Activation of cellular functions in macrophages by venom

secretory Asp-49 and Lys-49 phospholipases A2

Juliana Pavan Zuliania, Jose Marıa Gutierrezb, Luciana Lyra Casais e Silvaa,

Sandra Coccuzzo Sampaioc, Bruno Lomonteb, Catarina de Fatima Pereira Teixeiraa,*

aLaboratorio de Farmacologia, Instituto Butantan, Av.Vital Brazil, 1500-CEP 05503-900 Sao Paulo, SP, BrazilbInstituto Clodomiro Picado, Facultad de Microbiologıa, Universidad de Costa Rica, San Jose, Costa Rica

cLaboratorio de Fisiopatologia , Instituto Butantan, Sao Paulo, Brazil

Received 10 March 2005; revised 24 June 2005; accepted 29 June 2005

Available online 8 August 2005

Abstract

The in vitro effects of myotoxin III (MT-III), an Asp-49 catalytically-active phospholipase A2, and myotoxin II (MT-II), a

catalytically-inactive Lys-49 variant, isolated from Bothrops asper snake venom, on phagocytosis and production of hydrogen

peroxide (H2O2) by thioglycollate-elicited macrophages were investigated. MT-II and MT-III were cytotoxic to mouse

peritoneal macrophages at concentrations higher than 25 mg/ml. At non-cytotoxic concentrations, MT-II stimulated Fcg,

complement, mannose and b-glucan receptors-mediated phagocytosis, whereas MT-III stimulated only the mannose and b-

glucan receptors-mediated phagocytosis. Moreover, both myotoxins induced the release of H2O2 by thioglycollate-elicited

macrophages, MT-III being the most potent stimulator. MT-II induced the release of H2O2 only at a concentration of 3.2 mg/ml

(130% increment) while MT-III induced this effect at all concentrations tested (0.5–2.5 mg/ml; average of 206% increment). It

is concluded that, at non-cytotoxic concentrations, MT-II and MT-III activate defense mechanisms in macrophages

upregulating phagocytosis, mainly via mannose and b-glucan receptors, and the respiratory burst.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Venom myotoxic PLA2; Macrophages; Phagocytosis; Hydrogen peroxide; Inflammation

1. Introduction

Phospholipases A2 (PLA2; EC 3.1.1.4) represent a large

family of lipolytic enzymes that catalyse the hydrolysis of

the sn-2 fatty acyl ester bond of membrane glyceropho-

spholipids to release free fatty acids and lysophospholipids

such as arachidonic acid and lysophosphatidic acid,

respectively. PLA2s can be classified into several groups

based on cellular localization, amino acid sequence,

molecular mass, and calcium requirement for enzymatic

0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.toxicon.2005.06.017

* Corresponding author. Tel.: C55 11 3726 7222; fax: C55 11

3726 1505.

E-mail address: cteixeir@usp.br (C.F.P. Teixeira).

activity (Six and Dennis, 2000). The extracellular or

secreted PLA2s (sPLA2) are characterized by high Cys

content, low molecular mass (14–16 kD), the requirement of

millimolar concentration of calcium for catalysis, and a

wide substrate selectivity in vitro. Several mammalian and

venom sPLA2s have been identified. Among them, group IB

sPLA2 is higly expressed in porcine, bovine, and human

pancreas and in other tissues, whereas those of group IA are

found in elapid snake venoms. Group IIA sPLA2, which

includes inflammatory-type sPLA2, is highly expressed in

human synovial fluid, platelets and viperid snake venoms

and group III sPLA2s is found in bee, lizard, jellyfish and

scorpion venoms (Six and Dennis, 2000). In addition, a

group III sPLA2 human homologue has been cloned and

characterized (Valentin et al., 2000).

Toxicon 46 (2005) 523–532

www.elsevier.com/locate/toxicon

J.P. Zuliani et al. / Toxicon 46 (2005) 523–532524

Venoms from snakes of the family Viperidae contain

group II PLA2s, which share structural features with PLA2s

present in inflammatory exudates in mammals (Kaiser et al.,

1990; Kini, 1997). Two different types of sPLA2s have been

characterized in Bothrops asper venom: (i) Catalytically-

active PLA2s, which have the conserved residues at the

catalytic network and at the calcium-binding loop, including

Asp-49; and (ii) catalytically-inactive variants having Lys

instead of Asp at position 49 (Gutierrez and Lomonte, 1995,

1997). This modification together with other changes in the

calcium-binding loop precludes an effective calcium-

binding and, consequently, are responsible for the lack of

enzymatic activity described in these PLA2 variants (Francis

et al., 1991; Gutierrez and Lomonte, 1995; Ownby et al.,

1999; Lomonte et al., 2003). In addition to myotoxicity,

these PLA2s induce inflammatory events and release

important inflammatory mediators under both in vivo and

in vitro experimental conditions (Chaves et al., 1998;

Lomonte et al., 1993; Zuliani et al., 2005). In addition, it was

shown that various venom PLA2s are able to stimulate

neutrophil chemotaxis and to degranulate mast cells in vitro

(Gambero et al., 2002, 2004; Landucci et al., 1998).

However, the interference of snake venom PLA2 with

macrophage functions is still unknown.

Macrophages play a central role in a wide variety of

processes associated with tissue maintenance, antigen

presentation, inflammation and tissue repair (Naito, 1993).

Resident macrophages constitute one of the first lines of host

defense and are present in many tissues. Upon stimulation,

these quiescent cells become activated and display diverse

cellular functions such as the production of several active

substances and phagocytosis. Such macrophage activation

may play beneficial and deleterious roles in the tissues, and

these phenomena might play a relevant role in the scenario

of local pathological alterations induced by snake venoms.

Phagocytosis is the first step in triggering host defense

and inflammation. This event is initiated by engagement of

receptors on the surface of the phagocytes after recognition

of cognate ligands on the particle (Aderem and Underhill,

1999). There are four major classes of receptor-mediated

phagocytosis: (a) Fcg receptors, constitutively active for

phagocytosis of IgG-coated particles; (b) complement

receptors that recognize complement-coated particles; (c)

mannose receptors that recognize mannose and fucose on

the surface of pathogens, and (d) b-glucan receptors or

Dectin-1 which recognize b-glucans-bearing ligands

(Aderem and Underhill, 1999; Brown and Gordon, 2001;

Brown et al., 2002; Herre et al., 2004a,b). During this

process, the phagocytes are known to increase their oxygen

consumption, and oxygen is utilized by a specialized

membrane-bound NADPH-oxidase, a multicomponent

enzyme system, to produce superoxide anion and hydrogen

peroxide (H2O2). These reactive oxygen species (ROS) are

used by phagocytes to kill ingested microorganisms, thus

being an essential component of the innate immune system.

In resting cells, the oxidase is dormant and its components

exist separatedly in the membrane and cytosol. When

phagocytes are stimulated, these components associate on

the plasma or phagosomal membranes to form the active

oxidase complex (Babior, 1999, 2004; Babior et al., 2002).

This reaction parallels the release of a variety of mediators

ranging from hydrolytic enzymes to growth factors. All

these products play crucial roles in the host defense by

microbial killing, but may also cause injury to surrounding

tissues (Babior, 1999, 2004; Forman and Torres, 2002;

Naito, 1993).

The present study was therefore designed to evaluate the

effects of Lys-49 and Asp-49 venom PLA2s on selected

inflammatory functions of isolated peritoneal macrophages.

Our results indicate that these PLA2s, at non-cytotoxic

doses, are able to directly stimulate macrophages in various

ways, i.e. MT-II Lys-49 PLA2 stimulated phagocytosis

mediated by Fcg, complement, mannose and b-glucan

receptors, whereas MT-III Asp-49 PLA2 stimulated phago-

cytosis only via mannose and b-glucan receptors. Both

myotoxins induced H2O2 production, MT-III being the most

potent stimulator. These findings support the hypothesis that

venom PLA2s are able to directly act on macrophages

through mechanisms unrelated to phospholipid enzymatic

degradation.

2. Materials and methods

2.1. Chemicals and reagents

Heparin was obtained from Prod. Roche Quim. Farm.

S.A. (Rio de Janeiro, Brazil) and Hema3 stain from

Biochemical Sciences Inc (USA). Rabbit antiserum against

sheep erythrocytes was kindly provided by Dr Lourdes Isaac

(Departamento de Imunologia, Instituto de Ciencias

Biomedicas, Universidade de Sao Paulo, Sao Paulo,

Brazil). Trypan blue, BSA (bovine serum albumim),

RPMI-1640, L-glutamine, penicillin G, streptomycin,

phorbol myristate acetate (PMA), phenol red, horseradish

peroxidase, zymosan A, laminarin and mannan were

purchased from Sigma (MO, USA). Fetal calf serum was

obtained from Cultilab (Brazil). Sabouraud’s dextrose broth

was purchased from Difco (France). All salts used were

obtained from Merck (Darmstadt, Germany).

2.2. Animals

Male Swiss mice (18–20 g) were used. These animals

were housed in temperature-controlled rooms and received

water and food ad libitum until used. These studies were

approved by the Experimental Animals Committee of

Butantan Institute in accordance with the procedures laid

down by the Universities Federation for Animal Welfare.

J.P. Zuliani et al. / Toxicon 46 (2005) 523–532 525

2.3. Phospholipases A2

The myotoxic Asp-49 PLA2 (MT-III) and Lys-49 PLA2

(MT-II) from Bothrops asper snake venom were purified

according to Kaiser et al. (1990) and Lomonte and Gutierrez

(1989), respectively. Homogeneity was demonstrated by

SDS-polyacrylamide gel electrophoresis under reducing

conditions.

2.4. Harvesting of macrophages

Thioglycollate-elicited macrophages (TG-elicited

macrophages) were harvested 4 days after intraperitoneal

(i.p.) injection of 1 ml of 3% thioglycollate. Animals were

killed under halothane and exsanguined. Then, peritoneal

lavage was performed, after a gentle massage of the

abdominal wall, using 3 ml of cold phosphate-buffered

saline (PBS, 14 mM NaCl, 2 mM NaH2PO4H2O, 7 mM

Na2HPO412H2O) pH 7.2, which contained 10 U/ml heparin.

The peritoneal fluid, containing TG-elicited macrophages,

was collected. Total peritoneal cell counts were determined

in a Neubauer’s chamber, and the differential counts were

performed in smears stained with Hema3. The cell

population consisted of more than 95% macrophages, as

determined by morphological and phagocytic criteria.

2.5. Cytotoxic assay

Cell viability was measured by Trypan blue exclusion. In

brief, peritoneal lavage was centrifuged at 500 g for 6 min at

4 8C and the cell pellet was resuspended in 1 ml of RPMI-

1640 medium supplemented with 100 mg/ml penicillin,

100 mg/ml streptomycin and 2 mM L-glutamine. After

counting, 2!105 TG-elicited macrophages/80 ml were

added to plastic vials and incubated with 20 ml of different

concentrations of MT-II or MT-III (5, 25, 50, 100 and

200 mg/ml) diluted in assay medium or RPMI (control), for

1 h at 37 8C in a humidified atmosphere (5% CO2). Then,

20 ml 0.1% Trypan blue was added to 100 ml of TG-elicited

macrophage suspension. Viable cell index was determined

in a Neubauer’s chamber by counting a total number of 100

cells. Results were expressed as percentage of viable cells.

2.6. Phagocytic activity of peritoneal macrophages

2.6.1. Phagocytosis of opsonized sheep erythrocytes

TG-elicited macrophages were plated on glass coverslips

(Glass Tecnica, Brazil) in glass plate dishes at a density of

1!106 cells per coverslip, and allowed to attach for 30 min

at 37 8C under a 5% CO2 atmosphere. Nonadherent cells

were removed by washing with PBS. Cell monolayers were

cultured for 1 h with RPMI-1640 supplemented with

100 mg/ml penicillin, 100 mg/ml streptomycin and 2 mM

L-glutamine at 37 8C and 5% CO2, and then challenged with

PBS (control) or different concentrations of MT-II or MT-III

(1.5, 3.2, 6.3 and 12.5 mg/ml) diluted in PBS. After washing

in PBS, the monolayers were incubated for 1 h at 37 8C and

5% CO2 with opsonized sheep erythrocytes, prepared as

described below, and unbound particles were removed by

washing with PBS. Cells were fixed with 2.5% glutaralde-

hyde for 15 min at room temperature and the coverslips

were mounted in microscope slides. The extent of

phagocytosis was quantified by phase contrast microscopic

observation. At least 200 macrophages were counted in each

determination and those containing three or more inter-

nalized particles were considered positive for phagocytosis.

Results were presented as the percentage of cells positive for

phagocytosis.

To prepare opsonized erythrocytes, a suspension of

sheep erythrocytes was diluted in PBS (5%) and mixed with

rabbit antiserum against sheep erythrocytes. The mixture

was then incubated for 30 min at 37 8C. The opsonized

erythrocytes were centrifuged twice, at 200 g for 15 min,

washed with PBS and suspended with RPMI-1640 medium

supplemented with 100 mg/ml penicillin, 100 mg/ml strepto-

mycin and 2 mM L-glutamine for the phagocytosis assay.

2.6.2. Phagocytosis of opsonized zymosan

TG-elicited macrophages were resuspended in 1 ml of a

solution containing 5 mM glucose, 5 mM glutamine, 10%

BSA, in order to obtain 1!107 TG-elicited macropha-

ges/ml, as previously described by Costa Rosa et al. (1994).

Then, macrophages were incubated with PBS (control) or

selected concentrations of MT-II or MT-III (1.5, 3.2, 6.3 and

12.5 mg/ml) diluted in PBS, challenged with opsonized

zymosan particles, prepared as described below, and

incubated for 1 h at 37 8C. The extent of phagocytosis was

quantified by counting in a Neubauer’s chamber under light

microscopic observation. At least 200 macrophages were

counted in each determination, and those containing more

than four particles of opsonized zymosan were considered

positive for phagocytosis. Results were presented as the

percentage of cells positive for phagocytosis.

To prepare opsonized zymosan, the zymosan particles,

obtained from yeast cell walls, were suspended in PBS at a

concentration of particles of 5.7 mg/ml. For opsonization,

2 ml zymosan particles were mixed with 2 ml normal mouse

serum and incubated for 30 min at 37 8C (Costa Rosa et al.,

1994). The opsonized zymosan particles were then

centrifuged at 200 g for 15 min and suspended in PBS for

the phagocytosis assay.

2.6.3. Phagocytosis of Candida albicans

TG-elicited macrophages were collected in 1 ml of

Dulbecco’s PBS, centrifuged and resuspended in order to

obtain 2.5!105 TG-elicited macrophages/ml. Then, the

cells were incubated with PBS (control) or different

concentrations of MT-II or MT-III (1.5, 3.2, 6.3 and

12.5 mg/ml) diluted in Dulbecco’s PBS, and challenged

with Candida albicans particles, prepared as described

below, followed by incubation for 1 h at 37 8C. The extent of

phagocytosis was quantified by counting in a Neubauer’s

J.P. Zuliani et al. / Toxicon 46 (2005) 523–532526

chamber under light microscopic observation. At least 200

macrophages were counted in each determination, and those

containing more than four internalized particles were

considered positive for phagocytosis. Results were pre-

sented as the percentage of cells positive for phagocytosis.

Candida albicans (ATCC Y-537) were cultured in 20%

Sabouraud dextrose broth (Laboratorio de Microbiologia e

Micologia, Departamento de Analises Clinicas, Faculdade

de Ciencias Farmaceuticas, Universidade de Sao Paulo) at

30 8C, for 24 h. The fungi were suspended in 3 ml of

Dulbecco’s PBS, pH 7.4 and counted in a Neubauer’s

chamber. The Candida particles were then suspended in

RPMI-1640 medium for the phagocytosis assay. Fungi

viability was determined by exclusion of 0.01% methylene

blue and preparations had more than 95% viability. The

ratio of Candida particles per macrophage was 1:4.

2.6.4. Phagocytosis of non-opsonized zymosan

TG-elicited macrophages were plated on 13 mm diam-

eter glass coverslips (Glass Tecnica, Brazil) in 24-well

plates at a density of 2!105 cells per coverslip and allowed

to attach for 30 min at 37 8C under a 5% CO2 atmosphere.

Nonadherent cells were removed by washing with PBS. Cell

monolayers were cultured for 1 h with RPMI-1640

supplemented with 100 mg/ml penicillin, 100 mg/ml strepto-

mycin and 2 mM L-glutamine at 37 8C and 5% CO2, and

then challenged with PBS (control) or different concen-

trations of MT-II or MT-III (1.5, 3.2, 6.3 and 12.5 mg/ml)

diluted in PBS. After washing in cold PBS the monolayers

were incubated for 1 h at 37 8C and 5% CO2 with non-

opsonized zymosan, prepared as described below, and

unbound particles were removed by washing with PBS.

Cells were fixed with 2.5% glutaraldehyde for 15 min at

room temperature and the coverslips were mounted in

microscope slides. The extent of phagocytosis was

quantified by contrast phase microscopic observation. At

least 200 macrophages were counted in each determination

and those containing three or more internalized particles

were considered positive for phagocytosis. Results were

presented as the percentage of cells positive for

phagocytosis.

The zymosan particles, obtained from yeast cell walls,

were suspended in PBS providing a concentration of

3 mg/ml. After that, the zymosan suspension was sonicated

for 15 min and total zymosan particles were determined in a

Neubauer’s chamber. The ratio of zymosan per macrophage

was 1:10.

An additional set of experiments was carried out in order

to determine the specifity of non-opsonized zymosan

interaction with mannose or b-glucan receptors. For this

purpose, macrophages were incubated either with 8 mg/ml

of the soluble polyosides mannan (a-mannan) or laminarin

(b-glucan), specific ligands for mannose and b-glucan

receptors, respectively (Giaimis et al., 1993), for 30 min

before adding MT-II or MT-III (6.3 mg/ml), or

supplemented RPMI. Then, the assay was carried out as

decribed above.

2.7. H2O2 production by TG-elicited macrophages.

TG-elicited macrophages were resuspended in 1 ml of

phenol red solution (140 mM NaCl, 10 mM potassium-

phosphate buffer, pH 7.0, 5.5 mM dextrose, 0.56 mM

phenol red) containing 0.6 mg/ml of horseradish peroxidase,

in order to obtain 2!106 TG-elicited macrophages/ml, as

described by Pick and Mizel (1981). Using 96-well flat-

bottom tissue culture plates (Corning, USA), 2!105 TG-

elicited macrophages/100 ml were incubated with PBS

(control) or 10 ml of different concentrations of MT-II or

MT-III (0.5, 1.5, 3.2, 6.3 and 12.5 mg/ml), diluted in PBS,

together with 10 ml of phorbol myristate acetate solution

(PMA, 10 ng/well), and incubated for 1 h at 37 8C in a

humidified atmosphere (5% CO2). At the end of the

incubation period, 10 ml of 1 N NaOH were added to each

well to stop the reaction. H2O2-dependent phenol red

oxidation was spectrophotometrically determined by

recording the absorbance at 620 nm (Titertek Multiscan

reader, Labsystems, USA). H2O2 concentration values were

extrapolated from a standard H2O2 calibration curve and

expressed as nmoles H2O2/2!105 cells.

2.8. Statistical analyses

Means and S.E.M. of all data were obtained and

compared by two way ANOVA, followed by Tukey test

with significance probability levels of less than 0.05.

3. Results

3.1. Effect of myotoxins II and III on macrophage viability

To test the toxicity of myotoxins on isolated TG-elicited

macrophages, the effect of 1 h incubation of several

concentrations of MT-II or MT-III was investigated. As

shown in Fig. 1, incubation of both myotoxins at a

concentration of 5 mg/ml did not affect macrophage

viability. At a concentration of 25 mg/ml, both toxins

partially decreased macrophage viability. Incubation with

concentrations of 50 mg/ml and higher significantly reduced

the viability of macrophages.

3.2. Effect of MT-II and MT-III on phagocytosis

by macrophages

3.2.1. Phagocytosis via Fcg receptor

To investigate the effect of MT-II or MT-III on Fcgreceptors-mediated phagocytosis, the uptake of opsonized

sheep erythrocytes was evaluated. Thus, phagocytosis was

determined in adherent TG-elicited peritoneal macrophages

treated with PBS (control) or non-cytotoxic concentrations

0

20

40

60

80

100

**

**

#*

*

**

20010050255

ControlMT-IIMT-III

Via

ble

cells

(%

)

Myotoxin (µg/mL)

Fig. 1. Effect of MT-II and MT-III on cell viability. The cells were

incubated with various concentrations of MT-II and MT-III or PBS

(control) for 60 min, after which cytotoxicity was assessed by

trypan blue exclusion. Values represent the meanGS.E.M. from

four animals. *P!0.01 compared with control, # P!0.05

compared with MT-II (ANOVA).

MT- III

Control

MT- II

0

20

40

60

80

Pha

gocy

tosi

s (%

)

****

Concentration (µg/mL)

12.56.33.21.5 12.56.33.21.5

80

Fig. 3. Effects of MT-II and MT-III on phagocytosis of opsonized

zymosan particles. The phagocytosis of zymosan particles opso-

nized with normal serum was determined by phase-contrast

microscopy. TG-elicited macrophages were incubated with various

concentrations of myotoxins or PBS (control) during 60 min before

addition of opsonized zymosan particles. Values represent the

meanGS.E.M. from five animals. *P!0.05 compared with control

(ANOVA).

J.P. Zuliani et al. / Toxicon 46 (2005) 523–532 527

of MT-II or MT-III. As shown in Fig. 2, 47.6G2.8%

ingestion of opsonized sheep erythrocytes per macrophage

was observed in control cells treated with PBS. Pre-

incubation of adherent cells with MT-II at concentrations

of 6.3 up to 12.5 mg/ml resulted in 32 and 21% increase in

the phagocytic index, respectively, which were significantly

higher than controls. However, pre-incubation of adherent

macrophages with MT-III did not modify the control

phagocytic index of opsonized erythrocytes.

0

20

40

60

80

Pha

gocy

tosi

s (%

)

12.56.33.21.5

Concentration (µg/mL)

MT- III

Control

MT- II

*

*

12.56.33.21.5 12.56.33.21.5

*

*

Fig. 2. Effect of MT-II and MT-III on phagocytosis of sheep

erythrocytes. The phagocytosis of sheep erythrocytes opsonized

with rabbit antiserum against sheep erythrocytes was determined by

phase-contrast microscopy. Macrophages harvested 96 h after

the i.p. injection of thioglycollate were incubated with various

concentrations of myotoxins or RPMI (control) during 60 min

before addition of opsonized sheep erythrocytes. Values represent

the meanGS.E.M. from five animals. *P!0.05 compared with

control (ANOVA).

3.2.2. Phagocytosis via complement receptor

In order to assess the ability of MT-II and MT-III to

stimulate complement receptor-mediated phagocytosis, the

uptake of opsonized zymosan particles was determined in

adherent TG-elicited macrophages treated with non-cyto-

toxic concentrations of MT-II or MT-III, or with PBS

(control). As shown in Fig. 3, elicited macrophages

incubated with PBS showed an average of phagocytosis of

opsonized zymosan particles of 50.4G0.7%. Incubation of

macrophages with MT-II at concentrations of 1.5, 3.2, 6.3

and 12.5 mg/ml resulted in phagocytic indexes of 56.4G1.8,

58.5G1.1, 61.3G2.0 and 62.0G1.9% of phagocytosis of

opsonized zymosan, respectively. These values were

significantly higher than controls at all myotoxin concen-

trations studied. On the other hand, MT-III, did not affect the

control percentage of phagocytosis of opsonized zymosan

particles at all concentrations tested.

3.2.3. Phagocytosis via mannose receptor

To investigate the effect of MT-II or MT-III on mannose

receptor-mediated phagocytosis, the uptake of Candida

albicans was evaluated. Thus, phagocytosis was determined

in TG-elicited peritoneal macrophages treated with PBS

(control) or sub-cytotoxic concentrations of MT-II or MT-

III. As shown in Fig. 4, an index of 6.3G0.6% ingestion of

Candida albicans was observed in control cells treated with

PBS. Pre-incubation of isolated cells with MT-II or MT-III

at 12.5 mg/ml resulted in 15G1.2 and 15.6G2.7% of

phagocytosis of Candida albicans, respectively, in com-

parison to controls. Incubation of cells with the lower

concentrations of both myotoxins did not significantly

modify control phagocytic index.

MT- III

Control

MT- II

0

10

20

30

Pha

gocy

tosi

s (%

)

*

*

12.56.33.21.5

Concentration (µg/mL)

12.56.33.21.5

*

Fig. 4. Effects of MT-II and MT-III on phagocytosis of Candida

albicans yeast particles. The phagocytosis of Candida albicans yeast

particles was determined by phase-contrast microscopy. Macro-

phages harvested 96 h after the i.p. injection of thioglycollate, were

incubated with various concentrations of MT-II or MT-III or

Dulbecco PBS (control) during 60 min before addition of Candida

albicans yeast particles. Values represent the mean GS.E.M. from

five animals. *P!0.05 compared with control (ANOVA).

J.P. Zuliani et al. / Toxicon 46 (2005) 523–532528

3.2.4. Phagocytosis via b-glucan receptor

In order to assess the ability of MT-II and MT-III to

stimulate b-glucan receptor-mediated phagocytosis, the

uptake of non-opsonized zymosan particles was determined

in adherent TG-elicited macrophages treated with non-

cytotoxic concentrations of MT-II or MT-III, or with PBS

MT- III

Control

MT- II

0

20

40

60

#

#

**

Concentration (µg/mL)

6.33.21.56.33.21.5

*

**

*

Pha

gocy

tosi

s (%

)

12.5

#*

#*

12.5

Fig. 5. Effect of MT-II and MT-III on phagocytosis of non-

opsonized zymosan particles. The phagocytosis of non-opsonized

zymosan particles was determined by phase-contrast microscopy.

Macrophages harvested 96 h after the i.p. injection of thioglycollate

were incubated with selected concentrations of MT-II or MT-III or

RPMI (control) during 60 min before addition of non-opsonized

zymosan particles. Values represent the meanGS.E.M. from five

animals. *P!0.05 compared with control, # P!0.05 compared

with MT-II (1.5 mg/ml) (ANOVA).

(control). As shown in Fig. 5, elicited macrophages

incubated with PBS showed an average of phagocytosis of

non-opsonized zymosan particles of 15.6G1.5%. Incu-

bation of macrophages with MT-II at concentrations of 1.5,

3.2, 6.3 and 12.5 mg/ml resulted in phagocytic indexes of

21.1G0.7, 30.9G3.6, 34.2G5.3 and 37G5.3% of phago-

cytosis of non-opsonized zymosan particles, respectively.

These values were significantly higher than controls in all

the concentrations used. Similarly, incubation of macro-

phages with MT-III significantly increased the phagocytosis

of non-opsonized zymosan particles in all the concentrations

used. These results were significantly higher than control in

all the concentrations tested.

To assess the specifity of phagocytosis of non-opsonized

zymosan particles by TG-elicited macrophages, the cells

were incubated with laminarin or mannan before adding the

toxins or RPMI (control) and the uptake of non-opsonized

zymosan particles was determined. As shown in Fig. 6,

TG-elicited macrophages incubated with PBS showed an

average of phagocytosis of non-opsonized zymosan par-

ticles of 9.2G0.9%. Incubation of macrophages with

mannan did not modify this percentage of phagocytosis,

whereas incubation of cells with laminarin or with the

association of both mannan and laminarin resulted in a

significant reduction in phagocytosis in comparison to

control. Pre-treatment of macrophages with mannan did not

modify the stimulatory effect of both myotoxins on uptake

of non-opsonized zymosan particles. However, pre-

Fig. 6. Effect of laminarin and mannan on phagocytosis of non-

opsonized zymosan particles induced by MT-II and MT-III.

Macrophages harvested 96 h after the i.p. injection of thioglycollate

were treated with laminarin and/or mannan (8 mg/ml) or RPMI

(control) during 30 min followed by incubation with MT-II or MT-

III (6.3 mg/ml) or RPMI (control) for 1 h before addition of non-

opsonized zymosan particles. Values represent the meanGS.E.M.

from six animals. *P!0.05 compared with respective control, #

P!0.05 compared with respective control myotoxins (ANOVA).

MT- III

Control

MT- II

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4# #

*

*

**

*

*

12.56.30.5 3.21.5

H2O

2 nm

oles

/ 2

×10

5 ce

lls

Concentration (µg/mL)12.56.30.5 3.21.512.56.30.5 3.21.5 12.56.30.5 3.21.5

Fig. 7. Effects of MT-II and MT-III on H2O2 production by isolated

macrophages. Macrophages harvested 96 h after the i.p. injection of

thioglycollate, were incubated with various concentrations of

myotoxins or PBS (control) during 60 min in the absence or presence

of PMA. Values represent the meanGS.E.M. from five animals. *P!0.05 compared with control (ANOVA).

J.P. Zuliani et al. / Toxicon 46 (2005) 523–532 529

treatment of macrophages with laminarin or with the

association of both mannan and laminarin resulted in a

significant reduction of the stimulatory effect of MT-II and

MT-III on non-opsonized zymosan particles phagocytosis.

3.3. Effect of MT-II and MT-III on H2O2 production

by macrophages

To investigate the ability of MT-II and MT-III to induce

the production of H2O2 by TG-elicited macrophages, the

cells were incubated with non-cytotoxic concentrations of

both toxins, in the presence of PMA (10 ng/ml), and H2O2

production was measured. As shown in Fig. 7, MT-II, at

3.2 mg/ml, caused a 130% increase of H2O2 production in

PMA-stimulated macrophages, which was significantly

higher than production in controls (PBS). At the other

studied concentrations, MT-II did not affect H2O2 pro-

duction. In contrast, MT-III in all studied concentrations

induced a significant increase (average of 206%) of H2O2

production by PMA-stimulated cells.

4. Discussion

The entry of snake venom components into tissues affects

resident cells in various ways. Direct cytotoxicity, leading to

necrosis, represents one output of envenomation. However,

other cells may be reached by non-cytotoxic concentrations

of toxins; in these cases, other cellular responses, distinct

from cell death, may develop, and they may contribute to the

overall tissue alterations observed. Macrophages exert their

host defense function through various activities, which

include phagocytosis, production of microbicidal agents,

secretion of cytokines and other inflammatory mediators, and

antigen processing and presentation. In this study we

examined the effects of MT-II and MT-III on selected

functions of TG-elicited peritoneal macrophages. Our data

clearly showed that, at non-cytotoxic concentrations, these

PLA2s exert a number of stimulatory actions on macrophage

functions. These toxins affected macrophage viability only at

concentrations higher than 25 mg/ml, indicating their low

toxicity on this cell type.

Phagocytosis involves particle internalization after an

initial binding to one of various membrane receptors

(Aderem and Underhill, 1999). Our data evidenced that

MT-II and MT-III are able to directly stimulate phagocy-

tosis by isolated TG-elicited macrophages. MT-II signifi-

cantly increased phagocytosis mediated by all of the studied

receptors, whereas MT-III increased phagocytosis only via

mannose and b-glucan receptors. Therefore, mannose and

b-glucan receptors may be potential sites of PLA2

interaction with the membrane of macrophages. Interest-

ingly, M-type PLA2 receptors and mannose receptors have

high structural homology, both having carbohydrate-

recognition domains of the C-type lectin superfamily

(Lambeau and Lazdunski, 1999). Binding of bee venom

PLA2 to macrophage mannose receptor has been described

(Mukhopadhyay and Stahl, 1995), thus raising the possi-

bility that B. asper myotoxins interact with these receptors

in macrophages, a hypothesis that requires further explora-

tion. Similarly to mannose receptors, b-glucan receptors

also have carbohydrate-recognition domains (Brown et al.,

2002; Brown and Gordon, 2001). Our data showing that

laminarin, but not mannan, affected the uptake of non-

opsonized zymosan particles, strongly suggest that b-glucan

are the receptors involved in the uptake of these particles in

our experimental conditions; this is in agreement with the

results of Brown and Gordon (2001). Moreover, blockade of

the stimulatory effect of both myotoxins by laminarin

reinforces our hypothesis that b-glucan receptors are

potential sites of their interaction with the membrane of

macrophages.

MT-II and MT-III clearly differed in their effect on

phagocytosis by macrophages, as MT-II, which lacks

catalytic activity, activated all types of phagocytic receptors

whereas MT-III, being catalytically-active and displaying a

stronger proinflammatory effect (Chaves et al., 1998;

Zuliani et al., 2005), stimulated only phagocytosis via

mannose receptors and b-glucan receptors. This suggests

that the catalytic activity is not an essential requirement to

enhance macrophage phagocytosis by the four receptors

evaluated. Thus, it is likely that molecular regions distinct

from the catalytic network may be involved in this effect.

These molecular regions, in turn, may differ among both

myotoxins since MT-II does not have a complete sequence

identity with MT-III (Kaiser et al., 1990; Francis et al.,

1991). In the case of MT-II, previous experimental

evidences suggested that a stretch of residues, located at

the C-terminus of the molecule and involving cationic and

J.P. Zuliani et al. / Toxicon 46 (2005) 523–532530

hydrophobic amino acids, are responsible for the myotoxic

and cytotoxic effects of this PLA2 homologue (Lomonte

et al., 1994b; Calderon and Lomonte, 1998). Therefore the

molecular regions of each myotoxin involved in activation

of phagocytosis remain to be established.

Participation of mammalian intracellular PLA2s on

phagocytosis by macrophages induced by several agents

has already been described (Lennartz, 1999; Iyer et al.,

2004; Hiller and Sundler, 2002). However, to our knowl-

edge, this report represents the first demonstration that

venom secretory PLA2s have the ability to stimulate

macrophages and to activate phagocytosis. The cellular

mechanisms involved in this process are being investigated

in our laboratory.

Since phagocytosis mediated by Fcg, mannose and

b-glucan receptors is tightly coupled to the production of a

variety of proinflammatory and microbicidal molecules,

such as reactive oxygen species (Aderem and Underhill,

1999; Herre et al., 2004b; Park, 2003), we investigated the

effect of MT-II and MT-III on production of H2O2 by

isolated macrophages. Our results showed that both

myotoxins significantly stimulated the production of H2O2

by these cells, indicating their ability to prime leukocytes for

the respiratory burst. However, these effects did not

correlate very well with the magnitude of phagocytosis

induced by each PLA2, as MT-III was more potent than MT-

II. Since arachidonic acid, the main product of secretory

PLA2s in many cell types, has been implicated as a second

messenger in the activation of NADPH oxidases (Bromberg

and Pick, 1985; Zhao et al, 2002; Shmelzer et al., 2003),

membrane phospholipid hydrolysis by MT-III may contrib-

ute for H2O2 production. However, a role of cytosolic PLA2

activated by MT-III cannot be ruled out. In addition, some

known stimulants of the production of H2O2, such as PMA,

were shown to bypass phagocytosis and to stimulate

NADPH-oxidases through direct activation of protein

kinase C (PKC) (Clark et al., 1990; DeLeo et al., 1999).

Therefore, our data also suggest that MT-III may induce

H2O2 formation by stimulating PKC. This hypothesis is

under investigation in our laboratory. On the other hand, the

observation that catalytically-inactive MT-II also elicits an

increment in H2O2 production supports the concept of non-

catalytic molecular regions in PLA2s are capable of

activating the respiratory burst in macrophages.

Release of ROS by activated phagocytic cells into the

extracellular environment has been implicated in microbial

killing (Jiang et al., 1997) as well as in the damage to host

surrounding tissue (Clark and Klebanoff, 1975). Consider-

ing previous studies evidencing that MT-II and MT-III

display a broad cytolytic activity, affecting a variety of cell

types in culture (Bultron et al., 1993; Lomonte et al., 1994a),

our findings suggest a role for H2O2 in the cytotoxicity

induced by these myotoxins, a hypothesis that can be

addressed with the use of antioxidant agents. On the other

hand, more recently, a role for ROS as physiologic signal

transduction agents has been advocated (Wolin, 1996).

Signaling properties include activation of transcription

factor NFkB (Ginn-Pease and Whisler, 1998) and several

studies have now suggested that ROS can regulate the

production of cytokines, particularly TNF-a, in macro-

phages through mechanisms that are dependent on NFkB.

The ability of MT-II and MT-III to induce in vivo release of

cytokines has been reported (Lomonte et al., 1993; Zuliani

et al., 2005). Based on these findings, it is suggested that

increased levels of H2O2 play an important role in the

regulation of TNF-a production after incubation with these

myotoxic PLA2s, and ultimately in the development of the

inflammatory response evoked by these toxins. Additional

studies are necessary to reveal the exact contribution of ROS

in the in vitro and in vivo inflammatory effects elicited by

these myotoxins.

Secretory PLA2s of various sources have been shown to

protect human leukocytes from the replication of various

HIV-1 strains (Fenard et al., 1999). Although the precise

mechanisms behind this activity are not known, it was

observed that they are not related to a virucidal nor a

cytotoxic effect, and that they do not depend on enzymatic

phospholipid hydrolysis. Instead, they seem to be related to

activation of intracellular events after the binding to

membrane receptors (Fenard et al., 1999). Interestingly, B.

asper myotoxin IV, a Lys-49 variant having high sequence

identity with MT-II (Dıaz et al., 1995; Lizano et al., 2001),

was able to exert this protective effect (Fenard et al., 1999).

Since macrophages are infected by HIV and constitute

important sites of viral replication in HIV infections

(Fernard et al. 1999), it would be relevant to assess if

subcytotoxic concentrations of PLA2s would halt HIV

infection in macrophages. This phenomenon might be

related to the activation of cellular processes in macro-

phages such as those described in this study, thereby

resulting in the inhibition of viral replication. This

possibility illustrates the potential value of PLA2s in

manipulating macrophage functions.

Finally, our results have implications on the under-

standing of the inflammatory events occurring in muscle

tissue after injection of myotoxic PLA2s. There is a

population of resident macrophages in skeletal muscle

(McLennan, 1996), and their reaction to myotoxic PLA2s

may affect not only the local inflammatory response, but

also the extent of tissue damage inflicted by the toxins.

Thus, our results suggest that myotoxic PLA2s may affect

macrophages in various ways: at high concentrations they

may exert cytotoxic effects, whereas at non-cytotoxic

concentrations they may activate macrophages to exert a

number of inflammatory roles such as enhancement of

phagocytic activity and production of various mediators like

H2O2. This activation, in turn, may exert a dual role: it might

contribute to the inactivation of the toxins and the removal of

necrotic tissue, paving the way for orderly tissue remodeling

and muscle regeneration (Grounds, 1991). Alternatively, if

exaggerated, these processes may result in further tissue

damage through the action of reactive oxygen species,

J.P. Zuliani et al. / Toxicon 46 (2005) 523–532 531

peroxynitrites, matrix metalloproteinases and other products

secreted by macrophages. The precise role of tissue

macrophages in the local pathological alterations associated

with myotoxic PLA2s remains to be investigated.

Acknowledgements

The authors thank to Wanda Carrela (Instituto Butantan,

Brazil) for technical assistance. This project was supported by

Grants 02/13863-2 from FAPESP-Brazil and 301199/91-4

from CNPq and from Vicerrectorıa de Investigacion,

Universidad de Costa Rica. Juliana P Zuliani was the

beneficiary of FAPESP fellowship 98/1565-7 and

02/01009-7.

References

Aderem, A., Underhill, D.M., 1999. Mechanisms of phagocytosis in

macrophages. Ann. Rev. Immunol. 17, 593–623.

Babior, B.M., 1999. NADPH oxidase: an update. Blood 93, 1464–

1476.

Babior, B.M., 2004. NADPH oxidase. Curr. Opin. Immunol. 16,

42–47.

Babior, B.M., Lambeth, J.D., Nauseef, W., 2002. The neutrophil

NADPH oxidase. Arch. Biochem. Biophys. 397, 342–344.

Bromberg, Y., Pick, E., 1985. Activation of NADPH-dependent

superoxide production in a cell-free system by sodium dodecyl

sulfate. J. Biol. Chem. 260, 13539–13545.

Brown, G.D., Gordon, S., 2001. Immune recognition. A new

receptor for beta-glucans. Nature 413, 36–37.

Brown, G.D., Taylor, P.R., Reid, D.M., Willment, J.A., Williams,

D.L., Martinez-Pomares, L., Wong, S.Y., Gordon, S., 2002.

Dectin-1 is a major beta-glucan receptor on macrophages.

J. Exp. Med. 196, 407–412.

Bultron, E., Thelestam, M., Gutierrez, J.M., 1993. Effects on

cultured mammalian cells of myotoxin III, a phospholipase A2

isolated from Bothrops asper (tercioeplo) venom. Biochim.

Biophys. Acta 1179, 253–259.

Calderon, L., Lomonte, B., 1998. Immunochemical characterization

and role in toxic activities of region 115–129 of myotoxin II, a

Lys49 phospholipase A2 from Bothrops asper snake venom.

Arch. Biochem. Biophys. 358, 343–350.

Chaves, F., Leon, G., Alvarado, V.H., Gutierrez, J.M., 1998.

Pharmacological modulation of edema induced by Lys-49 and

Asp-49 myotoxic phospholipases A2 isolated from the venom of

the snake Bothrops asper (terciopelo). Toxicon 36, 1861–1869.

Clark, R.A., Klebanoff, S.J., 1975. Neutrophil-mediated tumor cell

cytotoxicity: role of the peroxidase system. J. Exp. Med. 141,

1442–1447.

Clark, R.A., Volpp, B.D., Leidal, K.G., Nauseef, W.M., 1990. Two

cytosolic components of the human neutrophil respiratory burst

oxidase translocate to the plasma membrane during cell

activation. J. Clin. Invest. 85, 714–721.

Costa Rosa, L.F., Safi, D.A., Curi, R., 1994. Effect of thioglycollate

and BCG stimuli on glucose and glutamine metabolism in rat

macrophages. J. Leukoc. Biol. 56, 10–14.

DeLeo, F.R., Allen, L.A.H., Apicella, M., Nauseef, W.M., 1999.

NADPH Oxidase activation and assembly during phagocytosis.

J. Immnuol. 163, 6732–6740.

Dıaz, C., Lomonte, B., Zamudio, F., Gutierrez, J.M., 1995.

Purification and characterization of myotoxin IV, a phospho-

lipase A2 variant, from Bothrops asper snake venom. Nat.

Toxins 3, 26–31.

Fenard, D., Lambeau, G., Valentin, E., Lefebvre, J.C., Lazdunski,

M., Doglio, A., 1999. Secreted phospholipases A2, a new class

of HIV inhibitors that block virus entry into host cells. J. Clin.

Invest. 104, 611–618.

Forman, H.J., Torres, M., 2002. Reactive oxygen species and cell

signaling: Respiratory burst in macrophage signaling. Am.

J. Respir. Crit. Care Med. 166, S4–S8.

Francis, B., Gutierrez, J.M., Lomonte, B., Kaiser, I.I., 1991.

Myotoxin II from Bothrops asper (terciopelo) venom is a lysine-

49 phospholipase A2. Arch. Biochem. Biophys. 284, 352–359.

Gambero, A., Landucci, E.C., Toyama, M.H., Marangoni, S.,

Giglio, J.R., Nader, H.B., Dietrich, C.P., De Nucci, G., Antunes,

E., 2002. Human neutrophil migration in vitro induced by

secretory phospholipases A2: a role for cell surface glycosami-

noglycans. Biochem. Pharmacol. 63, 65–72.

Gambero, A., Thomazzi, S.M., Cintra, A.C., Landucci, E.C., De

Nucci, G., Antunes, E., 2004. Signalling pathways regulating

human neutrophil migration induced by secretory phospho-

lipases A2. Toxicon 44, 473–481.

Giaimis, J., Lombard, Y., Fonteneau, P., Muller, C.D., Levy, R.,

Makaya-Kumba, M., Lazdins, J., Poindron, P., 1993. Both

mannose and beta-glucan receptors are involved in phagocytosis

of unopsonized, heat-killed Saccharomyces cerevisiae by

murine macrophages. J. Leukoc. Biol. 54, 564–571.

Ginn-Pease, M.E., Whisler, R.L., 1998. Redox signals and NFkB

activation in T cells. Free Radic. Biol. Med. 25, 346–361.

Grounds, M., 1991. Towards understanding skeletal muscle

regeneration. Path. Res. Pract. 187, 1–22.

Gutierrez, J.M., Lomonte, B., 1995. Phospholipase A2 myotoxins

from Bothrops snake venoms. Toxicon 33, 1405–1424.

Gutierrez, J.M., Lomonte, B., 1997. Phospholipase A2 myotox-

ins from Bothrops snake venoms, Venom Phospholipase

A2 Enzymes, Structure, Function and Mechanisms. Wiley,

New York pp. 321–352.

Herre, J., Gordon, S., Brown, G.D., 2004a. Dectin-1 and its role in

the recognition of beta-glucans by macrophages. Mol. Immunol.

40, 869–876.

Herre, J., Marshall, A.S., Caron, E., Edwards, A.D., Williams, D.L.,

Schweighoffer, E., Tybulewicz, V., Sousa, C.R., Gordon, S.,

Brown, G.D., 2004b. Dectin-1 uses novel mechanisms for yeast

phagocytosis in macrophages. Blood 104, 4038–4045.

Hiller, G., Sundler, R., 2002. Regulation of phospholipase

C-gamma 2 via phosphatidylinositol 3-kinase in macrophages.

Cell Signal. 14, 169–173.

Iyer, S.S., Barton, J.A., Bourgoin, S., Kusner, D.J., 2004.

Phospholipases D1 and D2 coordinately regulate macrophage

phagocytosis. J. Immunol. 173, 2615–2623.

Jiang, X., Wu, T.H., Rubin, R.L., 1997. Bridging of neutrophil to

target cells by opsonized zymosan enhances the cytotoxicity of

neutrophil-produced H2O2. J. Immunol. 159, 2468–2475.

Kaiser, I.I., Gutierrez, J.M., Plummer, D., Aird, S.D., Odell, G.V.,

1990. The amino acid sequence of a myotoxic phospholipase

from the venom of Bothrops asper. Arch. Biochem. Biophys.

278, 319–325.

J.P. Zuliani et al. / Toxicon 46 (2005) 523–532532

Kini, R.M., 1997. Venom Phospholipase A2 Enzymes, Structure,

Function and Mechanisms. Wiley, New York.

Lambeau, G., Lazdunski, M., 1999. Receptors for a growing

family of secreted phospholipases A2. Trends Pharmacol. Sci.

20, 162–170.

Landucci, E.C., Castro, R.C., Pereira, M.F., Cintra, A.C., Giglio,

J.R., Marangoni, S., Oliveira, B., Cirino, G., Antunes, E., De

Nucci, G., 1998. Mast cell degranulation induced by two

phospholipase A2 homologues: dissociation between enzy-

matic and biological activities. Eur. J. Pharmacol. 343, 257–

63.

Lennartz, M.R., 1999. Phospholipases and phagocytosis: the role of

phospholipid-derived second messengers in phagocytosis. Int.

J. Biochem. Cell Biol. 31, 415–430.

Lizano, S., Lambeau, G., Lazdunski, M., 2001. Cloning and cDNA

sequence analysis of Lys(49) and Asp(49) basic phospholipase

A(2) myotoxin isoforms from Bothrops asper. Int. J. Biochem.

Cell Biol. 33, 127–132.

Lomonte, B., Gutierrez, J.M., 1989. A new muscle damaging toxin,

myotoxin II, from the venom of the snake Bothrops asper

(terciopelo). Toxicon 27, 725–733.

Lomonte, B., Tarkowski, A., Hanson, L.A., 1993. Host response to

Bothrops asper snake venom. Analysis of edema formation,

inflammatory cells, and cytokine release in a mouse model.

Inflammation 17, 93–105.

Lomonte, B., Tarkowski, A., Hanson, L.A., 1994a. Broad

cytolytic specificity of myotoxin II, a lysine-49 phospho-

lipase A2 of Bothrops asper snake venom. Toxicon 32,

1359–1369.

Lomonte, B., Moreno, E., Tarkowski, A., Hanson, L.A.,

Maccarana, M., 1994b. Neutralizing interaction between

heparins and myotoxin II, a Lys-49 phospholipase A2 from

Bothrops asper snake venom: Identification of a heparin-

binding and cytolytic toxin region by the use of synthetic

peptides and molecular modeling. J. Biol. Chem. 269,

29867–29873.

Lomonte, B., Angulo, Y., Calderon, L., 2003. An overview of

Lysine-49 phospholipase A2 myotoxins from crotalid snake

venoms and their structural determinants of myotoxic action.

Toxicon 42, 885–901.

McLennan, I.S., 1996. Degenerating and regenerating skeletal

muscles contain several subpopulations of macrophages

with distinct spatial and temporal distributions. J. Anat.

188, 17–28.

Mukhopadhyay, A., Stahl, P., 1995. Bee venom phospholipase A2 is

recognized by the macrophage mannose receptor. Arch.

Biochem. Biophys. 324, 78–84.

Naito, M., 1993. Macrophage heterogeneity in development and

differentiation. Arch. Histol. Cytol. 56, 331–351.

Ownby, C.L., Araujo, H.S.S., White, S.P., Fletcher, J.E., 1999.

Lysine 49 phospholipase A2 proteins. Toxicon 37, 411–445.

Park, J.B., 2003. Phagocytosis induces superoxide formation and

apoptosis in macrophages. Exp. Mol. Med. 35, 325–335.

Pick, E., Mizel, D., 1981. Rapid microassays for the measurement

of superoxide and hydrogen peroxide production by macro-

phages in culture using an automatic enzyme immunoassay

reader. J. Immunol. Methods 46, 211–226.

Shmelzer, Z., Haddad, N., Admon, E., Pessach, I., Leto, T.L., Eitan-

Hazan, Z., Hershfinkel, M., Levy, R., 2003. Unique targeting of

cytosolic phospholipase A2 to plasma membranes mediated by

the NADPH oxidase in phagocytes. J. Cell Biol. 162, 683–692.

Six, D.A., Dennis, E.A., 2000. The expanding superfamily of

phospholipase A(2) enzymes: Classification and characteriz-

ation. Biochim. Biophys. Acta. 1488, 1–19.

Valentin, E., Ghomashchi, F., Gelb, M.H., Lazdunski, M.,

Lambeau, G., 2000. Novel human secreted phospholipase

A(2) with homology to the group III bee venom enzyme.

J. Biol. Chem. 275, 7492–7496.

Wolin, M.S., 1996. Reactive oxygen species and vascular signal

transduction mechanisms. Microcirculation 3, 1–17.

Zhao, X., Bey, E.A., Wientjes, F.B., Cathcart, M.K., 2002.

Cytosolic phospholipase A2 (cPLA2) regulation of human

monocyte NADPH oxidase activity. cPLA2 affects translocation

but not phosphorylation of p67(phox) and p47(phox). J. Biol.

Chem. 277, 25385–25392.

Zuliani, J.P., Fernandes, C.M., Zamuner, S.R., Gutierrez, J.M.,

Teixeira, C.F.P., 2005. Inflammatory events induced by Lys-

49 and Asp-49 phospholipases A2 isolated from bothrops

asper snake venom: Role of catalytic activity. Toxicon 45,

335–346.