Accepted Manuscript Bothrops jararacussu snake venom-induces a local inflammatory response in a Prostanoid- And Neutrophil-Dependent manner C.W.S. Wanderley, C.M.S. Silva, D.T. Wong, R.M. Ximenes, D.F.C. Moreo, F. Cosker, K.S. Aragão, C. Fernandes, R.C. Palheta-Júnior, A. Havt, G.A.C. Brito, F.Q. Cunha, R.A. Ribeiro, R.C.P. Lima-Júnior PII:
S0041-0101(14)00224-4
DOI:
10.1016/j.toxicon.2014.08.001
Reference:
TOXCON 4893
To appear in:
Toxicon
Received Date: 20 May 2014 Revised Date:
29 July 2014
Accepted Date: 5 August 2014
Please cite this article as: Wanderley, C.W.S., Silva, C.M.S., Wong, D.T., Ximenes, R.M., Moreo, D.F.C, Cosker, F., Aragão, K.S., Fernandes, C., Palheta-Júnior, R.C., Havt, A., Brito, G.A.C., Cunha, F.Q., Ribeiro, R.A., Lima-Júnior, R.C.P., Bothrops jararacussu snake venom-induces a local inflammatory response in a Prostanoid- And Neutrophil-Dependent manner, Toxicon (2014), doi: 10.1016/j.toxicon.2014.08.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT Bothrops jararacussu SNAKE VENOM-INDUCES A LOCAL INFLAMMATORY
2
RESPONSE IN A PROSTANOID- AND NEUTROPHIL-DEPENDENT MANNER
3
WANDERLEY, C.W.S.1; SILVA, C.M.S.1; WONG, D.T. 1; XIMENES, R.M. 1; MOREO, D.F.C2;
4
COSKER, F.1; ARAGÃO, K.S.1; FERNANDES, C. 1; PALHETA-JÚNIOR, R.C.3; HAVT, A.1;
5
BRITO, G.A.C.4, CUNHA, F.Q.2; RIBEIRO, R.A.1; LIMA-JÚNIOR, R.C.P.1
RI PT
1
6
1
7
Ceará, Brazil;
8
University of São Paulo, Brazil; 3Colege of Veterinary Medicine, Federal University of Vale do
9
São Francisco; 4Department of Morphology, Faculty of Medicine, Federal University of
2
SC
Department of Pharmacology, School of Medicine of Ribeirão Preto,
M AN U
10
Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of
Ceará, Brazil.
11
*To whom correspondence should be addressed:
13
Roberto César Pereira Lima Júnior
14
Departamento de Fisiologia e Farmacologia
15
Faculdade de Medicina
16
Universidade Federal do Ceará
17
Rua Cel Nunes de Melo, 1315, Rodolfo Teófilo, 60430-270,
18
Fortaleza, Ceará, Brazil.
19
Contact: +55 85 3366 8588 (Phone) +55 85 3366 8333 (Fax)
20
Email: [email protected], [email protected] (Corresponding author)
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ACCEPTED MANUSCRIPT ABSTRACT
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Local tissue reactions provoked by Bothrops venoms are characterized by edema,
25
hemorrhage, pain, and inflammation; however, the mechanisms of tissue damage vary
26
depending upon the species of snake. Here, we investigated the mechanisms involved in the
27
local inflammatory response induced by the Bothrops jararacussu venom (BjcuV). Female
28
Swiss mice were injected with either saline, BjcuV (0.125-8 µg/paw) or loratadine (an H1
29
receptor antagonist), compound 48/80 (for mast cell depletion), capsaicin (for C-fiber
30
desensitization), infliximab (an anti-TNF-α antibody), indomethacin (a non-specific COX
31
inhibitor), celecoxib (a selective COX-2 inhibitor) or fucoidan (a P- and L-selectins modulator)
32
given before BjcuV injection. Paw edema was measured by plethysmography. In addition,
33
paw tissues were collected for the measurement of myeloperoxidase activity, TNF-α and IL-1
34
levels, and COX-2 immunoexpression. The direct chemotactic effect of BjcuV and the in vitro
35
calcium dynamic in neutrophils were also investigated. BjcuV caused an edematogenic
36
response with increased local production of TNF-α and IL-1β as well as COX-2 expression.
37
Both edema and neutrophil migration were prevented by pretreatment with indomethacin,
38
celecoxib or fucoidan. Furthermore, BjcuV induced a direct in vitro neutrophil chemotaxis by
39
increasing intracellular calcium. Therefore, BjcuV induces an early onset edema dependent
40
upon prostanoid production and neutrophil migration.
41
Keywords: Bothrops jararacussu venom; edema; prostanoids; neutrophil; inflammation;
42
selectins.
44
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1 INTRODUCTION Clinical observations of local effects provoked by Bothrops venoms are characterized
50
by marked edema, pain, erythema, ecchymosis, bullae with a clear serum transudate or
51
hemorrhagic content, cyanosis, extensive hemorrhage resulting in the sloughing of
52
superficial tissues, and tissue necrosis (Amaral et al., 1985). Currently, studies in the
53
literature suggest that these pathological effects result from the direct action of venoms on
54
tissues and are associated with an inflammatory response that amplifies the damage
55
(Trebien and Calixto, 1989; de Faria et al., 2001; Barbosa et al., 2003; Olivo et al., 2007;
56
Nascimento et al., 2010).
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At present, antivenom treatment is the recommended therapeutic approach for
58
snakebite envenoming. However, Bothrops venom-related local damage can be only partially
59
neutralized by a specific or the polyvalent antivenom (Gutierrez et al., 1986; Chaves et al.
60
2003; Zammuner et al. 2004; da Silva et al 2007). A probable explanation is based on the
61
fact that snake venoms are complex mixtures of toxins, enzymes and biologically active
62
peptides and that the compositions vary among families, genera and species (Leite, 1992;
63
El-Din and Omar, 2013). Within the Bothrops genus, a broad range of variation exist in the
64
composition and biological activity of the venoms (Kamiguti and Cardoso, 1989; Queiroz et
65
al., 2008). Therefore, understanding the pathophysiological mechanisms involved in the
66
tissue damage resulting from Bothrops snake envenoming would contribute to a more
67
effective therapy.
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68
TE D
57
Several studies have demonstrated the role of inflammatory mediators activated
69
following intraplantar injection of venoms from different Bothrops snakes (Trebien and
70
Calixto, 1989; Gonçalves and Mariano, 2000; Barbosa et al., 2003; Araújo et al., 2000). For
71
instance, the production of cyclooxygenase and lipoxygenase by-products and the
72
recruitment of α1- and α2-adrenoceptors were reported as major mediators of the
73
edematogenic actions of B. jararaca venom, whereas hemorrhagic events have been
4
ACCEPTED MANUSCRIPT partially attributed to serotonin and neuro-humoral mediators. Histamine was characterized
75
as a weak mediator in these studies (Trebien and Calixto, 1989; Gonçalves and Mariano,
76
2000). In contrast, histamine, nitric oxide (NO) and cyclooxygenase products, but not
77
serotonin, were imputed in the edematogenic actions caused by B. insularis (Barbosa et al.,
78
2003). Cyclooxygenase (COX) and lipoxygenase products, with minor contributions by
79
histamine and serotonin, appear to participate of the injury induced by B. lanceolatus venom
80
(Araújo et al., 2000). According to these studies, the association between edema and the
81
inflammatory response is evident and appears to be species-specific.
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In addition, the biological actions of Bothrops venoms can be related to direct
83
interactions with immune cells. Moreira et al. (2009) showed that the incubation of crude
84
venom from Bothrops with neutrophils or macrophages induces the expression of COX-2.
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Elifio-esposito et al. (2011) demonstrated that a lectin purified from B. jararacussu venom
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recognizes glycoligands on the neutrophil surface and promotes the polarization and
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migration of neutrophils in vitro.
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Thus, considering the diversity of the intraspecific pathophysiological responses
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induced by Bothrops venoms, we investigate the mechanisms and mediators involved in the
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local inflammatory response induced by crude venom obtained from Bothrops jararacussu,
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one of the most fearsome snakes native to South-America.
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2 MATERIAL AND METHODS
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2.1 Animals
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Experiments were conducted using female Swiss mice (25 ± 2 g, n=7 per group)
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provided by the vivarium maintained at the Department of Physiology and Pharmacology of
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the Faculty of Medicine of the Federal University of Ceará. The animals were housed under a
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natural light/dark 12/12 h cycle with food and water available ad libitum.
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2.2 Venom and Drugs The lyophilized Bothrops jararacussu venom (BjcuV) used in this experiment was
101
obtained from the Butantan Institute, São Paulo, Brazil. The BjcuV was kept at -20 °C and
102
diluted in 0.9 % sterile saline for use. The drugs used were dexamethasone (Decadron®,
103
Pharmaceutical laboratories Aché S.A, Guarulhos, SP, Brazil, 2 mg ampoules), loratadine
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(Loratadina®, New Pharmaceutical Chemicals LTDA., Barueri, SP, Brazil, 10 mg tablet),
105
compound 48/80 (Sigma Chemical Co., St. Louis, MO, USA), capsaicin (Sigma Chemical
106
Co., St. Louis, MO, USA), infliximab (Remicade®, Schering-Plough Co., County Cork, IE,
107
100 mg ampoules), indomethacin (Sigma Chemical Co., St. Louis, MO, USA), celecoxib
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(Celebra®, Pfizer Pharmaceuticals LLC, PR, Brazil, 100 mg capsule), fucoidan (Sigma
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Chemical Co., St. Louis, MO, USA), carrageenan (Sigma Chemical Co., St. Louis, MO,
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USA), f-Met-Leu-Phe (fMLP) (Sigma Chemical Co., St. Louis, MO, USA) and IL-8 (Sigma
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Chemical Co., St. Louis, MO, USA).
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2.3 In vivo experiments
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To investigate the local effects of BjcuV, mice were divided into groups of 7 animals
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each and injected in the subplantar region of the right hind-paw (i.pl) with 30 µl of 0.9 %
116
saline (control group) or BjcuV (0.125, 0.5, 2 and 8 µg/paw). In other experimental groups,
117
dexamethasone (1 mg/kg, i.p., adopted as a positive control) was administered 1 h before
118
the injection of BjcuV (8 µg/paw) or carrageenan (300 µg/paw). To investigate the
119
mechanisms and mediators involved in the inflammatory response, additional groups of
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animals received loratadine (5 mg/kg, p.o., 1 h pretreatment), compound 48/80 (6x0.6 +
121
2x1.2 mg/kg, i.p., 36, 24 and 12 h pretreatment), capsaicin (2x 25 + 50 mg/kg, i.p., 7 days
122
pretreatment), infliximab (5 mg/kg, i.v. 1 h pretreatment), indomethacin (10 mg/kg, i.p., 30
123
min pretreatment), celecoxib (5 mg/kg, i.p., 30 min pretreatment) or fucoidan (25 mg/kg, i.p.,
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ACCEPTED MANUSCRIPT 30 min pretreatment) before being injected with BjcuV. Paw edema was measured by
125
plethysmography (UgoBasile 7140 water plethysmometer) immediately before (basal
126
volume) and hourly (0.5 - 24 h) after BjcuV, carrageenan or saline injection. The results are
127
expressed as the difference between the final and basal paw volumes [variation of paw
128
volume (%)]. Paw tissues were collected for the measurement of myeloperoxidase (MPO,
129
U/mg tissue), IL-1β and TNF-α levels (pg/mg tissue), histopathological analysis, and COX-2
130
immunohistochemistry, as described below.
2.4 Myeloperoxidase assay
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MPO is an enzyme present in the azurophilic granules of neutrophils that can be
134
colorimetrically detected as previously described (Bradley et al., 1982). MPO was extracted
135
from paw samples in a suspension containing 0.5 % hexadecyltrimethyl-ammonium bromide
136
(HTAB) mixed with potassium buffer (50 mg of tissue/ml, pH 6.0). The homogenate was
137
centrifuged at 1500 x g for 15 min at 4 °C resultin g in supernatant and a pellet. For MPO
138
activity measurements, 7 µl of the supernatant as mixed with 3 ml of a phosphate buffer
139
solution containing 5 mg of O-dianisidine, 15 µl of 1 % H2O2, and 27 ml of H2O. The MPO
140
was assayed spectrophotometrically by measuring the change in absorbance at 450 nm.
141
One unit of MPO was defined as the amount of MPO capable of degrading 1 µmol of
142
peroxide/min and the obtained data were expressed as MPO activity (MPO U/mg of tissue).
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2.5 Histopathological analysis
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The paw samples were formalin-fixed, embedded in paraffin, and cut into 4-µm-thick
146
sections using a microtome. The specimens were stained with hematoxylin-eosin for
147
examination by light microscopy at 100x and 1000x magnification The numbers of infiltrating
148
inflammatory cells and the extent of edema were analyzed.
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2.6 Immunohistochemical reaction to cyclooxygenase-2 (COX-2) Immunohistochemical analysis for COX-2 expression was performed using the
151
streptavidin-biotin-peroxidase method (Hsu and Raine, 1981). Paraffin-embedded specimens
152
were cut into 5-µm-thick tissue sections and mounted on poly-L-lysine-coated microscope
153
slides. Samples were sequentially incubated in xylene and graded alcohols, heated in citrate
154
buffer for antigen retrieval, and treated with 3 % (v/v) hydrogen peroxide for the blockade of
155
endogenous peroxidases (15 min). The slides were incubated overnight (4 °C) with a primary
156
goat anti-COX-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:200 in
157
5 % bovine serum albumin (BSA 5 %). After washing in phosphate-buffered saline (PBS), the
158
samples were incubated with a secondary biotinylated rabbit anti-goat antibody (sc-2018 kit,
159
Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:400 in 5 % BSA. Then, the
160
streptavidin-biotin-peroxidase complex (sc-2017, ABC Staining System Santa Cruz
161
Biotechnology,
162
diaminobenzidine (reference K3468, DAKO liquid DAB + substrate chromogen system, CA,
163
USA). The slides were counterstained with Harry’s hematoxylin, dehydrated in a graded
164
alcohol series, cleared in xylene, and cover-slipped.
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added, followed by the chromogen 3,3’-
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Santa Cruz,
2.7 Detection of cytokines (IL-1β and TNF-α) by ELISA
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The concentrations of IL-1β and TNF-α in paw samples were measured by ELISA, as
168
described previously (Melo et al., 2008). Briefly, primary anti-IL1β and anti-TNF-α antibodies
169
(2 µg/ml) were incubated overnight at 4 °C in 96 we ll microtiter plates. After blocking the
170
plates, several dilutions of the samples and standard curve were added and incubated at 4
171
°C for 24 h. After washing the plates with buffer, secondary anti-IL-1β or anti-TNF-α
172
polyclonal biotinylated sheep antibodies (diluted 1:1000 with 1 % BSA) were added to the
173
wells. After a further incubation at room temperature for 1 h, the plates were washed, and
8
ACCEPTED MANUSCRIPT 100 µl of Streptavidin-HRP that was diluted 1:200 as added. Then, 100 µl of substrate
175
solution (1:1 mixture of H2O2 and tetramethylbenzidine; R&D System, USA) was added, and
176
the plate was incubated in the dark at room temperature for 20 min. The enzyme reaction
177
was stopped with 2N H2SO4, and the absorbance was measured at 450 nm. The results are
178
expressed as pg/mg tissue and are reported as the mean ± S.E.M.
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179 2.8 Human Neutrophil Isolation
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Heparinized human blood from healthy volunteers was collected by venipuncture in
182
15 ml Falcon tubes containing a four-layer percoll gradient (72, 63, 54, and 45 %, 2 ml each).
183
After centrifugation at 650 x g for 30 min at 25 °C , the lower layer containing the neutrophils
184
was collected and washed in Hank’s balanced salt solution (HBSS) by centrifugation at 450 x
185
g for 8 min at 25 °C. The pellet, free of red blood cells, was resuspended in RPMI 1640
186
containing 0.1 % BSA. Total counts were performed with a Neubauer chamber, and
187
differential cell counts were carried out on cytocentrifuge slides. Neutrophil preparation that
188
were 98% pure were considered ideal for use in the experimental chemotaxis assay and for
189
intracellular Ca2+ measurements (Itakura et al., 2013; Souto et al., 2011).
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2.9 Migration Assays in Boyden Chamber
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The chemotaxis assay was performed in a 48-well microchamber (Boyden chamber, Neuro
193
Probe Inc., Gaithersburg, MD, USA) as previously described (Souto et al., 2011). BjcuV (10,
194
30, 100 ng/well, diluted in sterile saline) or IL-8 (100 ng/well) as a positive control or RPMI as
195
negative control were added to the bottom of the wells. A polycarbonate membrane (Neuro
196
Probe, 5 µm pore) was placed over the bottom of the plate. In the upper wells 5 x 104
197
neutrophils were added and the chamber was incubated for 1 h at 37 °C and 5 % CO 2.
198
Following incubation, the membrane was washed in PBS, fixed in methanol 80 %, and
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ACCEPTED MANUSCRIPT stained using a Romanowsky staining system (Dade Behring, Inc., Newark, DE). The
200
number of cells counted in five random fields per well was determined using light microscopy
201
(1000x magnification) in triplicate. The results are expressed as the number of neutrophils
202
per field.
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2.10 Neutrophil Activation by Measurement of Intracellular Calcium Dynamics
The intracellular calcium dynamic in BjcuV-treated neutrophils was investigated as
206
previously reported (Huang et al., 2013; Itakura et al., 2013). Briefly, a human neutrophil
207
suspension was mixed with fluo-4 (0.2% final concentration) and incubated for 30 min at 37
208
°C with 5 % CO 2. To remove the excess of fluo-4, the cells were washed with RPMI 1640 by
209
centrifugation at 450 x g for 8 min at 25 °C. The p ellet was resuspended in RPMI 1640
210
containing 0.1 % BSA. Intracellular Ca2+ spikes in neutrophils were monitored for 10 min at
211
488 nm using laser scanning confocal microscopy (FLUOVIEW® FV1200, Olympus). After
212
basal recordings were taken, BjcuV (30 ng/ml, diluted in sterile saline), fMLP (10 nM), IL-8
213
(100 ng/ml) or Tyrode´s solution was added in the cube. The images obtained were analyzed
214
using the FIJI 1.47v Software (NIH, USA). The results are expressed as fluorescence
215
intensity (F/F0) and the number of cells activated per field.
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All procedures were performed after approval by the local Ethics Committee for
219
Animal Experiments (protocol number 62/12) according to the guidelines outlined by the
220
National Institutes of Health for use of laboratory animals (NIH publication No. 85-23, revised
221
1985). The isolation of human neutrophils was performed in accordance with the Human
222
Ethics Committee of Federal University of Ceará (protocol 543.774).
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ACCEPTED MANUSCRIPT 223
2.12 Statistical Analyses Data are expressed as the means ± standard errors of the means (S.E.M.), with the
225
exception of the immunohistochemical score analysis, which is reported as the median
226
values (non-parametric data). The data were analyzed using either one- or two-way ANOVA
227
followed by Bonferroni’s test (parametric data) or the Kruskal-Wallis test followed by Dunn’s
228
test (non-parametric data), as appropriate. Statistical significance was confirmed when
229
P
S0041-0101(14)00224-4
DOI:
10.1016/j.toxicon.2014.08.001
Reference:
TOXCON 4893
To appear in:
Toxicon
Received Date: 20 May 2014 Revised Date:
29 July 2014
Accepted Date: 5 August 2014
Please cite this article as: Wanderley, C.W.S., Silva, C.M.S., Wong, D.T., Ximenes, R.M., Moreo, D.F.C, Cosker, F., Aragão, K.S., Fernandes, C., Palheta-Júnior, R.C., Havt, A., Brito, G.A.C., Cunha, F.Q., Ribeiro, R.A., Lima-Júnior, R.C.P., Bothrops jararacussu snake venom-induces a local inflammatory response in a Prostanoid- And Neutrophil-Dependent manner, Toxicon (2014), doi: 10.1016/j.toxicon.2014.08.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT Bothrops jararacussu SNAKE VENOM-INDUCES A LOCAL INFLAMMATORY
2
RESPONSE IN A PROSTANOID- AND NEUTROPHIL-DEPENDENT MANNER
3
WANDERLEY, C.W.S.1; SILVA, C.M.S.1; WONG, D.T. 1; XIMENES, R.M. 1; MOREO, D.F.C2;
4
COSKER, F.1; ARAGÃO, K.S.1; FERNANDES, C. 1; PALHETA-JÚNIOR, R.C.3; HAVT, A.1;
5
BRITO, G.A.C.4, CUNHA, F.Q.2; RIBEIRO, R.A.1; LIMA-JÚNIOR, R.C.P.1
RI PT
1
6
1
7
Ceará, Brazil;
8
University of São Paulo, Brazil; 3Colege of Veterinary Medicine, Federal University of Vale do
9
São Francisco; 4Department of Morphology, Faculty of Medicine, Federal University of
2
SC
Department of Pharmacology, School of Medicine of Ribeirão Preto,
M AN U
10
Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of
Ceará, Brazil.
11
*To whom correspondence should be addressed:
13
Roberto César Pereira Lima Júnior
14
Departamento de Fisiologia e Farmacologia
15
Faculdade de Medicina
16
Universidade Federal do Ceará
17
Rua Cel Nunes de Melo, 1315, Rodolfo Teófilo, 60430-270,
18
Fortaleza, Ceará, Brazil.
19
Contact: +55 85 3366 8588 (Phone) +55 85 3366 8333 (Fax)
20
Email: [email protected], [email protected] (Corresponding author)
21
22
AC C
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12
2
ACCEPTED MANUSCRIPT ABSTRACT
24
Local tissue reactions provoked by Bothrops venoms are characterized by edema,
25
hemorrhage, pain, and inflammation; however, the mechanisms of tissue damage vary
26
depending upon the species of snake. Here, we investigated the mechanisms involved in the
27
local inflammatory response induced by the Bothrops jararacussu venom (BjcuV). Female
28
Swiss mice were injected with either saline, BjcuV (0.125-8 µg/paw) or loratadine (an H1
29
receptor antagonist), compound 48/80 (for mast cell depletion), capsaicin (for C-fiber
30
desensitization), infliximab (an anti-TNF-α antibody), indomethacin (a non-specific COX
31
inhibitor), celecoxib (a selective COX-2 inhibitor) or fucoidan (a P- and L-selectins modulator)
32
given before BjcuV injection. Paw edema was measured by plethysmography. In addition,
33
paw tissues were collected for the measurement of myeloperoxidase activity, TNF-α and IL-1
34
levels, and COX-2 immunoexpression. The direct chemotactic effect of BjcuV and the in vitro
35
calcium dynamic in neutrophils were also investigated. BjcuV caused an edematogenic
36
response with increased local production of TNF-α and IL-1β as well as COX-2 expression.
37
Both edema and neutrophil migration were prevented by pretreatment with indomethacin,
38
celecoxib or fucoidan. Furthermore, BjcuV induced a direct in vitro neutrophil chemotaxis by
39
increasing intracellular calcium. Therefore, BjcuV induces an early onset edema dependent
40
upon prostanoid production and neutrophil migration.
41
Keywords: Bothrops jararacussu venom; edema; prostanoids; neutrophil; inflammation;
42
selectins.
44
45
46
47
SC
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TE D
EP
AC C
43
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23
3
ACCEPTED MANUSCRIPT 48
1 INTRODUCTION Clinical observations of local effects provoked by Bothrops venoms are characterized
50
by marked edema, pain, erythema, ecchymosis, bullae with a clear serum transudate or
51
hemorrhagic content, cyanosis, extensive hemorrhage resulting in the sloughing of
52
superficial tissues, and tissue necrosis (Amaral et al., 1985). Currently, studies in the
53
literature suggest that these pathological effects result from the direct action of venoms on
54
tissues and are associated with an inflammatory response that amplifies the damage
55
(Trebien and Calixto, 1989; de Faria et al., 2001; Barbosa et al., 2003; Olivo et al., 2007;
56
Nascimento et al., 2010).
M AN U
SC
RI PT
49
At present, antivenom treatment is the recommended therapeutic approach for
58
snakebite envenoming. However, Bothrops venom-related local damage can be only partially
59
neutralized by a specific or the polyvalent antivenom (Gutierrez et al., 1986; Chaves et al.
60
2003; Zammuner et al. 2004; da Silva et al 2007). A probable explanation is based on the
61
fact that snake venoms are complex mixtures of toxins, enzymes and biologically active
62
peptides and that the compositions vary among families, genera and species (Leite, 1992;
63
El-Din and Omar, 2013). Within the Bothrops genus, a broad range of variation exist in the
64
composition and biological activity of the venoms (Kamiguti and Cardoso, 1989; Queiroz et
65
al., 2008). Therefore, understanding the pathophysiological mechanisms involved in the
66
tissue damage resulting from Bothrops snake envenoming would contribute to a more
67
effective therapy.
EP
AC C
68
TE D
57
Several studies have demonstrated the role of inflammatory mediators activated
69
following intraplantar injection of venoms from different Bothrops snakes (Trebien and
70
Calixto, 1989; Gonçalves and Mariano, 2000; Barbosa et al., 2003; Araújo et al., 2000). For
71
instance, the production of cyclooxygenase and lipoxygenase by-products and the
72
recruitment of α1- and α2-adrenoceptors were reported as major mediators of the
73
edematogenic actions of B. jararaca venom, whereas hemorrhagic events have been
4
ACCEPTED MANUSCRIPT partially attributed to serotonin and neuro-humoral mediators. Histamine was characterized
75
as a weak mediator in these studies (Trebien and Calixto, 1989; Gonçalves and Mariano,
76
2000). In contrast, histamine, nitric oxide (NO) and cyclooxygenase products, but not
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serotonin, were imputed in the edematogenic actions caused by B. insularis (Barbosa et al.,
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2003). Cyclooxygenase (COX) and lipoxygenase products, with minor contributions by
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histamine and serotonin, appear to participate of the injury induced by B. lanceolatus venom
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(Araújo et al., 2000). According to these studies, the association between edema and the
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inflammatory response is evident and appears to be species-specific.
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In addition, the biological actions of Bothrops venoms can be related to direct
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interactions with immune cells. Moreira et al. (2009) showed that the incubation of crude
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venom from Bothrops with neutrophils or macrophages induces the expression of COX-2.
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Elifio-esposito et al. (2011) demonstrated that a lectin purified from B. jararacussu venom
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recognizes glycoligands on the neutrophil surface and promotes the polarization and
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migration of neutrophils in vitro.
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Thus, considering the diversity of the intraspecific pathophysiological responses
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induced by Bothrops venoms, we investigate the mechanisms and mediators involved in the
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local inflammatory response induced by crude venom obtained from Bothrops jararacussu,
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one of the most fearsome snakes native to South-America.
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2 MATERIAL AND METHODS
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2.1 Animals
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Experiments were conducted using female Swiss mice (25 ± 2 g, n=7 per group)
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provided by the vivarium maintained at the Department of Physiology and Pharmacology of
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the Faculty of Medicine of the Federal University of Ceará. The animals were housed under a
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natural light/dark 12/12 h cycle with food and water available ad libitum.
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2.2 Venom and Drugs The lyophilized Bothrops jararacussu venom (BjcuV) used in this experiment was
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obtained from the Butantan Institute, São Paulo, Brazil. The BjcuV was kept at -20 °C and
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diluted in 0.9 % sterile saline for use. The drugs used were dexamethasone (Decadron®,
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Pharmaceutical laboratories Aché S.A, Guarulhos, SP, Brazil, 2 mg ampoules), loratadine
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(Loratadina®, New Pharmaceutical Chemicals LTDA., Barueri, SP, Brazil, 10 mg tablet),
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compound 48/80 (Sigma Chemical Co., St. Louis, MO, USA), capsaicin (Sigma Chemical
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Co., St. Louis, MO, USA), infliximab (Remicade®, Schering-Plough Co., County Cork, IE,
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100 mg ampoules), indomethacin (Sigma Chemical Co., St. Louis, MO, USA), celecoxib
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(Celebra®, Pfizer Pharmaceuticals LLC, PR, Brazil, 100 mg capsule), fucoidan (Sigma
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Chemical Co., St. Louis, MO, USA), carrageenan (Sigma Chemical Co., St. Louis, MO,
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USA), f-Met-Leu-Phe (fMLP) (Sigma Chemical Co., St. Louis, MO, USA) and IL-8 (Sigma
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Chemical Co., St. Louis, MO, USA).
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2.3 In vivo experiments
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To investigate the local effects of BjcuV, mice were divided into groups of 7 animals
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each and injected in the subplantar region of the right hind-paw (i.pl) with 30 µl of 0.9 %
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saline (control group) or BjcuV (0.125, 0.5, 2 and 8 µg/paw). In other experimental groups,
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dexamethasone (1 mg/kg, i.p., adopted as a positive control) was administered 1 h before
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the injection of BjcuV (8 µg/paw) or carrageenan (300 µg/paw). To investigate the
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mechanisms and mediators involved in the inflammatory response, additional groups of
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animals received loratadine (5 mg/kg, p.o., 1 h pretreatment), compound 48/80 (6x0.6 +
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2x1.2 mg/kg, i.p., 36, 24 and 12 h pretreatment), capsaicin (2x 25 + 50 mg/kg, i.p., 7 days
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pretreatment), infliximab (5 mg/kg, i.v. 1 h pretreatment), indomethacin (10 mg/kg, i.p., 30
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min pretreatment), celecoxib (5 mg/kg, i.p., 30 min pretreatment) or fucoidan (25 mg/kg, i.p.,
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plethysmography (UgoBasile 7140 water plethysmometer) immediately before (basal
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volume) and hourly (0.5 - 24 h) after BjcuV, carrageenan or saline injection. The results are
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expressed as the difference between the final and basal paw volumes [variation of paw
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volume (%)]. Paw tissues were collected for the measurement of myeloperoxidase (MPO,
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U/mg tissue), IL-1β and TNF-α levels (pg/mg tissue), histopathological analysis, and COX-2
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immunohistochemistry, as described below.
2.4 Myeloperoxidase assay
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MPO is an enzyme present in the azurophilic granules of neutrophils that can be
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colorimetrically detected as previously described (Bradley et al., 1982). MPO was extracted
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from paw samples in a suspension containing 0.5 % hexadecyltrimethyl-ammonium bromide
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(HTAB) mixed with potassium buffer (50 mg of tissue/ml, pH 6.0). The homogenate was
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centrifuged at 1500 x g for 15 min at 4 °C resultin g in supernatant and a pellet. For MPO
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activity measurements, 7 µl of the supernatant as mixed with 3 ml of a phosphate buffer
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solution containing 5 mg of O-dianisidine, 15 µl of 1 % H2O2, and 27 ml of H2O. The MPO
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was assayed spectrophotometrically by measuring the change in absorbance at 450 nm.
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One unit of MPO was defined as the amount of MPO capable of degrading 1 µmol of
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peroxide/min and the obtained data were expressed as MPO activity (MPO U/mg of tissue).
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2.5 Histopathological analysis
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The paw samples were formalin-fixed, embedded in paraffin, and cut into 4-µm-thick
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sections using a microtome. The specimens were stained with hematoxylin-eosin for
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examination by light microscopy at 100x and 1000x magnification The numbers of infiltrating
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inflammatory cells and the extent of edema were analyzed.
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2.6 Immunohistochemical reaction to cyclooxygenase-2 (COX-2) Immunohistochemical analysis for COX-2 expression was performed using the
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streptavidin-biotin-peroxidase method (Hsu and Raine, 1981). Paraffin-embedded specimens
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were cut into 5-µm-thick tissue sections and mounted on poly-L-lysine-coated microscope
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slides. Samples were sequentially incubated in xylene and graded alcohols, heated in citrate
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buffer for antigen retrieval, and treated with 3 % (v/v) hydrogen peroxide for the blockade of
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endogenous peroxidases (15 min). The slides were incubated overnight (4 °C) with a primary
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goat anti-COX-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:200 in
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5 % bovine serum albumin (BSA 5 %). After washing in phosphate-buffered saline (PBS), the
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samples were incubated with a secondary biotinylated rabbit anti-goat antibody (sc-2018 kit,
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Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:400 in 5 % BSA. Then, the
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streptavidin-biotin-peroxidase complex (sc-2017, ABC Staining System Santa Cruz
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Biotechnology,
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diaminobenzidine (reference K3468, DAKO liquid DAB + substrate chromogen system, CA,
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USA). The slides were counterstained with Harry’s hematoxylin, dehydrated in a graded
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alcohol series, cleared in xylene, and cover-slipped.
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2.7 Detection of cytokines (IL-1β and TNF-α) by ELISA
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The concentrations of IL-1β and TNF-α in paw samples were measured by ELISA, as
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described previously (Melo et al., 2008). Briefly, primary anti-IL1β and anti-TNF-α antibodies
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(2 µg/ml) were incubated overnight at 4 °C in 96 we ll microtiter plates. After blocking the
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plates, several dilutions of the samples and standard curve were added and incubated at 4
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°C for 24 h. After washing the plates with buffer, secondary anti-IL-1β or anti-TNF-α
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polyclonal biotinylated sheep antibodies (diluted 1:1000 with 1 % BSA) were added to the
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wells. After a further incubation at room temperature for 1 h, the plates were washed, and
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solution (1:1 mixture of H2O2 and tetramethylbenzidine; R&D System, USA) was added, and
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the plate was incubated in the dark at room temperature for 20 min. The enzyme reaction
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was stopped with 2N H2SO4, and the absorbance was measured at 450 nm. The results are
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expressed as pg/mg tissue and are reported as the mean ± S.E.M.
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Heparinized human blood from healthy volunteers was collected by venipuncture in
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15 ml Falcon tubes containing a four-layer percoll gradient (72, 63, 54, and 45 %, 2 ml each).
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After centrifugation at 650 x g for 30 min at 25 °C , the lower layer containing the neutrophils
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was collected and washed in Hank’s balanced salt solution (HBSS) by centrifugation at 450 x
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g for 8 min at 25 °C. The pellet, free of red blood cells, was resuspended in RPMI 1640
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containing 0.1 % BSA. Total counts were performed with a Neubauer chamber, and
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differential cell counts were carried out on cytocentrifuge slides. Neutrophil preparation that
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were 98% pure were considered ideal for use in the experimental chemotaxis assay and for
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intracellular Ca2+ measurements (Itakura et al., 2013; Souto et al., 2011).
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2.9 Migration Assays in Boyden Chamber
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The chemotaxis assay was performed in a 48-well microchamber (Boyden chamber, Neuro
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Probe Inc., Gaithersburg, MD, USA) as previously described (Souto et al., 2011). BjcuV (10,
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30, 100 ng/well, diluted in sterile saline) or IL-8 (100 ng/well) as a positive control or RPMI as
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negative control were added to the bottom of the wells. A polycarbonate membrane (Neuro
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Probe, 5 µm pore) was placed over the bottom of the plate. In the upper wells 5 x 104
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neutrophils were added and the chamber was incubated for 1 h at 37 °C and 5 % CO 2.
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Following incubation, the membrane was washed in PBS, fixed in methanol 80 %, and
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number of cells counted in five random fields per well was determined using light microscopy
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(1000x magnification) in triplicate. The results are expressed as the number of neutrophils
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2.10 Neutrophil Activation by Measurement of Intracellular Calcium Dynamics
The intracellular calcium dynamic in BjcuV-treated neutrophils was investigated as
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previously reported (Huang et al., 2013; Itakura et al., 2013). Briefly, a human neutrophil
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suspension was mixed with fluo-4 (0.2% final concentration) and incubated for 30 min at 37
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°C with 5 % CO 2. To remove the excess of fluo-4, the cells were washed with RPMI 1640 by
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centrifugation at 450 x g for 8 min at 25 °C. The p ellet was resuspended in RPMI 1640
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containing 0.1 % BSA. Intracellular Ca2+ spikes in neutrophils were monitored for 10 min at
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488 nm using laser scanning confocal microscopy (FLUOVIEW® FV1200, Olympus). After
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basal recordings were taken, BjcuV (30 ng/ml, diluted in sterile saline), fMLP (10 nM), IL-8
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(100 ng/ml) or Tyrode´s solution was added in the cube. The images obtained were analyzed
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using the FIJI 1.47v Software (NIH, USA). The results are expressed as fluorescence
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intensity (F/F0) and the number of cells activated per field.
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All procedures were performed after approval by the local Ethics Committee for
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Animal Experiments (protocol number 62/12) according to the guidelines outlined by the
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National Institutes of Health for use of laboratory animals (NIH publication No. 85-23, revised
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1985). The isolation of human neutrophils was performed in accordance with the Human
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Ethics Committee of Federal University of Ceará (protocol 543.774).
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2.12 Statistical Analyses Data are expressed as the means ± standard errors of the means (S.E.M.), with the
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exception of the immunohistochemical score analysis, which is reported as the median
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values (non-parametric data). The data were analyzed using either one- or two-way ANOVA
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followed by Bonferroni’s test (parametric data) or the Kruskal-Wallis test followed by Dunn’s
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test (non-parametric data), as appropriate. Statistical significance was confirmed when
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P
