular DNA (ng/mL)
Control
Warieine 0.02 𝜇M
Warieine 0.2 𝜇M
Warieine 2𝜇M
∗∗
∗∗
∗∗
∗
(a)
fMLP
Control
fMLP + warieine
(b)
F : Extracellular DNA traps (NETs) induction in warieine-treated neutrophil cultures. (a) Neutrophils were preincubated with the
indicated doses of warieine. e cells were then stimulated with fMLP. e supernatants were then harvested and the amount of extracellular
DNA was measured. (b) Neutrophils were transferred to coverslips, treated with warieine ( 𝜇M),andthenstimulatedwithfMLP.e
coverslips were xed and stained with DAPI. is was followed by analysis by phase contrast and uorescence microscopy. Plots show
representative elds recorded in light (right) and dark (le) elds where DAPI labeling was determined. Data are representative of three
independent experiments.
support. e authors thank Dr. Leonardo Nimrichter for
providing the CHO-transfected cells, Dr. Adriana Bonomo
for supplying the anti-Gr antibody, and Dr. Marcelo Bozza
for providing the ROS detection probe.
References
[] M. Correa, Dicion´
ario de plantas ´
uteis do Brasil e ex´
oticas
cultivadas (Dictionary of Brasilian useful plants and exotics
cultivated), Minist´
erio da Agricultura, Rio de Janeiro, Brazil,
.
[] J. M. Barbosa-Filho, M. F. Agra, and G. omas, “Botanical,
chemical and pharmacological investigation on C. sympodialis
from Paraiba (Brazil),” JournaloftheBrazilianAssociationFor
the Advancement of Science,vol.,pp.–,.
[] M. R. Piuvezam, L. M. T. Pec¸anha, J. Alexander, and G.
omas, “Cissampelos sympodialis Eichl. leaf extract increases
the production of IL- by concanavalin-A-treated BALB/c
spleen cells,” Journal of Ethnopharmacology,vol.,no.,pp.
–, .
[]M.S.Alexandre-Moreira,M.R.Piuvezam,andL.M.T.
Pec¸anha, “Modulation of B lymphocyte function by an aqueous
fraction of the ethanol extract of Cissampelos sympodialis Eichl
(Menispermaceae),” Brazilian Journal of Medical and Biological
Research, vol. , no. , pp. –, .
[] M. S. Alkexandre-Moreira, C. G. Freire-de-Lima, M. N.
Trindade,H.C.Castro-Faria-Neto,M.R.Piuvezam,andL.M.
T. Pe c¸anha, “Cissampelos sympodialis Eichl (Menispermaceae)
leaf extract induces interleukin--dependent inhibition of
Tryp ano s oma c r uzi killingbymacrophages,”Brazilian Journal
of Medical and Biological Research,vol.,no.,pp.–,
.
[] C. R. Bezerra-Santos, F. M. P. Balestieri, B. Rossi-Bergmann, L.
M. T. Pec¸anha,andM.R.Piuvezam,“Cissampelos sympodialis
Eichl. (Menispermaceae): oral treatment decreases IgE levels
and induces a -skewed cytokine production in ovalbumin-
sensitized mice,” Journal of Ethnopharmacology,vol.,no.-,
pp.–,.
[] C. R. Bezerra-Santos, A. Vieira-de-Abreu, J. M. Barbosa-Filho,
C. Bandeira-Melo, M. R. Piuvezam, and P. T. Bozza, “Anti-
allergic properties of Cissampelos sympodialis and its isolated
alkaloid warieine,” International Immunopharmacology,vol.,
no.,pp.–,.
[] K. V. Batista-Lima, R. A. Ribeiro, F. M. P. Balestieri, G.
omas, and M. R. Piuvezam, “Anti-inammatory activity of
Cissampelos sympodialis Eichl. (Menispermaceae) leaf extract,”
Acta Farmaceutica Bonaerense,vol.,no.,pp.–,.
[] P. S. Melo, H. M. de Medeiros Cavalcante, J. M. Barbosa-Filho,
M. de F´
atima Formiga Melo Diniz, I. A. de Medeiros, and
M. Haun, “Warieine and milonine, alkaloids isolated from

Journal of Immunology Research 
Cissampelos sympodialis Eichl: cytotoxicity on rat hepatocyte
culture and in V cells,” To x i c olog y L etters,vol.,no.-,pp.
–, .
[] F.S.deCortes,J.L.deAlencar,G.omas,andJ.M.Barbosa
Filho, “Spasmolytic actions of warieine, a bisbenzylisoquino-
line alkaloid isolated from the root bark of Cissampelos sympo-
dialis Eichl. (Menispermaceae),” Phytotherapy Research,vol.,
no. , pp. –, .
[] M. R. de Freitas, S. F. Cortes, G. omas, and J. M. Barbosa-
Filho, “Modication of Ca2+ metabolism in the rabbit aorta
as a mechanism of spasmolytic action of warieine, a bisben-
zylisoquinoline alkaloid isolated from the leaves of Cissampelos
sympodialis Eichl. (Menispermaceae),” Journal of Phamaceutics
and Pharmacology,vol.,no.,pp.–,.
[] H. F. Costa, C. R. Bezerra-Santos, J. M. Barbosa Filho, M.
A. Martins, and M. R. Piuvezam, “Warieine, a bisbenzyliso-
quinoline alkaloid, decreases immediate allergic and thermal
hyperalgesic reactions in sensitized animals,” International
Immunopharmacology,vol.,no.,pp.–,.
[] H.F.Costa,F.C.Leite,A.F.Alves,J.M.Barbosa-Filho,C.R.dos
Santos, and M. R. Piuvezam, “Managing murine food allergy
with Cissampelos sympodialis Eichl (Menispermaceae) and its
alkaloids,” International Immunophamacology,vol.,no.,pp.
–, .
[] J. D. B. Ro cha, D. Decot´
e-Ricardo, P. Redner et al., “Inhibitory
eect of the alkaloid warieine puried from Cissampelos
sympodialis on B lymphocyte function in vitro and in vivo,”
Planta Medica,vol.,no.,pp.–,.
[] M. R. Piuvezam, C. B. Bezerra-Santos, P. T. Bozza, C. Bandeira-
Melo,G.Vieira,andH.F.Costa,“Cissampelos sympodialis
(Menispermaceae): a novel phytotherapic weapon against aller-
gic diseases?” in Allergic Diseases: Highlights in Clinic, Mecha-
nisms and Treatment,C.Pereira,Ed.,vol.,InTech,Rijeka,
Croatia.
[]E.J.Kunkel,J.L.Dunne,andK.Ley,“Leukocytearrest
during cytokine-dependent inammation in vivo,” Journal of
Immunology,vol.,no.,pp.–,.
[] C. W. Smith, “. Adhesion molecules and receptors,” Journal of
Allergy and Clinical Immunology,vol.,no.,pp.S–S,
.
[] A. Woodn, M.-B. Voisin, and S. Nourshargh, “Recent develop-
ments and complexities in neutrophil transmigration,” Current
Opinion in Hematology,vol.,no.,pp.–,.
[] A. Etzioni, “Defects in the leukocyte adhesion cascade,” Clinical
Reviews in Allergy and Immunology,vol.,no.,pp.–,
.
[] J. Herter and A. Zarbock, “Integrin regulation during leukocyte
recruitment,” Journal of Immunology,vol.,no.,pp.–
, .
[] C. Dahlgren and A. Karlsson, “Respiratory burst in human
neutrophils,” Journal of Immunological Methods,vol.,no.-
, pp. –, .
[] V. Brinkmann, U. Reichard, C. Goosmann et al., “Neutrophil
extracellular traps kill bacteria,” Science,vol.,no.,pp.
–, .
[] C. F. Urban, D. Ermert, M. Schmid et al., “Neutrophil extra-
cellular traps contain calprotectin, a cytosolic protein com-
plex involved in host defense against Candida albicans,” PLoS
Pathogens,vol.,no.,ArticleIDe,.
[] V. Brinkmann and A. Zychlinsky, “Neutrophil extracellular
traps: is immunity the second function of chromatin?” Journal
of Cell Biology,vol.,no.,pp.–,.
[] A. B. Guimar˜
aes-Costa, M. T. C. Nascimento, G. S. Froment
et al., “Leishmania amazonensis promastigotes induce and are
killed by neutrophil extracellular traps,” Proceedings of the
National Academy of Sciences of the United States of America,
vol. , no. , pp. –, .
[] A. B. Guimar˜
aes-Costa, M. T. C. Nascimento, A. B. Wardini, L.
H. Pinto-Da-Silva, and E. M. Saraiva, “ETosis: a microbicidal
mechanism beyond cell death,” Journal of Parasitology Research,
vol.,ArticleID,pages,.
[] T. Saitoh, J. Komano, Y. Saitoh et al., “Neutrophil extracellular
traps mediate a host defense response to human immunode-
ciency virus-,” Cell Host & Microbe,vol.,no.,pp.–,
.
[]R.Dworski,H.-U.Simon,A.Hoskins,andS.Youse,
“Eosinophil and neutrophil extracellular DNA traps in human
allergic asthmatic airways,” Journal of Allergy and Clinical
Immunology,vol.,no.,pp.–,.
[] D. Simon, H.-U. Simon, and S. Youse, “Extracellular DNA
traps in allergic, infectious, and autoimmune diseases,” Allergy,
vol.,no.,pp.–,.
[] M. Saarzadeh and K. T. Preissner, “Fighting against the dark
side of neutrophil extracellular traps in disease: manoeuvres for
host protection,” Current Opinion in Hematology,vol.,no.,
pp.–,.
[] M. J. Kaplan and M. Radic, “Neutrophil extracellular traps:
double-edged swords of innate immunity,” Journal of Immunol-
ogy,vol.,no.,pp.–,.
[] P. C. Wilkinson and G. R. Bradley, “Chemotactic and enzyme-
releasing activity of amphipathic proteins for neutrophils. A
possible role for proteases in chemotaxis on substratum-bound
protein gradients,” Immunology,vol.,no.,pp.–,.
[] D. E. van Epps, A. D. Bankhurst, and R. C. Williams Jr., “Casein-
mediated neutrophil chemotaxis: a parallel between surface
binding and chemotaxis,” Inammation,vol.,no.,pp.–
, .
[] S.Devi,J.Laning,Y.Luo,andM.E.Do,“Biologicactivities
of the 𝛽-chemokine TCA on neutrophils and macrophages,”
Journal of Immunology,vol.,no.,pp.–,.
[]S.Tansho,S.Abe,T.Tansho,andH.Yamaguchi,“Eective
inhibition of Candida albicans growth by the combination
of murine peritoneal neutrophils and activated macrophages,”
Microbiology and Immunology,vol.,no.,pp.–,.
[] G.A.deLira,L.M.deAndrade,K.C.Flor
ˆ
encio, M. S. da Silva,
J.M.Barbosa-Filho,andE.V.Leit
˜
ao da-Cunha, “Roraimine:
a bisbenzylisoquinoline alkaloid from Cissampelos sympodialis
roots,” Fitoterapia,vol.,no.,pp.–,.
[] Y. Luo and M. E. Dorf, “Isolation of mouse neutrophils,” in
Current Protocols in Immunology, chapter , unit . , .
[] N. W. Roehm, G. H. Rodgers, S. M. Hateld, and A. L. Glase-
brook,“Animprovedcolorimetricassayforcellproliferation
and viability utilizing the tetrazolium salt XTT,” Journal of
Immunological Methods,vol.,no.,pp.–,.
[] B. Furie and B. C. Furie, “P-selectin induction of tissue factor
biosynthesis and expression,” Haemostasis,vol.,no.,pp.–
, .
[] A. G. Gilman, “A protein binding assay for adenosine 󸀠:󸀠-
cyclic monophosphate,” Proceedings of the National Academy of
Sciences of the United States of America,vol.,no.,pp.–
, .
[] M. H. Varella, F. G. de Mello, and R. Linden, “Evidence for
an antiapoptotic role of dopamine in developing retinal tissue,”
Journal of Neurochemistry, vol. , no. , pp. –, .

 Journal of Immunology Research
[] C. R. Bezerra-Santos, A. Vieira-de-Abreu, G. C. Vieira et al.,
“Eectiveness of Cissampelos sympodialis and its isolated alka-
loid warieine in airway hyperreactivity and lung remodelingin
a mouse model of asthma,” International Immunopharmacology,
vol.,no.,pp.–,.
[] R. C. Furze and S. M. Rankin, “Neutrophil mobilization and
clearance in the bone marrow,” Immunology,vol.,no.,pp.
–, .
[] W.K.Seow,A.Ferrante,A.Summors,andY.H.ong,“Com-
parative eects of tetrandrine and berbamine on production of
the inammatory cytokines interleukin- and tumor necrosis
factor,” Life Sciences,vol.,no.,pp.PL–PL,.
[] S.-Y.Li,L.-H.Ling,B.S.Teh,W.K.Seow,andY.H.ong,
“Anti-inammatory and immunosuppressive properties of the
bis-benzyisoquinolines: in vitro comparisons of tetrandrine and
berbamine,” International Journal of Immunopharmacology,vol.
, no. , pp. –, .
[]W.K.Seow,L.Si-Ying,andY.H.ong,“Inhibitoryeects
of tetrandrine on human neutrophil and monocyte adherence,”
Immunology Letters,vol.,no.-,pp.–,.
[] L. J. Ho, T. Y. Juan, P. Chao et al., “Plant alkaloid tetran-
drine downregulates IkappaBalpha kinases-IkappaBalpha-NF-
kappaB signaling pathway in human peripheral blood T cell,”
British Journal of Pharmacology,vol.,no.,pp.–,
.
[] J.-H. Lai, “Immunomodulatory eects and mechanisms of plant
alkaloid tetrandrine in autoimmune diseases,” Acta Pharmaco-
logica Sinica,vol.,no.,pp.–,.
[] Y. Yang, J. Qi, Q. Wang et al., “Berberine suppresses 
and dendritic cell responses,” Investigative Ophthalmology and
Visual Science,vol.,no.,pp.–,.
[]F.Q.He,B.Y.Qiu,T.K.Lietal.,“Tetrandrinesuppresses
amyloid-𝛽-induced inammatory cytokines by inhibiting NF-
𝜅B pathway in murine BV microglial cells,” International
Immunopharmacology, vol. , no. , pp. –, .
[] Y. Ren, L. Lu, T. B. Guo et al., “Novel immunomodulatory
properties of berbamine through selective down-regulation
of STAT and action of IFN-𝛾in experimental autoimmune
encephalomyelitis,” Journal of Immunology,vol.,no.,pp.
–, .
[] Q.-Z. Fang, N. Zhong, Y. Zhang, and Z.-N. Zhou, “Tetrandrine
inhibits Ca2+-activated chloride channel in cultured human
umbilical vein endothelial cells,” Acta Pharmacologica Sinica,
vol. , no. , pp. –, .
[] R.Anderson,A.GoolamMahomed,A.J.eron,G.Rama,
and C. Feldman, “Eect of rolipram and dibutyryl cyclic AMP
on resequestration of cytosolic calcium in FMLP-activated
human neutrophils,” British Journal of Pharmacology,vol.,
no. , pp. –, .
[] T. A. Fuchs, U. Abed, C. Goosmann et al., “Novel cell death
program leads to neutrophil extracellular traps,” Journal of Cell
Biology,vol.,no.,pp.–,.
[] V. Papayannopoulos, K. D. Metzler, A. Hakkim, and A. Zych-
linsky, “Neutrophil elastase and myeloperoxidase regulate the
formation of neutrophil extracellular traps,” Journal of Cell
Biology,vol.,no.,pp.–,.
[] I. Neeli, S. N. Khan, and M. Radic, “Histone deimination as
a response to inammatory stimuli in neutrophils,” Journal of
Immunology,vol.,no.,pp.–,.
[] Y. Wang, M. Li, S. Stadler et al., “Histone hypercitrullination
mediates chromatin decondensation and neutrophil extracellu-
lar trap formation,” Journal of Cell Biolog y,vol.,no.,pp.
–, .
[] B.-B. Cui, C.-Y. Tan, C. Schorn, H.-H. Tang, Y. Liu, and Y. Zhao,
“Neutrophilextracellular traps in sterile inammation: the story
aer dying?” Autoimmunity,vol.,no.,pp.–,.

Submit your manuscripts at
http://www.hindawi.com
Stem Cells
International
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
M E D I ATO R S
INFLA M MAT I O N
of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Behavioural
Neurology
Endocrinology
International Journal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Disease Markers
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
BioMed
Research International
Oncology
Journal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Oxidative Medicine and
Cellular Longevity
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
PPAR Research
The Scientic
World Journal
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Immunology Research
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Journal of
Obesity
Journal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Computational and
Mathematical Methods
in Medicine
Ophthalmology
Journal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Diabetes Research
Journal of
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Research and Treatment
AIDS
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Gastroenterology
Research and Practice
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Parkinson’s
Disease
Evidence-Based
Complementary and
Alternative Medicine
Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com