Research Article Activation of J77A.1 Macrophages by Three ...downloads.hindawi.com/journals/bmri/2014/683123.pdf · Venom. Bothrops atrox specimens collected around the cityofPortoVelho,StateofRond

Research ArticleActivation of J77A1 Macrophages by ThreePhospholipases A2 Isolated from Bothrops atrox Snake Venom

Juliana L Furtado1 George A Oliveira2 Adriana S Pontes1

Sulamita da S Setuacutebal1 Caroline V Xavier1 Fabianne Lacouth-Silva1 Beatriz F Lima1

Kayena D Zaqueo2 Anderson M Kayano2 Leonardo A Calderon2 Rodrigo G Staacutebeli12

Andreimar M Soares2 and Juliana P Zuliani12

1 Laboratorio de Imunofarmacologia Aplicada a Saude Fundacao Oswaldo Cruz FIOCRUZ-Rondonia Rua da Beira7671 Br364 Km 35 76812-245 Porto Velho RO Brazil

2 Centro de Biomoleculas Aplicadas a Saude FIOCRUZ-Rondonia e Departamento de MedicinaUniversidade Federal de Rondonia (UNIR) Campus BR 364 Km 95 76801-059 Porto Velho RO Brazil

Correspondence should be addressed to Juliana P Zuliani jzulianiipepatroorgbr

Received 4 September 2013 Revised 18 November 2013 Accepted 18 November 2013 Published 27 January 2014

Academic Editor Saulo Luıs da Silva

Copyright copy 2014 Juliana L Furtado et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In the present study we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2) BaTX-I a catalyticallyinactive Lys-49 variant and BaTX-II a catalytically active Asp-49 and of one acidic myotoxic PLA2 BaPLA2 a catalytically activeAsp-49 isolated from Bothrops atrox snake venom on the activation of J774A1 macrophages At noncytotoxic concentrations thetoxins did not affect the adhesion of the macrophages nor their ability to detach The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis However BaTX-I BaTX-II and BaPLA2 induced the release of thesuperoxide anion by J774A1 macrophages Additionally only BaTX-I raised the lysosomal volume of macrophages after 15minof incubation After 30min all the phospholipases increased this parameter which was not observed within 60min MoreoverBaTX-I BaTX-II and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submittedto phagocytosis However BaTX-II and BaPLA2 induced the release of TNF-120572 by J774A1 macrophages Taken together the datashow that despite differences in enzymatic activity the three toxins induced inflammatory events and whether the enzyme is acidicor basic does not seem to contribute to these effects

1 Introduction

Bothrops atrox is responsible for most cases of envenoming inthe Northern region of Brazil [1] Its venom contains amongothers proteins serine proteinases metalloproteinases L-amino acid oxidases C-type lectin-like enzymes and phos-pholipases A

2 Phospholipases A

2(PLA2 EC 3114) belong

to a diverse super family of lipolytic enzymes that catalyse thehydrolysis of the sn-2 ester bond of membrane glycerophos-pholipids to produce free fatty acids and lysophospholipidsThese enzymes can differ in structure molecular weight sub-strate specificity requirement for Ca2+ cell localization andmechanism of action [2] PLA

2s are classified into five major

families extracellular or secretory PLA2s (sPLA

2) intra-

cellular cytosolic PLA2s (cPLA

2) Ca2+-independent PLA

2s

(iPLA2) PAF acetylhydrolases and lysossomal PLAs (lPLAs)

[3ndash6]Snake venom PLA

2s of the Viperidae family belong to

the subgroup IIA which share structural features with PLA2s

present in inflammatory exudates in mammals [7 8] Thesesnake venom PLA

2s display a variety of pharmacological

activities such asmyotoxic neurotoxic anticoagulant hypot-ensive hemolytic platelet aggregation inhibiting bacterici-dal and proinflammatory activities [9ndash12] Proteins of thissubgroup can be further subdivided into two types (i) cata-lytically active PLA

2s which have conserved residues at

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 683123 13 pageshttpdxdoiorg1011552014683123

2 BioMed Research International

the catalytic network and at the calcium-binding loop includ-ing Asp49 and (ii) catalytically inactive variants having a Lysinstead of Asp at position 49 [10 13 14]

In addition among the pro inflammatory activities it wasshown that various venom PLA

2s are able to stimulate neu-

trophil chemotaxis degranulate mast cells in vitro induceprostaglandin production in peritoneal leukocytes and acti-vate macrophage functions [12 15ndash19]

Macrophages play a central role in a wide variety ofprocesses associatedwith tissuemaintenance antigen presen-tation inflammation and tissue repair [20] Resident macro-phages constitute one of the first lines of host defense and arepresent in many tissues Upon stimulation these quiescentcells are activated and display diverse cellular functions suchas phagocytosis This process consists of the uptake anddestruction of invading microorganisms an essential part ofthe host response against infections It is initiated by engagingreceptors on the surface of the macrophages which expressvarious receptors that participate in target cell recognitionand internalization of ligands [21] There are four majorclasses of receptor-mediated phagocytosis (a) Fc120574 receptorswhich recognize the Fc120574 domain of IgG-coated particles (b)complement receptor type 3 (CR3 also referred to asCD11bCD18 Mac-1 and integrin 120572M1205732) which recognizescomplement-coated particles (c) mannose receptors whichrecognize mannose and fucose on the surface of pathogensand (d) 120573-glucan receptors or Dectin-1 which recognize 120573-glucans-bearing ligands [20 21]

Subsequent to phagocytosis an abrupt production of oxy-gen free radicals such as the superoxide anion occurs referredto as an oxidative burst The enzyme complex responsible forthe production of this reactive oxygen species is the NADPHoxidase complex When macrophages are stimulated themembrane and cytosolic components of this complex asso-ciate on the phagosomal membranes to form the active oxi-dase complex [22ndash25] The reactive oxygen species (ROS)formed such as superoxide anion and hydrogen peroxide areused by macrophages to kill ingested microorganisms [22ndash25] Parallel to this reaction occurs lysossomal membranedestabilization involving increased fusion of lysosomes andphagosomes deriving from cellular stress Lysossomal activ-ities mediate several processes in cell feeding homeostasisand antimicrobial defense which involve lysosome fusionwith endosomes and phagosomes [26] Furthermore acti-vated macrophages express a large amount of inclusionscalled lipid bodies (LBs) These LBs are neutral lipid storageorganelles with key roles in cell signaling intracellular traf-ficking cell activation and eicosanoid formation [27] LBswere shown to characteristically increase both the size andnumber of cells associated with the inflammatory processAdditionally macrophages upon stimulation release cytok-ines such as TNF-120572 an important inflammatory mediatorinvolved in many inflammatory reactions [28]

The present study was therefore designed to evaluate theeffects of Bothrops atrox Lys-49 andAsp-49 PLA

2s acidic and

basic proteins on J774A1 macrophage function Our resultsindicate that these PLA

2s at noncytotoxic concentrations are

able to directly stimulate macrophages Despite differences in

enzymatic activity the toxins induced inflammatory eventsand whether the enzyme is acidic or basic does not seem tocontribute to these effects

2 Materials and Methods

21 Chemicals andReagents Trypan blue RPMI-1640 L-glu-tamine gentamicin zymosan thiocarbohydrazide (TCH)nitroblue tetrazolium (NBT) mouse INF-120574 and neutral redwere purchased from Sigma (MO USA) Fetal bovine serumwas obtained from Cultilab (Brazil) Formaldehyde andOsO4

minus (osmium tetroxide) were purchased from ElectronMicroscopy Sciences (PA USA) DuoSet Elisa Mouse TNF-alphawas purchased fromRampDSystems (OxonUnitedKing-dom) All salts and reagents used were obtained from Merck(Darmstadt Germany) with low endotoxin or endotoxin-freegrades

22 Venom Bothrops atrox specimens collected around thecity of Porto Velho State of Rondonia Brazil were kept at theFiocruz Rondonia bioterium in order to be used for venomproduction under authorization from IBAMA (license num-ber 27131-1) and CGEN (license number 0106272011-1) Thecrude venom was dehydrated and stored at a temperature ofminus20∘C in the Amazon Venom Bank at CEBio

23 Cell Line and Culture The murine macrophage cell lineJ774A1 was obtained from the Rio de Janeiro Cell Bank Col-lection (Brazil) Briefly monolayers of J774A1 macrophageswere maintained in flasks in a RPMI-1640 medium supple-mented with 100 120583gmL gentamicin 2mM L-glutamine and10 heat-inactivated fetal bovine serum (FBS) in humidifiedair with 5 CO

2at 37∘C

24 Isolation and Biochemical Characterization of Phospholi-pases A

2 Lys49 (BaTX-I) and Asp49 (BaTX-II) basic PLA

2s

as well as Asp49 (BaPLA2) acidic PLA

2from Bothrops atrox

snake venom were purified according to the method previ-ously described [29 30] B atrox crude venom (100mg) wasdissolved in 10mL of 005M Tris-HCl buffer pH 74 cen-trifuged at 755timesg for 10min at room temperature and theclear supernatant applied on a CM-Sepharose FF column (1 times40 cm) which was previously equilibrated with the same buf-fer Elution was carried out with a continuous gradient up toa concentration of 10M of NaCl at a flow rate of 10mLmin(HPLC Akta Purifier GE) Absorbance of the effluent solu-tion was recorded at 280 nm The last fraction with phos-pholipase and myotoxic activity was collected and freeze-dried The acidic fraction (Peak 1) was freeze-dried and thendiluted with 005M Tris-HCl buffer pH 74 containing 4MNaCl prior to the next chromatographic step in a butyl-HP(Hitrap 5mL) column previously equilibrated with the samebuffer The chromatography was carried out at a flow rateof 25mLmin with a decreasing gradient of NaCl (4 to0M) followed by Milli-Q H

2OThe active fractions showing

PLA2activity were desalinized lyophilized and applied on a

C18 reverse-phase column (Supelco 250mm times 46mm)previously equilibrated with 01 TFA and then eluted with

BioMed Research International 3

an acetonitrile-TFA gradient (0 to 70) at a flow rate of10mLmin and monitored at 120582 = 280 nm

Homogeneity was demonstrated using SDS-polyacryl-amide gel electrophoresis under reducing conditions [31]PLA2activity of the proteins was evaluated in vitro by indirect

erythrocyte lysis in agar containing human erythrocytes andegg yolk as previously described [32] A prior agreementfrom all participants involved was made in order to beincluded in the study and the Center of Tropical MedicineResearch (Rondonia Brazil) Research Ethics Committees(number CAAE 14204413500000011) approved this studyThe assay for myotoxicity was carried out using the creatinekinase (CK)-UV kinetic kit from Bioclin Brazil [33]

25 Cytotoxic Assay Cell viability was measured by trypanblue exclusion In brief J774A1 macrophages were centri-fuged at 200timesg for 5min at 4∘C and the cell pellets wereresuspended in 1mL of RPMI-1640 medium supplementedwith 100 120583gmL gentamicin 2mML-glutamine and 10 fetalbovine serum (FBS) After counting 2 times 105 macrophages80 120583L were added to plastic vials and incubated with 20 120583Lof different concentrations of BaTX-I or BaTX-II or BaPLA

2

(15 3 and 6 120583gmL) diluted in assay medium or RPMI(control) for 1 h and 4 h at 37∘C in a humidified atmosphere(5 CO

2) Then 20120583L 01 trypan blue was added to 100

120583Lofmacrophage suspensionThe viable cell indexwas deter-mined in a Neubauerrsquos chamber by counting a total of 100cells Results were expressed as percentage of viable cells

26 Adhesion Assay Macrophage adhesion was assayedaccording to the procedure described by [34] The cells (2times 105 cellswell) were cultured for 24 h in a supplementedmediumAfter incubation the plateswerewashed three timeswith PBS and the adherent cells were fixed with methanolAfter staining with 10Giemsa solution for 10min the plateswere washed with water and the remaining dye was solu-bilized with methanol Adherence of control cells was con-sidered to be 100 Absorbance was determined spectropho-tometrically at 550 nm

27 Detachment Assay Macrophage detachment was assayedaccording to the procedure described by [34] In this testJ774A1macrophageswere plated in 96-well plates at a densityof 2 times 105 cellswell and allowed to attach for 24 h at 37∘Cin a 5 CO

2atmosphere Nonadherent cells were removed

by washing with PBS After 24 h the cells were incubatedwith 200120583L of supplemented RPMI (control) or BaTX-I orBaTX-II or BaPLA

2(6 120583gmL) diluted in RPMI at 37∘C in

a humidified atmosphere (5 CO2) for 1 h After another

washing with PBS the cells were stained with 50120583L of 01Giemsa for 40min Finally the cells were washed with PBSand 100 120583L of 100methanol was added to solubilize the dyeAdherence of the remaining control cells was considered tobe 100 Absorbance was read at 550 nm The results wereexpressed as absorbance

28 Phagocytic Activity of J774A1 Macrophages This test wasperformed according to [12] In brief J774A1 macrophages

were plated on 13mm diameter glass coverslips (Glass Tec-nica Brazil) in 24-well plates at a density of 2 times 105 cells percoverslip and allowed to attach for 2 h at 37∘Cunder a 5CO

2

atmosphere Nonadherent cells were removed by washingwith PBS Cell monolayers were cultured for 1 h with RPMI-1640 supplemented with 100 120583gmL gentamicin 2mM L-glutamine and 10 FBS at 37∘C and 5 CO

2and then incu-

bated with RPMI (control) or different concentrations ofBaTX-I or II or BaPLA

2(6 120583gmL) diluted in RPMI After

washing in cold PBS the monolayers were incubated for 40min at 37∘C and 5 CO

2with serum-opsonized zymosan

prepared as described below and unbound particles wereremoved by washing with PBS Cells were fixed with 25glutaraldehyde for 15min at room temperature and the cov-erslips were mounted on microscope slides The extent ofphagocytosis was quantified by contrast phase microscopicobservation At least 200 macrophages were counted in eachdetermination and those containing three or more interna-lized particles were considered positive for phagocytosisResults were presented as the percentage of cells positive forphagocytosis

To prepare serum-opsonized zymosan zymosan parti-cles obtained from yeast cell walls were suspended in PBS ata concentration of particles of 57mgmL For opsonization2mL of zymosan particles was mixed with 2mL normalmouse serum and incubated for 30min at 37∘C The serum-opsonized zymosan particles were then centrifuged at 200 gfor 10min and suspended in PBS in preparation for thephagocytosis assay Mouse serum from swiss mice used inthe present study was approved by the Tropical PathologyResearch Institute Ethics Committee onAnimalUse (number082008)

29 Staining andCounting of Lipid Bodies Macrophageswereplated on 13mm diameter glass coverslips (Glass TecnicaBrazil) in 24-well plates at a density of 2 times 105 cells per cover-slip and allowed to attach for 2 h at 37∘C in a 5 CO

2atmos-

phere Nonadherent cells were removed bywashingwith PBSCell monolayers were incubated with supplemented RPMI(control) or BaTX-I or BaTX-II or BaPLA

2(6 120583gmL) diluted

in RPMI for 60min at 37∘C and 5 CO2 In a set of experi-

ments J774A1 macrophages were submitted to phagocytosisaccording to item 28 and then the lipid bodies were stainedAfter washing in cold PBS the monolayers were fixed with37 formaldehyde in PBS pH 74 for 15min at roomtemperature rinsed in 01M cacodylate buffer pH 74 stainedin 15 OsO

4(30min) rinsed in dH

2O immersed in 10

thiocarbohydrazide (5min) rinsed in 01Mcacodylate bufferrestained in 15 OsO


2O and then

dried andmounted onmicroscope slidesThemorphology offixed cells was observed and lipid bodies were counted usingphase contrast microscopy with an objective lens at a magni-fication of 100 in 50 consecutively scannedmacrophagesTheresults were expressed as the mean number of lipid bodiesper cell

210 Production of Superoxide Anion by J774A1 on Glass Cov-erslips In this test J774A1 the concentration ofmacrophages


was adjusted to 2 times 105100 120583L and they were incubated with100 120583L RPMI containing 01 NBT (control) or 100 120583L ofdifferent concentrations of BaTX-I or BaTX-II or BaPLA

2

(6 120583gmL) diluted in RPMI containing 01 NBT and incu-bated for 1 h at 37∘C and 5CO

2 At the end of the incubation

period the cells were centrifugated in a cytospin for 5minat 180timesg and then fixed with methanol for 5min Finallythe cells were stained with 01 safranin for 5min At least100 macrophages were counted and those containing blueformazan crystals were considered positive The results wereexpressed in a percentage

211 Lysosomal Volume This test which evaluates the vesi-cles of the endocytic compartment by lysosomal retentionof neutral red was performed according to [35] J774A1macrophages were incubated for 15 30 and 60minutes in thepresence of RPMI (control) or BaTX-I or BaTX-II or BaPLA

2

(6 120583gmL) at 37∘C in a humidified atmosphere (5 CO2)

Then the cells were washed by centrifugation and incubatedwith 004 neutral red (stock solution 2 = 20mg dissolvedin 1mL DMSO) After this the macrophages were washedagain by centrifugation and the dye was solubilized by adding200120583L of extraction solution (acetic acid 10 and 40ethanol in distilled water) The cells were incubated for 30minutes and then the samples were read on a spectropho-tometer at 550 nm The results were expressed in percentageconsidering the control group to have 100 absorbance

212 Tumor Necrosis Factor-120572 (TNF-120572) QuantificationJ774A1 macrophages (2 times 10550 120583L) were incubated withBaTX-I or BaTX-II or BaPLA

2(6 120583gmL) or RPMI (control)

or mouse INF-120574 (1 120583gmL positive control) for 4 hours at37∘C in a humidified atmosphere (5CO

2) After centrifuga-

tion the supernatant was used to determine TNF-120572 levels byspecific EIA as described by [36] Briefly 96-well plates werecoated with 100 120583L of the first capture monoclonal antibodyantitumor necrosis factor-120572 (4 120583gmL) and incubated for 18hours at 37∘C Following this period the plate was washedwith washer buffer (PBSTween20) After that 200120583L ofblocking buffer containing 5 bovine serum albumin (BSA)in PBSTween20 was added to the wells and the plates wereincubated for 1 hour at 37∘C Following this period the wellswere washed and 50 120583L of either samples or standard wasdispensed into each well and the plates incubated for 2hours at 37∘C After this period the plate was washed and100 120583L of detection antibody TNF-120572 (250 ngmL) wasadded for 2 hours at 37∘C After incubation and washing100 120583L of streptavidin-peroxidase was added followed byincubation and addition of the substrate (100 120583LmL 331015840551015840-tetramethylbenzidine) Finally sulfuric acid (50120583L) wasadded to stop the reaction Absorbances at 540 and 450 nmwere recorded and concentrations of TNF-120572 were estimatedfrom standard curves prepared with recombinant TNF-120572The results were expressed as pgmL of this cytokine

213 Statistical Analyses Means and SEM of all data wereobtained and compared using two way ANOVA followed bya Tukey test with significance probability levels of less than005

3 Results

31 Isolation and Biochemical Characterization of MyotoxinsBothrops atrox crude venom (100mg) was applied on a CM-Sepharose ion-exchange column previously equilibratedwith 005M Tris-HCl buffer pH 74 and then eluted witha continuous gradient up to a concentration of 10M ofNaCl Fraction CM-11 with myotoxic activity was namedBaTX-I (Figures 1(a) and 1(c)) The fractions CM-6 and CM-1 with phospholipase A

2activity were further fractionated

on reverse phase C18 (Figure 1(b)) and Butyl-Sepharose(Figure 2(a)) columns respectively The fraction CM-6-3(Figure 1(b)) with PLA

2and myotoxic activities was named

BaTX-II (Figure 1(d)) Fraction CM-1-6 was applied on a RP-HPLC C18 column and resolved into one main peak withonly PLA

2activity named BaPLA

2(Figures 2(b) and 2(c))

The homogeneity of these proteins was further demonstratedby SDS-PAGE The purified myotoxins consisted of a singlepolypeptide chain with an apparent approximate molecularweight of 14500Da

32 Effect of BaTX-I BaTX-II and BaPLA2on Macrophage

Viability To test the toxicityof PLA2s on J774A1 macropha-

ges the effect of 1 and 4 hours of incubation of several concen-trations of BaTX-I or BaTX-II or BaPLA

2was investigated

As shown in Figure 3 the incubation of secretory PLA2s at

concentrations of 15 3 and 6120583gmL for 1 h and 4 h (data notshown) did not affect J774A1 macrophage viability


Adhesion To investigate the effect of BaTX-I BaTX-II andBaPLA

2on macrophage adhesion the cells were incubated

with 6 120583gmL of either PLA2s (a noncytotoxic concentration)

or RPMI (control) As shown in Figure 4(a) the toxinsdid not affect the adhesion of the J774A1 macrophages


Detachment To test whether these secretory PLA2s of Both-

rops atrox snake venom affect the ability of J774A1 macro-phages to detach the cells were incubated with BaTX-I orBaTX-II or BaPLA

2at a noncytotoxic concentration (6 120583g

mL) or RPMI (control) As shown in Figure 4(b) the studiedPLA2s did not affect the ability of the macrophages to detach

35 Effect of BaTX-I BaTX-II andBaPLA2onPhagocytosis by

Macrophages In order to assess the ability of BaTX-I BaTX-II and BaPLA

2to stimulate complement receptor-mediated

phagocytosis the uptake of serum-opsonized zymosan parti-cles was determined in adherent J774A1macrophages treatedwith noncytotoxic concentration of toxins or with RPMI(control) As shown in Figure 5 macrophages incubated withRPMI showed an average phagocytosis of serum-opsonizedzymosan particles of 37 Incubation of macrophages withBaTX-I induced a significant increase in J774A1 macrophagephagocytosis of serum-opsonized zymosan at a concentra-tion of 6 120583gmL On the other hand incubation of macro-phages with BaTX-II and BaPLA

2 at a concentration of


BaTX-I

lowast

lowast

1

2 3 45 6

7

89 10 11 12

130

500

1000

1500

2000

2500

(mAU

)

UV 280nmConcentration

0 50 100 150 200 250 300 350

0

20

40

60

80

100

(min)

B(

)

(a)

BaTX-II

(mAU

)


0

20

40

60

80

100

50040030020010000

minus50

00

50

100

150

200

250

(min)

B(

)

(b)

17580

58

463023

17

1 2 3 4 5 6 7 8

(c)

1 2 3175

80

5846

30

23

177

(d)

Figure 1 (a) Chromatographic profile of Bothrops atrox venom The crude venom of B atrox after being solubilized was applied on CM-Sepharose (400mm times 10mm) previously equilibrated with Tris 50mMpH 74 and eluted under a gradient of NaCl (0ndash1M) in the samebuffer in 5 column volumesThe fractions with PLA

2activity are identified with (lowast) (b) High performance chromatographic profile Fraction

6 obtained from CM-Sepharose was solubilized in 01 TFA (solvent A) and applied on a C18 column (discovery 25mm times 46mm 5 120583300 A) previously equilibrated with the same buffer and eluted with 01 TFA in 999 acetonitrile (solvent B) under a gradient 0ndash70and flow of 1mLmin Both chromatograms were monitored with absorbance at 280 nm (c) SDS-PAGE 12 5 samples 1 molecular weightmarker 2 BthTX-I 3 fraction 8 4 fraction 9 5 fraction 10 6 11 fraction (BaTX-I) 7 fraction 12 (d) SDS-PAGE 125 in reducing conditionselectrophoretic analysis of basic Asp49 PLA

2from B atrox Samples 1 molecular weight 2 BthTX-I 3 BaTX-II

6 120583gmL resulted in 446plusmn38 and 413plusmn23 of phagocy-tosis of serum-opsonized zymosan respectivelyThese valueswere not significant compared to control

36 Effect of BaTX-I BaTX-II and BaPLA2on Superoxide

Anion (O2

minus) Production by Macrophages To investigate theability of BaTX-I BaTX-II and BaPLA

2to induce the pro-

duction of the superoxide anion by J774A1 macrophages thecells were incubated with a noncytotoxic concentration ofBaTX-I BaTX-II or BaPLA

2 or with RPMI (control) in the

presence of NBT As shown in Figure 6 J774A1 macrophagesincubated with RPMI showed an average superoxide anionproduction of 37 plusmn 28 Incubation of macrophageswith BaTX-I BaTX-II and BaPLA

2at a concentration of

6 120583gmL induced significant production of O2

minus by J774A1macrophages


Lipid Bodies To determine whether these enzymes induce

the formation of lipid bodies in J774A1 macrophages thecells were incubated with noncytotoxic BaTX-I BaTX-IIor BaPLA

2 or with RPMI (control) for 1 h After that the

cells were submitted to phagocytosis and lipid bodies werecounted As shown in Figure 7 the incubation of macropha-ges with the toxins increased the number of lipid bodies com-pared to the control In the cells submitted to phagocytosisthe amount of lipid bodies was significantly higher than cellsnot submitted to phagocytosis


Lysossomal Volume To assess the effect of these secretoryPLA2s on macrophage lysossomal volume the cells were

stimulated with noncytotoxic concentrations of BaTX-IBaTX-II or BaPLA

2 or with RPMI (control) for 15 30 and

60min As shown in Figure 8(a) incubation with the toxinBaTX-I at a concentration of 6120583gmL for 15min increasedthe lysossomal volume in J774A1 macrophages compared tothe control The toxins BaTX-II and BaPLA

2did not affect


0

100

200

300

400

500

600

(mAU

)

0 5 10 15 20 25

B (

)

1

23 4

56

280nmConcentrationConcentration

0

100

PLA2

(min)

(a)

(mA

U)

B (

)

0

50

100

150

200

250

300

350

400

(min)

0

20

40

60

80

100

280nmConcentration

BaPLA2-I

(b)

1 2 3 4

175

8058

46

30

23

17

7

(c)

Figure 2 (a) Chromatography profile on hydrophobic interaction resin (Butyl-Sepharose HP 50mm times 10mm) Fractions 1 2 and 3 obtainedfrom CM-Sepharose chromatography were pooled and solubilized in 20mM Ambic plus 4M NaCl (buffer A) and applied on a Butyl-Sepharose HP column previously equilibrated with the same buffer and eluted with 20mM Ambic (buffer B) using a step gradient of 025 50 75 and 100 Subsequently a step using Milli-Q water was performed (b) High performance chromatographic profile Fraction 6obtained from a Butyl-Sepharose HP column was solubilized in 01 TFA (solvent A) and applied on a C18 column (discovery 25mm times46mm 5 120583 300 A) previously equilibrated with the same buffer and eluted with 01 TFA in 999 acetonitrile (solvent B) under a gradientof 0ndash70 and flow of 1mLmin Both elutions were monitored with absorbance at 280 nm

macrophage lysossomal volume at this time However afterincubation for 30min the three toxins induced a significantincrease in macrophage lysossomal volume as shown inFigure 8(b) Incubation of the cells with secretory PLA

2s at a

concentration of 6120583gmL for 1 h did not affect the lysossomalvolume in macrophages as shown in Figure 8(c)

39 Effect of BaTX-I BaTX-II andBaPLA2onTNF-120572Produc-

tion by Macrophages To investigate the effect of these secre-tory PLA

2s on TNF-120572 production by macrophages the cells

were stimulated with IFN-120574 (1 120583gmL positive control)or noncytotoxic concentrations of BaTX-I or BaTX-II orBaPLA

2or RPMI (control) for a period of 4 hours Soon after

the supernatant was collected and used to determine TNF-120572concentration using an enzyme immunoassay As shown inFigure 9 the incubation with the enzymatically active toxins

BaTX-II and BaPLA2induced an increase in TNF-120572 produc-

tion by macrophages although BaTX-II was more effectivethan BaPLA

2in maintaining this effect The enzymatically

inactive toxin BaTX-I did not induce any significant increasein the production of TNF-120572 in the studied period

4 Discussion

Both acidic and basic PLA2s can be found in snake venoms

in variable proportions depending on the snake species Thebasic PLA

2isoforms appear to have the highest toxicity and

the acidic PLA2isoform usually has higher catalytic activity

To date all acidic PLA2s purified from Viperidae venoms

present an Asp residue at position 49 [37 38] In this studywe purified two basic myotoxic PLA

2s from B atrox venom

named BaTX-I (Lys-49) and BaTX-II (Asp-49) and an acidic


0

20

40

60

80

100

Viab

le ce

lls (

)

Control

BaTX-I

BaTX-II

15 3 615 3 6 15 3 6(120583gmL)

BaPLA2

Figure 3 Effect of BaTX-I BaTX-II and BaPLA2on macrophage

viability 2 times 105 J774A1 macrophages were incubated with differentconcentrations of toxins or RPMI (control) for 60minutes at 37∘C ina humidified atmosphere of 5 CO

2 The viability of macrophages

was assessed by a Trypan blue exclusion test Values represent themean plusmn SEM from 3 independent experiments

PLA2named BaPLA

2(Asp-49) by one- two- or three-step

chromatography The quality of the final sample is crucialfor further studies involving structure and function SeveralPLA2s fromBothrops venoms have been exhaustively purified

using a combination of chromatographic methods gel filtra-tion ion exchange RP-HPLC and affinity chromatography[10 39]

Based on this we performed a study in order to evaluatethe effect of three sPLA

2s BaTX-I (Lys-49) BaTX-II (Asp-

49) and BaPLA2(Asp-49) on the functionality of the murine

macrophage cell line J774A1For this we assessed first the cytotoxic activity of these

phospholipases The viability assay using the trypan blueexclusionmethod demonstrated that toxins at concentrationsof 15 3 and 6 120583gmL showed no cytotoxic effect on macro-phages during 1 hour of incubation Thus for the realizationof the other proposed experiments the highest concentra-tion 6120583gmLwas chosen It is noteworthy that the same con-centration was used in the study of macrophage activation byZuliani et al [12] and Setubal et al [19]The three PLA

2s used

had the same effect on macrophages Still it is worth notingthat the acidic or basic character of these toxins did not influ-ence the results

Corroborating this data Lomonte and Gutierrez [40]showed that two basic PLA

2s isolated from the venom of

Bothrops asper (MT-II and MT-III) exert cytotoxic effectsonly at high concentrations Also in the study by Zulianiet al [12] it was shown that thioglycollate elicitedmurine per-itoneal macrophages incubated with concentrations less than25 120583gmL of MT-II and MT-III for a period of one hour didnot show compromised viability The study also showed thatthe enzymatically active toxin MT-III (Asp-49) is more toxicthan the enzymatically inactive toxin MT-II (Lys-49) Inaddition Setubal et al [19] examined the effects of BaltTX-I and BaltTX-II on thioglycollate elicited murine peritonealmacrophage viability These two basic PLA

2did not affect

macrophage viability indicating their low toxicity on this celltype

Macrophages a professional phagocyte play an impor-tant role in the innate immune response against invadingpathogens in the resolution of inflammation and mainte-nance of tissue homeostasis This cell type has a central rolein the inflammatory reaction producing microbicidal agentsand exerting its main function phagocytosis For this tooccur efficiently macrophages need to adhere to a substrateand then modify their cell morphology gaining a more flat-tened shape This step is very important and allows themacrophage to spread and increase its contact surface areawith the particle to be phagocytosed Thus our study eval-uated the effect of the three phospholipases on the ability ofmacrophages to adhere and to detachThe results showed thatBaTX-I BaTX-II and BaPLA

2did not affect the ability of

macrophages to adhere and to detach from support in theperiod studied

The literature reports that PLA2s have an important role

in cell adhesion The use of inhibitors of PLA2s such as p-

bromophenacyl bromide reduced monocyte adhesion andaffected the expression of Mac-1 in neutrophils [41 42] aswell as the spreading of these cells [43] On the other handtwo nontoxic enzymes with high PLA

2activity isolated from

Cerastes cerastes snake venomcalled CC-PLA2-I and CC-

PLA2-II inhibited the migration and adhesion of IGR39

melanoma and HT1080 fibrosarcoma cells to fibrinogen andfibronectin [44] Bazaa et al [45] demonstrated the action ofanother PLA

2 called MVL-PLA

2 isolated fromMacrovipera

lebetina Transmediterranea In this study the enzyme wasalso able to inhibit the adhesion and migration of tumor cellsto fibrinogen and fibronectin but not to collagen type ITheseeffects weremediated by the specific inhibition of12057251205731120572v1205736and 120572v1205733 integrins These data show that integrins may bea specific target for phospholipasesrsquo mechanism of action intumor cell lines Despite the ability of detachment our datashowed that cells remained viable after incubationwith toxinsremaining adhered to the plate surface It is important forthe process of phagocytosis to occur effectivelyThen macro-phages need to adhere to a substrate to subsequently modifytheir cellular morphology from a round shape into a flattenedshape [46]

Phagocytosis is an extremely complex process and maybe mediated by several receptors such as the receptor for theFc portion of IgG (Fc120574Rs) the mannose receptor (MR) the120573-glucan receptor (Dectin-1) and complement receptor type3 (CR3) Accordingly we evaluated the effect of PLA

2s on

phagocytosis via the complement receptor using opsonizedzymosan particles Our results showed that only BaTX-I aLys-49 toxin induced a significant increase in phagocytosisvia the complement receptor The others toxins BaTX-II andBaPLA

2 both Asp-49 did not affect this process The acidic

or basic character does not seem to affect this parameterThese results are in agreement with those obtained by Zulianiet al [12] The authors showed that MT-II a Lys-49 isolatedfrom the venomofBothrops asper also considerably increasesphagocytosis via the complement receptor while MT-III anAsp-49 only stimulates phagocytosis via mannose receptorsSimilar results were obtained by Setubal et al [19] These


Control BaTX-I BaTX-II00

01

02

03

04

05Ad

hesio

n (D

O)

BaPLA2

6120583gmL

(a)


01

02

03

04

05

Rem

aini

ng ce

lls (D

O)

BaPLA2

6120583gmL

(b)

Figure 4 Effect of BaTX-I BaTX-II and BaPLA2on J774A1 macrophage adhesion and detachment For the adhesion test (a) 2 times 105

macrophages were incubated for 60 minutes with toxins (6120583gmL) or RPMI (control) at 37∘C in a humidified atmosphere of 5 CO2 For

the detachment test (b) macrophages were incubated with RPMI for only 24 hours and then for 60 minutes with toxins (6120583gmL) or RPMI(control) at 37∘C in a humidified atmosphere of 5 CO

2 Adhered cell levels were determined by optical density (550 nm) being proportional

to the amount of incorporated Giemsa dye Values represent the mean plusmn SEM from 3 independent experiments


20

40

60

80

100

Phag

ocyt

osis

()

BaPLA2

6120583gmL

lowast

Figure 5 Effect of BaTX-I BaTX-II andBaPLA2on phagocytosis of

opsonized zymosan by J774A1 macrophages 2 times 105 macrophageswere incubated for 60 minutes with toxins (6120583gmL) or RPMI(control) at 37∘C in a humidified atmosphere of 5 CO

2before

addition of opsonized zymosan particles Values represent the meanplusmn SEM from 3 independent experiments lowast119875 le 005 compared withcontrol (ANOVA)

authors showed that BaltTX-I a Lys49 but not BaltTX-II anAsp-49 enzyme is able to directly stimulate phagocytosis viathe complement receptor Thus it is important to note thatenzymatic activity does not seem to be essential for increasingthe phagocytic capacity via CR3 since only catalytically inac-ive phospholipases were able to affect this parameter Themechanism by which Lys49 PLA

2homologs activate the

process of phagocytosis has not been clarified There aretwo hypotheses for the action of these enzymatically inactivetoxins One is the interaction of the enzymewith the receptorleading to its internalization The other hypothesis is that thePLA2s have the ability to induce perturbations in the cell

PMA Control BaTX-I BaTX-II0

20

40

60

80

100

Supe

roxi

de an

ion

prod

uctio

n (

)

BaPLA2

6120583gmL

lowastlowast

lowastlowast

Figure 6 Effect of BaTX-I BaTX-II and BaPLA2on superoxide

production by J774A1 macrophages 2 times 105 macrophages wereincubated with RPMI 01 NBT 10 uL of PMA (500 ngmL)(positive control) or toxins (6 120583gmL) for 60 minutes at 37∘C in ahumidified atmosphere of 5 CO

2for the formation of formazan

crystals resulting from the reduction of NBT by superoxide Thecrystals were solubilized and the absorbance of the supernatant wasdetermined at 620 nm in a spectrophotometer Data were expressedas OD Values represent the mean plusmn SEM from 3 independentexperiments lowast119875 le 005 compared with control (ANOVA)

membrane which activate signaling pathways culminating inan increase of phagocytosis Thus further studies are neededto elucidate themechanism of action of these enzymes whichmakes them extremely interesting from a scientific stand-point

Concomitantly with phagocytosis there is an increase inthe oxidative metabolism of macrophages called a respira-tory burst that is mediated by the NADPH oxidase enzymecomplex The high O

2consumption associated with rapid


00

05

10

15

20

25

30

35

Lipi

d bo

dies

()

Zymosan

ControlBaTX-I

BaTX-IIBaPLA2

minusminusminusminus

lowastlowast

lowast

lowastlowast

lowast

Figure 7 Effect of BaTX-I BaTX-II and BaPLA2on lipid body

formation by J774A1 macrophages 2 times 105 macrophages wereincubated with toxins (6120583gmL) or RPMI (control) for 60 minutesat 37∘C in a humidified atmosphere of 5 CO

2before the addition

of opsonized zymosan particles The number of lipid bodies stainedwith osmium tetroxide within macrophages was determined usingphase-contrast microscopy Values represent the mean plusmn SEMfrom 3 independent experiments lowast119875 le 005 compared with controland 119875 le 005 compared with control submitted to phagocytosis

(ANOVA)

activation of NADPH oxidase triggers the production ofreactive oxygen species (ROS) such as the superoxide anion(O2

minus) and hydrogen peroxide (H2O2) which are potent

microbicidal agents [47] Therefore this study evaluated thePLA2action on superoxide anion production by J774A1

macrophages The results showed that all three toxins sig-nificantly increased superoxide anion release compared withthe unstimulated control The increase was similar to thatobserved in cells incubated with PMA a positive controlThere was no difference between the Asp49 and Lys49 phos-pholipases A

2 suggesting that catalytic activity may not be

essential to superoxide anion production Once again it isclear that the enzymersquos acidic or basic character does notappear to contribute to this effect

According to Setubal et al [19] the excessive release ofsuperoxide can be harmful to tissues involved in inflamma-tion In this study the authors showed that BaltTX-I andBaltTX-II isolated from Bothrops alternatus venom induceda significant release of superoxide anion compared with thenegative control (RPMImedium) but there was no differencewhen compared to PMA (a positive control) indicating thatthemyotoxins are able to activate the respiratory burstMore-over Zuliani et al [12] showed that MT-II and MT-III iso-lated from B asper venom induce the release of H

2O2

another reactive oxygen intermediate produced from thesuperoxide anion In this case MT-IIIrsquos activity was morepronounced than that ofMT-IIThis increase inmicrobicidalagent production induced by PLA

2s may be considered one

of the key points for the development of the intense localinflammatory process observed in Bothrops envenoming

It is possible that the increase in phagocytic index is alsoassociated with an increase in the endocytic compartment

contributing to an increase in the microbicidal activity of themacrophages since destabilization of lysosomal membranesinvolves an increase in lysosome and phagolysosome fusionoften associated with stress derived from the action of pro-oxidant agents and pathological conditions Accordinglyexperiments were performed to assess whether the toxins areable to induce an increase in lysosomal volume The macro-phages were incubated with toxins for 15 30 and 60 minutesIn evaluating the time course of the effect of the three toxins itcan be seen that increasing the endocytic compartment ofmacrophages occurs quite sharply in the first 15 minutes afterstimulation and that enzymatic activity is not essential forthis effect since only BaTX-I which is enzymatically inac-tive changed this parameter The enzymatically active phos-pholipases BaTX-II and BaPLA

2were only able to induce an

increase in lysosomal volume after 30 minutes of incubationDuring this period BaTX-I was also effective in maintainingthis effect After being stimulated for 1 hour with phospho-lipases macrophages showed no change in their endocyticcompartment This may suggest that after a period of 30minutes the PLA

2action decreases and the volume of the

cellsrsquo lysosomal compartments returns to their baseline levelDespite the fact that the lipolytic activity of PLA

2often affects

several membrane structures including lysosomes in ourstudies enzymatic activity does not appear to interfere withthe increase in lysosomal volume nor does the acidic orbasic character of these enzymes Studies by Burlando et al[26] showed that a calcium-independent cPLA

2is one of

the agents responsible for the lysosomal membrane desta-bilization induced by estradiol The destabilization of themembrane leads to the fusion of the lysosome with otherorganelles such as the endosome This process as evidencedbyMayorga et al [48] involves the action of a PLA

2Thus the

data suggest that these enzymes can induce changes in thevacuole-lysosomal system

The literature shows that in the presence of inflamma-tion activated leukocytes induced an increase of organellescalled lipid bodies These organelles compartmentalize dif-ferent groups of proteins involved in lipid metabolism and incell signaling in addition to containing the complex enzymeCOX and 5-LO and even AA cPLA

2 MAPK and PI3K [49

50] Thus the actions of BaTX-I BaTX-II and BaPLA2on

lipid body formation in J774A1 macrophages were evaluatedThe data obtained show that all three phospholipases wereable to increase the number of lipid bodies present in themacrophages This action occurred independently of enzy-matic activity and no statistical difference was observedbetween the toxins Similar results were obtained by Leiguezet al [51] and Giannotti et al [52]These authors showed thatMT-III an Asp49 and MT-II a Lys49 both isolated fromB asper venom were able to induce a marked increase inlipid bodies formation in macrophages Therefore we eval-uated the action of these PLA

2s in macrophages undergoing

phagocytosis The results showed that the number of lipidbodies inside macrophages that phagocytosed was greaterthan that found in macrophages which were not subjectedto the process The BaTX-I was slightly more efficient thanthe others in the maintenance of this effect In relation toenzymatic activity the three phospholipases were able to


000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min

Control BaTX-I BaTX-II BAPLA2

6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

lowast lowast lowast

30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)

Figure 8 Effect of BaTX-I BaTX-II and BaPLA2on J774A1 macrophage lysosomal volume 2 times 105 macrophages were incubated with the

toxins (6 120583gmL) or RPMI (control) for 15 30 and 60 minutes at 37∘C in a humidified atmosphere of 5 CO2 After that the macrophages

were incubatedwith 004neutral redThe incorporated dyewas solubilized and the absorbance of the supernatant was determined at 505 nmin a spectrophotometer Values represent the mean plusmn SEM from 3 independent experiments lowast119875 le 005 compared with control (ANOVA)

induce these events regardless of catalytic ability Again therewas no difference in the acidic or basic character of theseenzymes The data suggest that the activation of complementreceptors during phagocytosis stimulates lipid body forma-tion in macrophages also induced by the action of sPLA

2s

Thus PLA2s in addition to releasing the AA of membrane

phospholipids can hydrolyze phosphatidylcholine and causelyso-PAF release which is the precursor of platelet activatingfactor (PAF) Data from the literature indicate that thisinflammatory mediator with platelet aggregating activity isinvolved in signaling pathways that favor the formation oflipid bodies and the release of mediators such as LTC

4and

PGE2[50] Additional studies to determine the role of lipid

bodies in the process of phagocytosis and the mechanisms offormation of these organelles are required These details alsohave significance for the functionality of the leucocytesduring inflammatory reactions

Besides the increase in the number of lipid bodies acti-vated macrophages show an increased production of cytok-ines with important roles in inflammatory responses One

of them is TNF-120572 a potent inflammatory mediator capa-ble of inducing proliferation of fibroblasts producing othercytokines activating leukocytes and stimulating the gen-eration of reactive oxygen species Thus we evaluated theaction of the three phospholipases BaTX-I BaTX-II andBaPLA

2 on TNF-120572 production by J774A1 macrophagesThe

data showed that BaTX-II and BaPLA2induced a signifi-

cant increase in TNF-120572 production by macrophages after 4hours of incubation and BaTX-II was somewhat more effec-tive than BaPLA

2in inducing the production of this cytokine

Both toxins are enzymatically active Asp49 homologswhereas the first is basic and the latter is acidic The resultsalso show that BaTX-I an enzymatically inactive Lys49 PLA

2

homolog did not induce any increase in the production ofthis cytokine in the studied period These data are in agree-ment with Zuliani et al [11] in which MT-III an Asp49 iso-lated from B asper induced an increase in the production ofTNF-120572 in the peritoneal fluid of mice 1 hour after injectionMT-II a Lys49 induced a small increase in this parameteronly after 6 hours of incubation Increased production of


Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast

Figure 9 Effect of BaTX-I BaTX-II and BaPLA2on J774A1macro-

phage TNF-120572 production 2 times 105 macrophages were incubatedwith RPMI (control) or INF-120574 (1 120583gmL) (positive control) ortoxins (6 120583gmL) for 4 hours The concentrations of TNF-120572 inthe supernatant were quantified by specific EIA The results wereexpressed as pgmL of TNF-120572 and represent the mean plusmn SEM from3 independent experiments lowast119875 le 005 compared to control and119875 le 005 compared to BaTX-II (ANOVA)

cytokines particularly TNF-120572 can be regulated by increasedproduction of reactive oxygen species in amechanismdepen-dent on NF-kB Zuliani et al [12] demonstrated the ability ofthese isolated myotoxins to induce a significant increase inH2O2production suggesting that high levels of this reactive

oxygen may play an important role in regulating the produc-tion of TNF-120572

It is important to note that TNF-120572 binds to its cellularTNF-120572 receptor 1 (TNFR1) which triggers signaling cascadesthat activate NF-120581B and AP-1 transcription factors [53 54]This cytokine has also been reported to increase reactive oxy-gen species (ROS) production from mitochondria plasma-membrane NADPH oxidase and lipoxygenase [55ndash58] It ispossible that BaTX-II and BaPLA

2stimulate ROS production

through TNF-120572 production probably through the engage-ment of a subset of Toll-like receptors (TLR1 TLR2 andTLR4) different from BaTX-I that stimulates ROS throughphagocytosis

Taken together our data indicate that the all studiedPLA2s do not interfere with adhesion or detachment of

J774A1 macrophages BaTX-I but not BaTX-II and BaPLA2

stimulates opsonized zymosan phagocytosis via a comple-ment receptor However all of the PLA

2s stimulated an

increase in superoxide production and lysosome volume byJ774A1 macrophages after 30min of incubation MoreoverBaTX-I BaTX-II and BaPLA

2increases the number of

lipid bodies on macrophages submitted to phagocytosis andnot submitted to phagocytosis Only BaTX-II and BaPLA

2

induced the release of TNF-120572 by J774A1 macrophages It isimportant to note that PLA

2activity is not essential for trig-

gering these effects Certain regions of the toxin moleculesother than the catalytic site can interact with cellmembranesleading to the activation of macrophages These studies have

added knowledge about the mechanisms of action of PLA2s

isolated from B atrox contributing to better characterizationof macrophage function

Regarding the acidic or basic character of these enzymesthere is no evident difference in the activity of phospholi-pases used in this study Many of the PLA

2s isolated from

Bothrops venoms are basic proteins Acidic PLA2s are still

being studied but the literature shows that these enzymescan induce various pharmacological effects such as plateletaggregation and hypotension Acidic phospholipases A

2 in

general are not myotoxic but this effect may appear on asmaller scale compared to basic phospholipases which showintense myotoxicity However acidic phospholipases havehigher catalytic activity [59] Additional studies with theseenzymes both acidic and basic are still needed so that we candetermine a possible correlation between toxicity and theirpharmacological potential

Conflict of Interests

There is no conflict of interests statement

Acknowledgments

This study was supported by Conselho Nacional de Desen-volvimento Cientııfico e Tecnoloogico (CNPq) Coordenacaode Aperfeicoamento de Nıvel Superior (CAPES NanoBi-otec) MCTI INCT-InpetAM and Secretaria de Desenvolvi-mento do Estado de Rondonia (SEPLAN-ROCNPq) BrazilJuliana Pavan Zuliani was a recipient of productivity Grant3039742008-7 fromConselhoNacional de DesenvolvimentoCientıfico e Tecnologico (CNPq) and Juliana Loca Furtadowas the beneficiary of CAPES by Masterrsquos fellowship AmyHamaker provided the English editing of the paper

References

[1] C A T Souza A M Kayano S S Setubal et al ldquoLocal and sys-temic biochemical alterations induced by Bothrops atrox snakevenom in micerdquo Journal of Venom Research vol 3 pp 28ndash342012

[2] J E Burke and E A Dennis ldquoPhospholipase A2biochemistryrdquo

Cardiovascular Drugs and Therapy vol 23 no 1 pp 49ndash592009

[3] D A Six and E A Dennis ldquoThe expanding superfamily ofphospholipase A

2enzymes classification and characterizationrdquo

Biochimica et Biophysica Acta vol 1488 no 1-2 pp 1ndash19 2000[4] M Murakami Y Taketomi C Girard K Yamamoto and G

Lambeau ldquoEmerging roles of secreted phospholipase A2

enzymes lessons from transgenic and knockout micerdquo Bio-chimie vol 92 no 6 pp 561ndash582 2010

[5] E Valentin and G Lambeau ldquoWhat can venom phospholipasesA2tell us about the functional diversity of mammalian secreted

phospholipases A2rdquo Biochimie vol 82 no 9-10 pp 815ndash831

2000[6] M Murakami and I Kudo ldquoPhospholipase A

2rdquo Journal of Bio-

chemistry vol 131 no 3 pp 285ndash292 2002[7] I I Kaiser J M Gutierrez D Plummer S D Aird and G V

Odell ldquoThe amino acid sequence of a myotoxic phospholipase


from the venomofBothrops asperrdquoArchives of Biochemistry andBiophysics vol 278 no 2 pp 319ndash325 1990

[8] R M Kini ldquoPhospholipase A2mdashA complex multifunctional

protein puzzlerdquo inVenom Phospholipase A2Enzymes Structure

Function and Mechanism R Manjunatha Kini Ed p 1 JohnWiley amp Sons New York NY USA 1997

[9] C F P Teixeira E C T Landucci E Antunes M Chacur andY Cury ldquoInflammatory effects of snake venom myotoxic phos-pholipases A

2rdquo Toxicon vol 42 no 8 pp 947ndash962 2003

[10] A M Soares M R M Fontes and J R Giglio ldquoPhospholipaseA2myotoxins fromBothrops snake venoms structure-function

relationshiprdquoCurrent Organic Chemistry vol 8 no 17 pp 1677ndash1690 2004

[11] J P Zuliani C M Fernandes S R Zamuner J M Gutierrezand C F P Teixeira ldquoInflammatory events induced by Lys-49and Asp-49 phospholipases A

2isolated from Bothrops asper

snake venom role of catalytic activityrdquo Toxicon vol 45 no 3pp 335ndash346 2005

[12] J P Zuliani J M Gutierrez L L Casais E Silva S C SampaioB Lomonte and C D F Pereira Teixeira ldquoActivation of cellularfunctions in macrophages by venom secretory Asp-49 and Lys-49 phospholipasesA

2rdquoToxicon vol 46 no 5 pp 523ndash532 2005

[13] J Gutierrez and B Lomonte ldquoPhospholipase A2myotoxins

from Bothrops snake venomsrdquo Toxicon vol 33 no 11 pp 1405ndash1424 1995

[14] J M Gutierrez and B Lomonte ldquoPhospholipase A2myotox-

ins from Bothropssnake venomsrdquo in Venom Phospholipase A2

Enzymes Structure Function and Mechanism R M Kini Edpp 321ndash352 1997

[15] A Gambero E C T Landucci M H Toyama et al ldquoHumanneutrophil migration in vitro induced by secretory phospholi-pases A

2 a role for cell surface glycosaminoglycansrdquo Biochemi-

cal Pharmacology vol 63 no 1 pp 65ndash72 2002[16] A Gambero S M Thomazzi A C O Cintra E C T

Landucci G De Nucci and E Antunes ldquoSignalling pathwaysregulating human neutrophil migration induced by secretoryphospholipases A


[17] E C T Landucci R C Castro M F Pereira et al ldquoMast celldegranulation induced by two phospholipase A

2homologues

dissociation between enzymatic and biological activitiesrdquo Euro-pean Journal of Pharmacology vol 343 no 2-3 pp 257ndash2631998

[18] V Moreira J M Gutierrez A M Soares S R Zamuner EPurgatto and C D F P Teixeira ldquoSecretory phospholipasesA2isolated from Bothrops asper and from Crotalus durissus

terrificus snake venoms induce distinctmechanisms for biosyn-thesis of prostaglandins E2 and D2 and expression of cyclooxy-genasesrdquo Toxicon vol 52 no 3 pp 428ndash439 2008

[19] S S Setubal A S Pontes JL Furtado et alldquoAction of twophos-pholipases A

2purified from Bothrops alternatus snake venom

on macrophagesrdquo Biochemistry vol 78 pp 194ndash203 2013[20] M Naito ldquoMacrophage heterogeneity in development and dif-

ferentiationrdquo Archives of Histology and Cytology vol 56 no 4pp 331ndash351 1993

[21] A Aderem andT AUnderhill ldquoThe cell biology ofmacrophageactivationrdquo Annual Review of Immunology vol 17 pp 593ndash6231999

[22] W J Brown K Chambers and A Doody ldquoPhospholipase A2

(PLA2) enzymes in membrane trafficking mediators of mem-

brane shape and functionrdquo Traffic vol 4 no 4 pp 214ndash2212003

[23] B M Babior ldquoNADPH oxidase an updaterdquo Blood vol 93 no5 pp 1464ndash1476 1999

[24] B M Babior ldquoNADPH oxidaserdquo Current Opinion in Immunol-ogy vol 16 no 1 pp 42ndash47 2004

[25] B M Babior J D Lambeth and W Nauseef ldquoThe neutrophilNADPH oxidaserdquo Archives of Biochemistry and Biophysics vol397 no 2 pp 342ndash344 2002

[26] B Burlando B Marchi I Panfoli and A Viarengo ldquoEssentialrole of Ca2+-dependent phospholipase A

2in estradiol-induced

lysosome activationrdquoAmerican Journal of PhysiologyndashCell Phys-iology vol 283 no 5 pp C1461ndashC1468 2002

[27] P T Bozza R C N Melo and C Bandeira-Melo ldquoLeukocytelipid bodies regulation and function contribution to allergy andhost defenserdquo Pharmacology andTherapeutics vol 113 no 1 pp30ndash49 2007

[28] C A Dinarello ldquoThe proinflammatory cytokines interleukin-1 and tumor necrosis factor and treatment of the septic shocksyndromerdquo Journal of Infectious Diseases vol 163 no 6 pp1177ndash1184 1991

[29] S H Andriao-Escarso A M Soares V M Rodrigues et alldquoMyotoxic phospholipases A

2in Bothrops snake venoms effect

of chemical modifications on the enzymatic and pharmacolog-ical properties of bothropstoxins from Bothrops jararacussurdquoBiochimie vol 82 no 8 pp 755ndash763 2000

[30] S H Andriao-Escarso A M Soares M R M Fontes et alldquoStructural and functional characterization of an acidic plateletaggregation inhibitor and hypotensive phospholipase A

2from

Bothrops jararacussu snake venomrdquo Biochemical Pharmacologyvol 64 no 4 pp 723ndash732 2002

[31] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[32] J M Gutierrez C L Ownby and G V Odell ldquoSkeletal muscleregeneration after myonecrosis induced by crude venom anda myotoxin from the snake Bothrops asper (Fer-de-Lance)rdquoToxicon vol 22 no 5 pp 719ndash731 1984

[33] A M Soares S H Andriao-Escarso Y Angulo et al ldquoStruc-tural and functional characterization of myotoxin I a Lys49phospholipaseA

2homologue fromBothropsmoojeni (Caissaca)

snake venomrdquo Archives of Biochemistry and Biophysics vol 373no 1 pp 7ndash15 2000

[34] S S Setubal A S Pontes J L Furtado A M Kayano R GStabeli and J P Zuliani ldquoEffect of Bothrops alternatus snakevenom on macrophage phagocytosis and superoxide produc-tion participation of protein kinase Crdquo Journal of VenomousAnimals and Toxins Including Tropical Diseases vol 17 no 4pp 430ndash441 2011

[35] R K Pipe J A Coles and S R Farley ldquoAssay for measuringimmune response in the mussel Mytilus edulisrdquo Techniques inthe Immunology vol 4 pp 93ndash100 1995

[36] J H Schumacher A OrsquoGarra B Shrader et al ldquoThe charac-terization of four monoclonal antibodies specific for mouse IL-5 and development of mouse and human IL-5 enzyme-linkedimmunosorbentrdquo Journal of Immunology vol 141 no 5 pp1576ndash1581 1988

[37] J Fernandez J M Gutierrez Y Angulo et al ldquoIsolation ofan acidic phospholipase A

2from the venom of the snake

Bothrops asper of Costa Rica biochemical and toxicologicalcharacterizationrdquo Biochimie vol 92 no 3 pp 273ndash283 2010

[38] S S Teixeira L B Silveira F M N da Silva et al ldquoMolecularcharacterization of an acidic phospholipase A

2from Bothrops


pirajai snake venom synthetic C-terminal peptide identifies itsantiplatelet regionrdquo Archives of Toxicology vol 85 no 10 pp1219ndash1233 2011

[39] R G Stabeli R Simoes-Silva and A M Kayano ldquoPurifica-tion of phospholipases A

2from American snake venomsrdquo in

Chromatographymdashthe Most Versatile Method of Chemical Anal-ysis A Calderon Ed pp 1ndash34 2012

[40] B Lomonte and J M Gutierrez ldquoA new muscle damagingtoxin myotoxin II from the venom of the snake Bothrops asper(terciopelo)rdquo Toxicon vol 27 no 7 pp 725ndash733 1989

[41] P B Jacobson and D J Schrier ldquoRegulation of CD11bCD18expression in human neutrophils by phospholipase A

2rdquo Journal

of Immunology vol 151 no 10 pp 5639ndash5652 1993[42] J B Lefkowith M R Lennartz M Rogers A R Morrison

and E J Brown ldquoPhospholipase activation during monocyteadherence and spreadingrdquo Journal of Immunology vol 149 no5 pp 1729ndash1735 1992

[43] S I Galkina G F Sudrsquoina and L B Margolis ldquoRegulation ofintracellular pHbyphospholipaseA

2andprotein kinaseCupon

neutrophil adhesion to solid substratardquo FEBS Letters vol 393no 1 pp 117ndash120 1996

[44] R Zouari-Kessentini J Luis A Karray et al ldquoTwo purified andcharacterized phospholipases A

2from Cerastes cerastes venom

that inhibit cancerous cell adhesion and migrationrdquo Toxiconvol 53 no 4 pp 444ndash453 2009

[45] A Bazaa N Marrakchi M El Ayeb L Sanz and J J CalveteldquoSnake venomics comparative analysis of the venomproteomesof the Tunisian snakes Cerastes cerastes Cerastes vipera andMacrovipera lebetinardquo Proteomics vol 5 no 16 pp 4223ndash42352005

[46] P R Taylor G D Brown D M Reid et al ldquoThe 120573-glucanreceptor dectin-1 is predominantly expressed on the surfaceof cells of the monocytemacrophage and neutrophil lineagesrdquoJournal of Immunology vol 169 no 7 pp 3876ndash3882 2002

[47] BM Babior R S Kipnes and J T Curnutte ldquoBiological defensemechanisms The production by leukocytes of superoxide apotential bactericidal agentrdquo The Journal of Clinical Investiga-tion vol 52 no 3 pp 741ndash744 1973

[48] L S Mayorga M I Colombo M Lennartz et al ldquoInhibition ofendosome fusion by phospholipase A

2(PLA2) inhibitors points

to a role for PLA2 in endocytosisrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 90 no21 pp 10255ndash10259 1993

[49] P T Bozza J L Payne S G Morham R Langenbach OSmithies and P F Weller ldquoLeukocyte lipid body formation andeicosanoid generation cyclooxygenase-independent inhibitionby aspirinrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 93 no 20 pp 11091ndash110961996

[50] P T Bozza W Yu and P F Weller ldquoMechanisms of formationand function of eosinophil lipid bodies inducible intracellularsites involved in arachidonic acid metabolismrdquo Memorias doInstituto Oswaldo Cruz vol 92 pp 135ndash140 1997

[51] E Leiguez J P Zuliani A M Cianciarullo C M Fernandes JM Gutierrez and C Teixeira ldquoA group IIA-secreted phospho-lipase A

2from snake venom induces lipid body formation in

macrophages the roles of intracellular phospholipases A2and

distinct signaling pathwaysrdquo Journal of Leukocyte Biology vol90 no 1 pp 155ndash166 2011

[52] K C Giannotti E Leiguez VMoreira et al ldquoA Lys49 phospho-lipase A

2 isolated from Bothrops asper snake venom induces

lipid droplet formation in macrophages which depends ondistinct signaling pathways and the C-terminal regionrdquo BioMedResearch International vol 2013 Article ID 807982 14 pages2013

[53] G Chen and D V Goeddel ldquoTNF-R1 signaling a beautifulpathwayrdquo Science vol 296 no 5573 pp 1634ndash1635 2002

[54] A Ashkenazi ldquoTargeting death and decoy receptors of thetumour-necrosis factor superfamilyrdquo Nature Reviews Cancervol 2 no 6 pp 420ndash430 2002

[55] O Micheau and J Tschopp ldquoInduction of TNF receptor I-mediated apoptosis via two sequential signaling complexesrdquoCell vol 114 no 2 pp 181ndash190 2003

[56] T Hennet C Richter and E Peterhans ldquoTumour necrosisfactor-120572 induces superoxide anion generation in mitochondriaof L929 cellsrdquo Biochemical Journal vol 289 no 2 pp 587ndash5921993

[57] BMeier HH Radeke S Selle et al ldquoHuman fibroblasts releasereactive oxygen species in response to interleukin-1 or tumournecrosis factor-120572rdquo Biochemical Journal vol 263 no 2 pp 539ndash545 1989

[58] V B OrsquoDonnell S Spycher and A Azzi ldquoInvolvement of oxi-dants and oxidant-generating enzyme(s) in tumour-necrosis-factor-120572-mediated apoptosis role for lipoxygenase pathway butnot mitochondrial respiratory chainrdquo Biochemical Journal vol310 no 1 pp 133ndash141 1995

[59] P Rosenberg E Condrea B E Rapuano K R Soons andC C Yang ldquoDissociation of pharmacological and enzymaticactivities of snake venom phospholipases A

2by modification of

carboxylate groupsrdquo Biochemical Pharmacology vol 32 no 23pp 3525ndash3530 1983

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


the catalytic network and at the calcium-binding loop includ-ing Asp49 and (ii) catalytically inactive variants having a Lysinstead of Asp at position 49 [10 13 14]

In addition among the pro inflammatory activities it wasshown that various venom PLA

2s are able to stimulate neu-

trophil chemotaxis degranulate mast cells in vitro induceprostaglandin production in peritoneal leukocytes and acti-vate macrophage functions [12 15ndash19]

Macrophages play a central role in a wide variety ofprocesses associatedwith tissuemaintenance antigen presen-tation inflammation and tissue repair [20] Resident macro-phages constitute one of the first lines of host defense and arepresent in many tissues Upon stimulation these quiescentcells are activated and display diverse cellular functions suchas phagocytosis This process consists of the uptake anddestruction of invading microorganisms an essential part ofthe host response against infections It is initiated by engagingreceptors on the surface of the macrophages which expressvarious receptors that participate in target cell recognitionand internalization of ligands [21] There are four majorclasses of receptor-mediated phagocytosis (a) Fc120574 receptorswhich recognize the Fc120574 domain of IgG-coated particles (b)complement receptor type 3 (CR3 also referred to asCD11bCD18 Mac-1 and integrin 120572M1205732) which recognizescomplement-coated particles (c) mannose receptors whichrecognize mannose and fucose on the surface of pathogensand (d) 120573-glucan receptors or Dectin-1 which recognize 120573-glucans-bearing ligands [20 21]

Subsequent to phagocytosis an abrupt production of oxy-gen free radicals such as the superoxide anion occurs referredto as an oxidative burst The enzyme complex responsible forthe production of this reactive oxygen species is the NADPHoxidase complex When macrophages are stimulated themembrane and cytosolic components of this complex asso-ciate on the phagosomal membranes to form the active oxi-dase complex [22ndash25] The reactive oxygen species (ROS)formed such as superoxide anion and hydrogen peroxide areused by macrophages to kill ingested microorganisms [22ndash25] Parallel to this reaction occurs lysossomal membranedestabilization involving increased fusion of lysosomes andphagosomes deriving from cellular stress Lysossomal activ-ities mediate several processes in cell feeding homeostasisand antimicrobial defense which involve lysosome fusionwith endosomes and phagosomes [26] Furthermore acti-vated macrophages express a large amount of inclusionscalled lipid bodies (LBs) These LBs are neutral lipid storageorganelles with key roles in cell signaling intracellular traf-ficking cell activation and eicosanoid formation [27] LBswere shown to characteristically increase both the size andnumber of cells associated with the inflammatory processAdditionally macrophages upon stimulation release cytok-ines such as TNF-120572 an important inflammatory mediatorinvolved in many inflammatory reactions [28]

The present study was therefore designed to evaluate theeffects of Bothrops atrox Lys-49 andAsp-49 PLA

2s acidic and

basic proteins on J774A1 macrophage function Our resultsindicate that these PLA

2s at noncytotoxic concentrations are

able to directly stimulate macrophages Despite differences in

enzymatic activity the toxins induced inflammatory eventsand whether the enzyme is acidic or basic does not seem tocontribute to these effects

2 Materials and Methods

21 Chemicals andReagents Trypan blue RPMI-1640 L-glu-tamine gentamicin zymosan thiocarbohydrazide (TCH)nitroblue tetrazolium (NBT) mouse INF-120574 and neutral redwere purchased from Sigma (MO USA) Fetal bovine serumwas obtained from Cultilab (Brazil) Formaldehyde andOsO4

minus (osmium tetroxide) were purchased from ElectronMicroscopy Sciences (PA USA) DuoSet Elisa Mouse TNF-alphawas purchased fromRampDSystems (OxonUnitedKing-dom) All salts and reagents used were obtained from Merck(Darmstadt Germany) with low endotoxin or endotoxin-freegrades

22 Venom Bothrops atrox specimens collected around thecity of Porto Velho State of Rondonia Brazil were kept at theFiocruz Rondonia bioterium in order to be used for venomproduction under authorization from IBAMA (license num-ber 27131-1) and CGEN (license number 0106272011-1) Thecrude venom was dehydrated and stored at a temperature ofminus20∘C in the Amazon Venom Bank at CEBio

23 Cell Line and Culture The murine macrophage cell lineJ774A1 was obtained from the Rio de Janeiro Cell Bank Col-lection (Brazil) Briefly monolayers of J774A1 macrophageswere maintained in flasks in a RPMI-1640 medium supple-mented with 100 120583gmL gentamicin 2mM L-glutamine and10 heat-inactivated fetal bovine serum (FBS) in humidifiedair with 5 CO

2at 37∘C

24 Isolation and Biochemical Characterization of Phospholi-pases A

2 Lys49 (BaTX-I) and Asp49 (BaTX-II) basic PLA

2s

as well as Asp49 (BaPLA2) acidic PLA

2from Bothrops atrox

snake venom were purified according to the method previ-ously described [29 30] B atrox crude venom (100mg) wasdissolved in 10mL of 005M Tris-HCl buffer pH 74 cen-trifuged at 755timesg for 10min at room temperature and theclear supernatant applied on a CM-Sepharose FF column (1 times40 cm) which was previously equilibrated with the same buf-fer Elution was carried out with a continuous gradient up toa concentration of 10M of NaCl at a flow rate of 10mLmin(HPLC Akta Purifier GE) Absorbance of the effluent solu-tion was recorded at 280 nm The last fraction with phos-pholipase and myotoxic activity was collected and freeze-dried The acidic fraction (Peak 1) was freeze-dried and thendiluted with 005M Tris-HCl buffer pH 74 containing 4MNaCl prior to the next chromatographic step in a butyl-HP(Hitrap 5mL) column previously equilibrated with the samebuffer The chromatography was carried out at a flow rateof 25mLmin with a decreasing gradient of NaCl (4 to0M) followed by Milli-Q H

2OThe active fractions showing

PLA2activity were desalinized lyophilized and applied on a

C18 reverse-phase column (Supelco 250mm times 46mm)previously equilibrated with 01 TFA and then eluted with






2













2


2and then incu-








2atmos-






2O immersed in 10



2O and then





2





2









3 Results












2was investigated



















BaTX-I

lowast

lowast

1

2 3 45 6

7

89 10 11 12

130

500

1000

1500

2000

2500

(mAU

)


0 50 100 150 200 250 300 350

0

20

40

60

80

100

(min)

B(

)

(a)

BaTX-II

(mAU

)


0

20

40

60

80

100

50040030020010000

minus50

00

50

100

150

200

250

(min)

B(

)

(b)

17580

58

463023

17

1 2 3 4 5 6 7 8

(c)

1 2 3175

80

5846

30

23

177

(d)







Anion (O2


2to induce the pro-

















2did not affect


0

100

200

300

400

500

600

(mAU

)

0 5 10 15 20 25

B (

)

1

23 4

56


0

100

PLA2

(min)

(a)

(mA

U)

B (

)

0

50

100

150

200

250

300

350

400

(min)

0

20

40

60

80

100

280nmConcentration

BaPLA2-I

(b)

1 2 3 4

175

8058

46

30

23

17

7

(c)



2s at a












4 Discussion










0

20

40

60

80

100

Viab

le ce

lls (

)

Control

BaTX-I

BaTX-II

15 3 615 3 6 15 3 6(120583gmL)

BaPLA2





PLA2named BaPLA









2s used





2did not affect












2 called MVL-PLA




2s on






01

02

03

04

05Ad

hesio

n (D

O)

BaPLA2

6120583gmL

(a)


01

02

03

04

05

Rem

aini

ng ce

lls (D

O)

BaPLA2

6120583gmL

(b)







20

40

60

80

100

Phag

ocyt

osis

()

BaPLA2

6120583gmL

lowast



2before






20

40

60

80

100

Supe

roxi

de an

ion

prod

uctio

n (

)

BaPLA2

6120583gmL

lowastlowast

lowastlowast









00

05

10

15

20

25

30

35

Lipi

d bo

dies

()

Zymosan

ControlBaTX-I

BaTX-IIBaPLA2


lowastlowast

lowast

lowastlowast

lowast





(ANOVA)








2O2










2often affects


2is one of


2Thus the



2 MAPK and PI3K [49






000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min


6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)


30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)





2s



4and










2



Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






2













2


2and then incu-








2atmos-






2O immersed in 10



2O and then





2





2









3 Results












2was investigated



















BaTX-I

lowast

lowast

1

2 3 45 6

7

89 10 11 12

130

500

1000

1500

2000

2500

(mAU

)


0 50 100 150 200 250 300 350

0

20

40

60

80

100

(min)

B(

)

(a)

BaTX-II

(mAU

)


0

20

40

60

80

100

50040030020010000

minus50

00

50

100

150

200

250

(min)

B(

)

(b)

17580

58

463023

17

1 2 3 4 5 6 7 8

(c)

1 2 3175

80

5846

30

23

177

(d)







Anion (O2


2to induce the pro-

















2did not affect


0

100

200

300

400

500

600

(mAU

)

0 5 10 15 20 25

B (

)

1

23 4

56


0

100

PLA2

(min)

(a)

(mA

U)

B (

)

0

50

100

150

200

250

300

350

400

(min)

0

20

40

60

80

100

280nmConcentration

BaPLA2-I

(b)

1 2 3 4

175

8058

46

30

23

17

7

(c)



2s at a












4 Discussion










0

20

40

60

80

100

Viab

le ce

lls (

)

Control

BaTX-I

BaTX-II

15 3 615 3 6 15 3 6(120583gmL)

BaPLA2





PLA2named BaPLA









2s used





2did not affect












2 called MVL-PLA




2s on






01

02

03

04

05Ad

hesio

n (D

O)

BaPLA2

6120583gmL

(a)


01

02

03

04

05

Rem

aini

ng ce

lls (D

O)

BaPLA2

6120583gmL

(b)







20

40

60

80

100

Phag

ocyt

osis

()

BaPLA2

6120583gmL

lowast



2before






20

40

60

80

100

Supe

roxi

de an

ion

prod

uctio

n (

)

BaPLA2

6120583gmL

lowastlowast

lowastlowast









00

05

10

15

20

25

30

35

Lipi

d bo

dies

()

Zymosan

ControlBaTX-I

BaTX-IIBaPLA2


lowastlowast

lowast

lowastlowast

lowast





(ANOVA)








2O2










2often affects


2is one of


2Thus the



2 MAPK and PI3K [49






000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min


6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)


30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)





2s



4and










2



Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



2





2









3 Results












2was investigated



















BaTX-I

lowast

lowast

1

2 3 45 6

7

89 10 11 12

130

500

1000

1500

2000

2500

(mAU

)


0 50 100 150 200 250 300 350

0

20

40

60

80

100

(min)

B(

)

(a)

BaTX-II

(mAU

)


0

20

40

60

80

100

50040030020010000

minus50

00

50

100

150

200

250

(min)

B(

)

(b)

17580

58

463023

17

1 2 3 4 5 6 7 8

(c)

1 2 3175

80

5846

30

23

177

(d)







Anion (O2


2to induce the pro-

















2did not affect


0

100

200

300

400

500

600

(mAU

)

0 5 10 15 20 25

B (

)

1

23 4

56


0

100

PLA2

(min)

(a)

(mA

U)

B (

)

0

50

100

150

200

250

300

350

400

(min)

0

20

40

60

80

100

280nmConcentration

BaPLA2-I

(b)

1 2 3 4

175

8058

46

30

23

17

7

(c)



2s at a












4 Discussion










0

20

40

60

80

100

Viab

le ce

lls (

)

Control

BaTX-I

BaTX-II

15 3 615 3 6 15 3 6(120583gmL)

BaPLA2





PLA2named BaPLA









2s used





2did not affect












2 called MVL-PLA




2s on






01

02

03

04

05Ad

hesio

n (D

O)

BaPLA2

6120583gmL

(a)


01

02

03

04

05

Rem

aini

ng ce

lls (D

O)

BaPLA2

6120583gmL

(b)







20

40

60

80

100

Phag

ocyt

osis

()

BaPLA2

6120583gmL

lowast



2before






20

40

60

80

100

Supe

roxi

de an

ion

prod

uctio

n (

)

BaPLA2

6120583gmL

lowastlowast

lowastlowast









00

05

10

15

20

25

30

35

Lipi

d bo

dies

()

Zymosan

ControlBaTX-I

BaTX-IIBaPLA2


lowastlowast

lowast

lowastlowast

lowast





(ANOVA)








2O2










2often affects


2is one of


2Thus the



2 MAPK and PI3K [49






000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min


6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)


30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)





2s



4and










2



Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


BaTX-I

lowast

lowast

1

2 3 45 6

7

89 10 11 12

130

500

1000

1500

2000

2500

(mAU

)


0 50 100 150 200 250 300 350

0

20

40

60

80

100

(min)

B(

)

(a)

BaTX-II

(mAU

)


0

20

40

60

80

100

50040030020010000

minus50

00

50

100

150

200

250

(min)

B(

)

(b)

17580

58

463023

17

1 2 3 4 5 6 7 8

(c)

1 2 3175

80

5846

30

23

177

(d)







Anion (O2


2to induce the pro-

















2did not affect


0

100

200

300

400

500

600

(mAU

)

0 5 10 15 20 25

B (

)

1

23 4

56


0

100

PLA2

(min)

(a)

(mA

U)

B (

)

0

50

100

150

200

250

300

350

400

(min)

0

20

40

60

80

100

280nmConcentration

BaPLA2-I

(b)

1 2 3 4

175

8058

46

30

23

17

7

(c)



2s at a












4 Discussion










0

20

40

60

80

100

Viab

le ce

lls (

)

Control

BaTX-I

BaTX-II

15 3 615 3 6 15 3 6(120583gmL)

BaPLA2





PLA2named BaPLA









2s used





2did not affect












2 called MVL-PLA




2s on






01

02

03

04

05Ad

hesio

n (D

O)

BaPLA2

6120583gmL

(a)


01

02

03

04

05

Rem

aini

ng ce

lls (D

O)

BaPLA2

6120583gmL

(b)







20

40

60

80

100

Phag

ocyt

osis

()

BaPLA2

6120583gmL

lowast



2before






20

40

60

80

100

Supe

roxi

de an

ion

prod

uctio

n (

)

BaPLA2

6120583gmL

lowastlowast

lowastlowast









00

05

10

15

20

25

30

35

Lipi

d bo

dies

()

Zymosan

ControlBaTX-I

BaTX-IIBaPLA2


lowastlowast

lowast

lowastlowast

lowast





(ANOVA)








2O2










2often affects


2is one of


2Thus the



2 MAPK and PI3K [49






000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min


6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)


30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)





2s



4and










2



Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

100

200

300

400

500

600

(mAU

)

0 5 10 15 20 25

B (

)

1

23 4

56


0

100

PLA2

(min)

(a)

(mA

U)

B (

)

0

50

100

150

200

250

300

350

400

(min)

0

20

40

60

80

100

280nmConcentration

BaPLA2-I

(b)

1 2 3 4

175

8058

46

30

23

17

7

(c)



2s at a












4 Discussion










0

20

40

60

80

100

Viab

le ce

lls (

)

Control

BaTX-I

BaTX-II

15 3 615 3 6 15 3 6(120583gmL)

BaPLA2





PLA2named BaPLA









2s used





2did not affect












2 called MVL-PLA




2s on






01

02

03

04

05Ad

hesio

n (D

O)

BaPLA2

6120583gmL

(a)


01

02

03

04

05

Rem

aini

ng ce

lls (D

O)

BaPLA2

6120583gmL

(b)







20

40

60

80

100

Phag

ocyt

osis

()

BaPLA2

6120583gmL

lowast



2before






20

40

60

80

100

Supe

roxi

de an

ion

prod

uctio

n (

)

BaPLA2

6120583gmL

lowastlowast

lowastlowast









00

05

10

15

20

25

30

35

Lipi

d bo

dies

()

Zymosan

ControlBaTX-I

BaTX-IIBaPLA2


lowastlowast

lowast

lowastlowast

lowast





(ANOVA)








2O2










2often affects


2is one of


2Thus the



2 MAPK and PI3K [49






000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min


6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)


30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)





2s



4and










2



Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

20

40

60

80

100

Viab

le ce

lls (

)

Control

BaTX-I

BaTX-II

15 3 615 3 6 15 3 6(120583gmL)

BaPLA2





PLA2named BaPLA









2s used





2did not affect












2 called MVL-PLA




2s on






01

02

03

04

05Ad

hesio

n (D

O)

BaPLA2

6120583gmL

(a)


01

02

03

04

05

Rem

aini

ng ce

lls (D

O)

BaPLA2

6120583gmL

(b)







20

40

60

80

100

Phag

ocyt

osis

()

BaPLA2

6120583gmL

lowast



2before






20

40

60

80

100

Supe

roxi

de an

ion

prod

uctio

n (

)

BaPLA2

6120583gmL

lowastlowast

lowastlowast









00

05

10

15

20

25

30

35

Lipi

d bo

dies

()

Zymosan

ControlBaTX-I

BaTX-IIBaPLA2


lowastlowast

lowast

lowastlowast

lowast





(ANOVA)








2O2










2often affects


2is one of


2Thus the



2 MAPK and PI3K [49






000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min


6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)


30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)





2s



4and










2



Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



01

02

03

04

05Ad

hesio

n (D

O)

BaPLA2

6120583gmL

(a)


01

02

03

04

05

Rem

aini

ng ce

lls (D

O)

BaPLA2

6120583gmL

(b)







20

40

60

80

100

Phag

ocyt

osis

()

BaPLA2

6120583gmL

lowast



2before






20

40

60

80

100

Supe

roxi

de an

ion

prod

uctio

n (

)

BaPLA2

6120583gmL

lowastlowast

lowastlowast









00

05

10

15

20

25

30

35

Lipi

d bo

dies

()

Zymosan

ControlBaTX-I

BaTX-IIBaPLA2


lowastlowast

lowast

lowastlowast

lowast





(ANOVA)








2O2










2often affects


2is one of


2Thus the



2 MAPK and PI3K [49






000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min


6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)


30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)





2s



4and










2



Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


00

05

10

15

20

25

30

35

Lipi

d bo

dies

()

Zymosan

ControlBaTX-I

BaTX-IIBaPLA2


lowastlowast

lowast

lowastlowast

lowast





(ANOVA)








2O2










2often affects


2is one of


2Thus the



2 MAPK and PI3K [49






000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min


6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)


30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)





2s



4and










2



Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


000

025

050

075

100

125

150Ly

soso

me v

olum

e (D

O)

lowast15min


6120583gmL

(a)

000

025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)


30min


6120583gmL

(b)


025

050

075

100

125

150

Lyso

som

e vol

ume (

DO

)

60min

BAPLA2

6120583gmL

(c)





2s



4and










2



Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Control

BaTX-I

BaTX-II

BaPLA2

lowast

lowast

TNF-120572

prod

uctio

n (p

gm

L)

6120583gmL0

100

200

300

400

500

600

700

INF-120574

lowast











2s stimulated an




2







2s isolated from



2 in




Acknowledgments


References








































2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


























2homologues
























2from














2from Bothrops








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








2rdquo Journal































2by modification of






Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Research Article Activation of J77A.1 Macrophages by Three ...downloads.hindawi.com/journals/bmri/2014/683123.pdf · Venom. Bothrops atrox specimens collected around the cityofPortoVelho,StateofRond