Inflammatory events induced by Lys-49 and Asp-49

phospholipases A2 isolated from Bothrops asper snake venom:

role of catalytic activity

Juliana P. Zuliania, Cristina M. Fernandesa, Stella R. Zamunera,Jose M. Gutierrezb, Catarina F.P. Teixeiraa,*

aLaboratorio de Farmacologia, Instituto Butantan, Sao Paulo, BrazilbInstituto Clodomiro Picado, Facultad de Microbiologıa, Universidad de Costa Rica, San Jose, Costa Rica

Received 23 July 2004; revised 27 October 2004; accepted 1 November 2004

Available online 5 January 2005

Abstract

The inflammatory events induced in the peritoneal cavity of mice by two PLA2s isolated from Bothrops asper snake venom

were investigated. MT-III, an Asp-49 catalytically active enzyme and MT-II, a catalytically inactive Lys-49 variant induced

increase in vascular permeability. Inhibition of enzymatic activity of MT-III with p-bromophenacyl bromide reduced this

effect. MT-III induced a larger neutrophil infiltrate than MT-II. This activity was significantly reduced after inhibition of

catalytic activity. Reduction in the number of neutrophils was observed when antibodies against L-selectin, CD18 or LFA-1

were used, suggesting the involvement of these adhesion molecules in the effects of both PLA2s. There was no effect with

antibodies against ICAM-1 and PECAM-1. Increase in the levels of LTB4 and TXA2, as well as of IL-1, IL-6 and TNF-a, were

observed in the peritoneal exudates induced by MT-III. MT-II did not enhance levels of eicosanoids but increased those of

cytokines. It is concluded that both PLA2s induce inflammatory events in this model. Since MT-III exerts a stronger

proinflammatory effect, the enzymatic phospholipid hydrolysis may be relevant for these phenomena. However, the fact that

MT-II induced inflammation suggests that molecular regions distinct from the catalytic site elicit inflammatory events perhaps

by interacting with specific cell membrane acceptors.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: Snake venom PLA2; Bothrops asper; Inflammation; Leukocytes; Adhesion molecules; Eicosanoids; Cytokines

1. Introduction

Venoms from snakes of the family Viperidae contain

class II PLA2s which share structural features with secretory

PLA2s of the class II-A present in inflammatory exudates in

mammals (Kini, 1997; Kaiser et al., 1990). A number of

venom PLA2s has been shown to induce edema

0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.toxicon.2004.11.004

* Corresponding author. Address: Laboratorio de Farmacologia,

Instituto Butantan, Av.Vital Brazil, 1500-CEP 05503-900-Sao

Paulo, SP, Brazil. Tel.: C55 11 3726 7222; fax: C55 11 3726 1505.

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

(Vishwanath et al., 1987; Lomonte et al., 1993; Lloret and

Moreno, 1993) and to promote inflammatory cell infiltration

(Zhang and Gopalakrishnakone, 1999; de Castro et al.,

2000), although comprehensive studies of the actions of

venom PLA2s on the various events of inflammation are

scarce. A particularly interesting subgroup of venom PLA2s

includes homologues having a number of amino acid

substitutions at the calcium-binding loop, especially lysine

substituting aspartate at position 49, resulting in the inability

of these enzymes to bind calcium and, consequently, in the

abrogation of their catalytic activity (Ownby et al., 1999).

Thus, these PLA2s homologues exert their activities

Toxicon 45 (2005) 335–346

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J.P. Zuliani et al. / Toxicon 45 (2005) 335–346336

independently of enzymatic phospholipid degradation

(Lomonte et al., 1994; Landucci et al., 1998).

Bothrops asper constitutes the most important poisonous

snake from a medical point of view in Central America

(Gutierrez and Lomonte, 1995), and four myotoxic PLA2s

have been isolated and characterized from this venom

(Gutierrez et al., 1984; Lomonte and Gutierrez, 1989; Kaiser

et al., 1990; Dıaz et al., 1995). Two of them, myotoxins II

and III have been sequenced, and the crystallographic

structure of the former has been determined (Arni et al.,

1995), paving the way for the performance of structure–

function analyses. MT-III is a catalytically-active Asp-49

variant (Kaiser et al., 1990), whereas MT-II is a Lys-49,

catalytically inactive homologue (Lomonte and Gutierrez,

1989). These proteins play a relevant role in the patho-

genesis of local tissue damage induced by Bothrops asper

venom, exerting myotoxic and edema-forming effects.

Moreover, a conspicuous inflammatory cell infiltrate has

been described histologically in muscle affected by these

PLA2s (Lomonte et al., 1993). However, the mechanisms

underlying the inflammatory cells tissue infiltration are still

unknown.

Inflammation is a defense mechanism characterized by

increase of vascular permeability, edema, and leukocyte

migration from the vasculature into damaged tissues to

destroy the injurious agent. During the acute phase of

inflammation, neutrophils are the first cells to accumulate in

tissues (Ryan and Majno, 1977). Migration of leukocytes

into tissues is a multistep process, which is characterized by

an initial weak interaction with the endothelium mediated

by selectins adhesion molecules (L-selectin on neutrophils,

E-selectin and P-selectin on endothelium) and their

carbohydrated ligands, giving a rolling motion to leukocytes

(Bevilacqua and Nelson, 1993). Rolling enables leukocyte

activaction by chemotactic agents associated with the

endothelial-cell membrane, inducing activation of b2

integrins (LFA-1 and MAC-1) on the white-cell surface.

These molecules interact with endothelial members of the

immunoglobulin superfamily (ICAM-1, -2 and -3) for firm

leuko-endothelial adhesion. Leukocytes then begin to cross

the endothelial layer through homologous interactions of

platelet endothelial-cell adhesion molecule (PECAM-1)

expressed in both leukocytes and the intercellular

membranes of endothelial cells, and migrate following a

chemoattractant gradient initiated in the injured tissue. The

sequence of expression and function of the adhesion

molecules is regulated by a range of inflammatory mediators

(Hubbard and Rothlein, 2000; Kaplanski et al., 2003). In the

present work, a comprehensive analysis of the inflammatory

reaction elicited by the two PLA2s, MT-II and MT-III, in the

mouse peritoneal cavity was performed, with special focus

on leukocyte migration. Results indicate that, although both

proteins are able to elicit an inflammatory reaction, the

profile of inflammatory events and mechanisms elicited by

these PLA2s differ. MT-III-induced cell influx is mediated

by eicosanoids and cytokines whereas that event triggered

by MT-II is mainly cytokine-dependent. However, both

PLA2s upregulate similar leukocyte adhesion molecules.

Finally, our findings suggest that, despite the fact that

catalytic activity is not essential for the induction of

inflammation in the case of a Lys-49 PLA2, it does play

an important role in the development of inflammatory

events after injection of an Asp-49 PLA2.

2. Materials and methods

2.1. Chemicals and reagents

Heparin was obtained from Prod. Roche Quim. Farm.

S.A. (Rio de Janeiro, Brazil) and Evans blue from Inlab

(Brazil). Murine capture antibody anti-IL-6 (clone

MP5-20F3), recombinant rIL-6 and detection antibody

anti-IL-6 (clone MP5-32C11) were purchased from Phar-

mingen (CA, USA). 2,2 0-azino-bis (3 ethylbenzthiazoline-6-

sulfonic acid) ABTS was purchased from Southern

Biotechnology Associates Inc. (AL, USA). Rat monoclonal

antibodies anti-mouse L-selectin (anti-mouse CD62L; clone

MEL-14), anti-mouse ICAM-1 (anti-mouse CD54; clone

3E2:), anti-mouse LFA-1 a chain (anti-mouse CD11a;

clone M17/4), anti-mouse PECAM-1 (anti-mouse CD31;

clone MEC 13.3:1), anti-mouse b2 integrin chain (anti-

mouse CD18; clone GAME 46) were purchased from

Pharmingen (CA, USA). Rat immunoglobulin (Ig)G anti-

horse IgG derived from mouse myeloma was kindly

provided by Dr Irene Fernandes (Immunophatology

Laboratory, Butantan Institute, Sao Paulo, Brazil). Murine

capture antibody anti-IL-1 (clone MP5-20F3), recombinant

rIL-1, detection antibody anti-IL-1 (clone MP5-32C11) and

streptoavidin peroxidase were purchased from R and D

Systems (MN, USA). Avidin-phosphatase, o-phenylenedia-

mine and p-bromophenacyl bromide (pBPB) were purchased

from Sigma (MO, USA). Murine antibody anti-TNF-a and

recombinant rTNF-a were purchased from Pharmingen

(CA, USA) and Hema3 stain from Biochemical Sciences Inc

(USA). 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 Commitee of

Butantan Institute in accordance with the procedures laid

down by the Universities Federation for Animal Welfare.

2.3. Phospholipases A2

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

(MT-II) from Bothrops asper venom were purified

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

J.P. Zuliani et al. / Toxicon 45 (2005) 335–346 337

(1989), respectively. Homogeneity was demonstrated by

SDS-polyacrylamide gel electrophoresis run under reducing

conditions. In some experiments, MT-III was inactivated

with pBPB. Briefly, 150 mL of a 3 mg/mL pBPB solution,

prepared in ethanol, were added to 1.0 mL of a solution of

3 mg MT-III dissolved in 0.1 M Tris, 0.7 mM EDTA, pH

8.0, and incubated for 20 h at room temperature. Abrogation

of PLA2 activity was demonstrated titrimetrically (Gutierrez

et al., 1986).

2.4. Induction of inflammatory reaction

Myotoxins MT-II, MT-III (0.5 or 1.0 or 2.0 mg/kg) or

inactivated MT-III (iMT-III), 1.0 mg/kg, dissolved in 1 mL

sterile saline (0.15 M NaCl) were injected by the intra-

peritoneal (i.p.) route. Control animals received 1 mL of

sterile saline alone. At selected time intervals after these

injections, the animals were killed under halothane

atmosphere and the inflammatory exudate was withdrawn

after washing the cavities with 2 mL phosphate-buffered

saline (PBS), pH 7.2. Aliquots of the washes were used to

determine total cell counts. The remaining volume was

centrifuged at 500g for 6 min (at 4 8C) and supernatants

were stored at K70 8C and later used for determination of

eicosanoid or cytokine concentration.

2.5. Evaluation of vascular permeability (VP)

Alterations in VP were evaluated by assessing the

extravasation of Evans blue dye (EB) into the mouse

peritoneal cavity at several time intervals (5, 15, 30 and

60 min) after i.p. injection of PLA2s, inactivated MT-III or

sterile saline (control). EB (20 mg/kg, i.v.) was injected

20 min before each time for assessment of extravasation,

when the animals were killed under halothane and

exsanguinated. The peritoneal cavity of each animal was

then washed with 1 mL of PBS, pH 7.4 and centrifuged at

2000g for 5 min. The concentration of EB in the supernatant

was determined by recording the absorbance at 620 nm

(Sirois et al., 1988). Results were expressed as mgEB/mL,

using a standard curve of EB (0.5–25 mg/mL).

2.6. Leukocyte harvesting and counting

Leukocytes were harvested by washing peritoneal

cavities with 2 mL of PBS containing heparin (10U/mL).

Aliquots of the washes were used to determine total cell

counts in a Neubauer chamber after dilution (1:20, v/v) in

Turk solution (0.2% crystal violet dye in 30% acetic acid).

For differential cell counts, cytospin preparations

were stained with Hema3 stain. Differential cell counts

were performed by counting at least 100 cells, which were

classified as either polymorphonuclear or mononuclear

cells, based on conventional morphological criteria.

2.7. Treatment with anti-adhesion molecules monoclonal

antibodies (mAb)

Groups of mice were injected i.v., in the tail vein, with

monoclonal antibodies against the adhesion molecules

L-selectin, LFA-1, CD18, ICAM-1 or PECAM-1

(1.0 mg/kg). Control animals received an injection of a

similar volume of either sterile saline solution or an

equivalent dose of normal rat IgG. After 30 min, mice

were injected i.p. with MT-II (2.0 mg/kg) or MT-III

(1.0 mg/kg), and the neutrophil influx was assessed after

6 h, as described above.

2.8. Eicosanoid assays

Concentrations of LTB4 and TXB2 (stable metabolite of

TXA2) were measured in the peritoneal washes at 30, 60 and

240 min after i.p. injections of PLA2s (1.0 mg/kg) or sterile

saline, by a specific enzymatic immunoassay (EIA)

previously described by Pradelles et al. (1985), using a

commercial kit (Cayman Chemicals, MI, USA), after

extraction of eicosanoids on Sep Pak C18 columns (Waters

Corporation) eluted with ethanol. In brief, 100 mL aliquots

of each extracted sample were incubated with the

eicosanoids conjugated with acetylcholinesterase and the

specific rabbit antiserum in 96-well microtitration plates,

coated with anti-rabbit IgG mouse monoclonal antibody.

After addition of the substrate, the absorbances of the

samples were recorded at 412 nm in a microplate reader, and

concentrations of the eicosanoids were estimated from

standard curves.

2.9. Quantification of IL-1 and IL-6

Peritoneal washes were collected 30, 60, 180 and

360 min after i.p. injection of the PLA2s (1.0 mg/kg) or

sterile saline. After centrifugation, the supernatants were

used for determination of IL-1 and IL-6 levels by a specific

EIA, as described by Schumaker et al. (1988). Briefly,

96-well plates were coated with 50 mL of the first capture

monoclonal antibody anti-IL-1 (2 mg/mL) or anti-IL-6

(2.5 mg/mL) and incubated for 18 h in the case of IL-1 or

2 h for IL-6 at 37 8C. Following this period, 200 mL of

blocking buffer, containing 5% bovine serum albumin

(BSA) in PBS/Tween20, were added to the wells and the

plates incubated for 2 h at 37 8C for IL-1 and overnight at

4 8C for IL-6. After washing, 50 mL of either samples or

standards were dispensed into each well and the plates

incubated for 2 h at 37 8C. Wells were washed and bound

IL-1 or IL-6 was detected by the addition of the biotinylated

monoclonal antibodies anti-IL-1 (5 mg/mL, 50 mL/well) or

anti-IL-6 (5 mg/mL, 50 mL/well). After incubation and

washing, 50 mL of streptoavidin-peroxidase in the case of

IL-1 or avidin-phosphatase in the case of IL-6 were

added, followed by incubation and addition of the substrate

(50 mL/mL of o-phenylenediamine in the case of IL-1 or

J.P. Zuliani et al. / Toxicon 45 (2005) 335–346338

200 mL/mL r-nitrophenylphosphate in the case of IL-6).

Absorbances at 450 nm were recorded and concentrations of

IL-1 and IL-6 were estimated from standard curves prepared

with recombinant IL-1 or IL-6.

2.10. Quantification of TNF-a

Peritoneal washes were collected 30, 60, 180 and

360 min after i.p. injection of the PLA2s (1.0 mg/kg) or

sterile saline. After centrifugation, the supernatants were

used for determination of TNF-a levels by a standard assay

using the fibroblast continuous cell line L-929, as described

by Ruff and Gifford (1981). Briefly, monolayers of L-929

cells grown in RPMI-1640 medium were seeded at 3.5!104

cells per well in 96 well plates and incubated in humidified

air with 5% CO2 at 37 8C for 18 h. Afterwards, 100 mL of

each serially diluted supernatant and actinomycin D

(2 mg/mL) were added. Then, 500 ng/well of anti-TNF-aantibody were added for determination of the assay

specificity. After incubation for 24 h at 37 8C, supernatants

were removed and viable cells were assessed after fixation

and staining with crystal violet dye (0.2% in 20% methanol).

Absorbances at 620 nm were recorded in a microtiter reader.

Citotoxicity, expressed as a percentage, was calculated as

follows (Abs control-Abs sample / Abs control)!100. TNF-alevels were then expressed as units/mL, using a standard

curve prepared with recombinant TNF-a.

Fig. 1. Plasma extravasation induced by MT-II and MT-III in the

mouse peritoneal cavity. MT-II, MT-III (0.5 or 1.0 mg/kg), pBPB-

treated MT-III (iMT-III) (1.0 mg/kg) or sterile saline alone (control)

were injected i.p. After the indicated time intervals vascular

permeability was evaluated by assessing the extravasation of Evans

2.11. Statistical analysis

Means and SEM of all data were obtained and compared

by two way ANOVA, followed by Tukey test with

significance probability levels of less than 0.05.

blue dye, as described in Materials and Methods. (A) dose-response;

(B) time-course of plasma extravasation. Values are mean GSEM

of 5-6 animals. *P!0.05 when compared with control group;

#P!0.05 when compared with corresponding MT-II group.

3. Results

3.1. Effects of PLA2s on vascular permeability

The increase of vascular permeability (VP) in the

peritoneal cavities of animals was determined 15 min after

the injection of two doses (0.5 and 1.0 mg/kg) of MT-II or

MT-III into the cavities. Fig. 1A shows that a significant

increase of VP was induced by 0.5 and 1.0 mg/kg of MT-II

or MT-III. Doses of 1.0 mg/kg caused the most pronounced

effect. Chemical inactivation of the catalytic site of MT-III

abrogated the effect of this toxin on VP. Time course of the

increase of VP induced by MT-II or MT-III in the peritoneal

cavity is shown in Fig. 1B. Both toxins caused a marked

increase of Evans blue extravasation from 5 to 30 min, with

highest values at 15 min, decreasing rapidly thereafter

reaching basal levels at 60 min. The effect of MT-III

was three times higher than that induced by MT-II between

15 and 30 min.

3.2. Cellular influx into the peritoneal cavity induced

by PLA2s

The number of leukocytes in the peritoneal washes was

determined 6 and 24 h after the injection of MT-II or MT-III

into the peritoneal cavity. MT-II at doses of 0.5 and

1.0 mg/kg, did not induce a significant leukocyte infiltration

into the peritoneal cavity; only a dose of 2.0 mg/kg of MT-II

promoted an inflammatory infiltrate (Fig. 2A). In contrast,

MT-III at a dose of 1.0 mg/kg, was able to induce an

inflammatory infiltrate (Fig. 2A), whereas the infiltrate

elicited with a dose of 0.5 mg/kg was not significantly

different from cell counts in mice receiving saline solution

(not shown). Differential counts showed that the predominant

cells in the infiltrate after MT-III and MT-II injection were

polymorphonuclear leukocytes (PMN), mainly neutrophils

(Fig. 2B), whereas the population of mononuclear leukocytes

Fig. 2. Leukocyte accumulation into the mouse peritoneal cavity after injection of MT-II and MT-III. MT-II (1.0 or 2.0 mg/kg), MT-III

(1.0 mg/kg), pBPB-treated MT-III (iMT-III) (1.0 mg/kg) or sterile saline alone (control) were injected into the mouse peritoneal cavity in a final

volume of 1 mL. Total leukocyte and polymorphonuclear (PMN) and mononuclear (MN) cell counts were determined in peritoneal washes

harvested 6 and 24 h after these injections as described in Materials and Methods. Values are mean GSEM of 5–7 animals. *P!0.05 when

compared with the corresponding control group. At the dose of 1.0 mg/kg, MT-II did not induce leukocyte accumulation (PO0.05).

J.P. Zuliani et al. / Toxicon 45 (2005) 335–346 339

(MN) did not show a significant increase (Fig. 2C). Maximal

total leukocyte and PMN counts were detected 6 h after

injection of MT-III. Moreover, iMT-III (1.0 mg/kg) did not

induce leukocyte influx into the mouse peritoneal cavity.

3.3. Effect of treatment with monoclonal antibodies against

adhesion molecules on neutrophil influx

To identify the critical adhesion molecules involved in

MT-III and MT-II-induced neutrophil influx, the effect of

neutralizing mAbs against selected adhesion molecules was

examined. Since maximal neutrophil influx was observed at

6 h after injection of both toxins, we evaluated the effects of

these mAbs at this time point. Pre-treatment of animals with

anti-L-selectin, anti-CD18 and anti-LFA-1 (CD11a/CD18)

mAbs strikingly decreased the neutrophil influx promoted

by both myotoxins, when compared with control animals

injected with control IgG (Fig. 3). In contrast, anti-ICAM-1

and anti-PECAM-1 mAbs did not inhibit neutrophil

infiltration by the toxins.

Fig. 3. Effects of treatments with anti-adhesion molecules

monoclonal antibodies (mAb) on PLA2s-induced neutrophil influx

into mouse peritoneal cavity. Groups of mice were injected (i.v. at a

dose of 1.0 mg/kg) with mAb against selected adhesion molecules

or with normal rat IgG (control), 30 min before i.p. injection of (A)

MT-III (1.0 mg/kg, i.p.) or (B) MT-II (2.0 mg/kg, i.p.). Cell counts

were performed in peritoneal washes collected 6 h after MT-III or

MT-II injection as described in Material and Methods. Values are

mean GSEM of 5–6 animals. *P!0.05 when compared with

control normal rat IgG-treated group; #P!0.05 when compared

with saline group.

J.P. Zuliani et al. / Toxicon 45 (2005) 335–346340

3.4. Eicosanoid concentration in the peritoneal cavity

To investigate the ability of both PLA2s to release

chemotactic lipid mediators in the local area of injection, the

concentrations of LTB4 and TXB2 in the peritoneal fluids of

these animals were measured. MT-III (1.0 mg/kg) induced a

marked increase in peritoneal TXB2 between 30 and

240 min, with maximum levels at 60 min (Fig. 4). On the

other hand, LTB4 levels after MT-III injection significantly

increased only 30 min after injection (Fig. 4). In contrast,

MT-II, at the same dose of MT-III (1.0 mg/kg), did not

cause a significant enhancement of peritoneal TXB2 and

LTB4 concentrations (Fig. 4).

3.5. IL-1, IL-6 and TNF-a concentrations in the

peritoneal cavity

To further analyse and compare the mechanisms of the

inflammatory events induced by these PLA2s, the concen-

trations of IL-1, IL-6 and TNF-a in the peritoneal fluid were

measured. MT-III caused a marked increase in both IL-1 and

IL-6 levels between 180 and 360 min, peaking at 180 min

(Fig. 5). MT-II induced a significant increase in the levels of

IL-6 only at 180 min (Fig. 5). MT-III caused a significant

increase in the TNF-a concentrations only at 1 h, whereas

MT-II significantly increased TNF-a concentrations at 6 h

(Fig. 5).

4. Discussion

PLA2s from snake venoms exert a large number of

pharmacological activities (Kini, 1997) due to a process of

accelerated evolution through which a high mutational rate

in the coding regions of their genes has allowed the

development of new functions, mainly associated with the

exposed regions of the molecules (Kini and Chan, 1999).

The integral analysis of the inflammation elicited by Asp-49

and Lys-49 PLA2s from B. asper venom in the mouse

peritoneal cavity performed in the present study allowed a

parallel evaluation of the increase in microvascular

permeability, the cellular infiltrate, the role of adhesion

molecules in cellular recruitment, and the production of

various inflammatory mediators.

Both Asp-49 and Lys-49 PLA2s induced an increase in

vascular permeability in peritoneal cavity of mice. This is in

agreement with previous observations on the edema-

forming activity of similar molecules in the rodent footpad

model (Lomonte et al., 1993; Landucci et al., 1998; Chaves

et al., 1998). The increase of vascular permeability was

detected early after MT-II and MT-III injection and

developed rapidly, indicating that the observed plasma

extravasation is primarily due to formation of endothelial

gaps in vessels of microcirculation. The mediators involved

in this effect of both myotoxins were not addressed in this

study. However, the immediate plasma extravasation in

response to both venom PLA2, strongly suggests the

involvement of vasoactive mediators derived from mast

cell granules. Previosly, the ability of venom PLA2 to

degranulate mast cells has been shown (Landucci et al.,

1998) and histamine receptor antagonists significantly

reduced footpad edema induced by MT-II and MT-III

(Chaves et al., 1998).

Our results also indicate that the catalytically active

Asp-49 MT-III is more potent than Lys-49, enzymatically

inactive MT-II, in enhancing vascular permeability. This

strongly suggests that enzymatic phospholipid hydrolysis

Fig. 4. TXB2 and LTB4 concentrations in the mouse peritoneal fluid after injection of MT-II and MT-III. Animals were injected i.p. with MT-II,

MT-III (1.0 mg/kg) or sterile saline alone (control) in a final volume of 1 mL. TXB2 and LTB4 were quantitated by specific EIA in peritoneal

fluids collected at the indicated time intervals after PLA2 or saline injection. Each bar represents mean GSEM of 4–5 animals. *P!0.05 when

compared with the corresponding control groups; #P!0.05 when compared with corresponding MT-II group.

J.P. Zuliani et al. / Toxicon 45 (2005) 335–346 341

plays a significant role in this event. This contention is

supported by the observation that chemical modification

with pBPB, which abolishes catalytic activity, greatly

reduced edema-forming activity of MT-III (Chaves et al.,

1998) and of other PLA2s (Cirino et al., 1989), and also

caused a drastic reduction in peritoneal vascular

permeability in our experiments. Despite a possible role of

enzymatic activity in enhancing vascular permeability, in

the case of MT-II the increase in vascular permeability may

occur by mechanisms unrelated to phospholipid hydrolysis.

Various enzymatically inactive Lys-49 PLA2s have been

shown to induce edema (Lomonte et al., 1993; Landucci

et al., 1998, 2000), clearly indicating the existence of

molecular regions, different from the catalytic site in these

PLA2s homologues, which are responsible for mast cell

degranulation and edema formation. Interestingly, a

synthetic 13mer peptide comprising a C-terminal region of

B. asper MT-II, rich in hydrophobic and cationic residues,

induces footpad edema in mice (Nunez et al., 2001).

Edema-forming activity of various Lys-49 myotoxic PLA2s

from Bothrops sp venoms is significantly reduced after

chemical modification of Lys residues (Soares et al., 2000a,b)

and after incubation with the polyanion heparin (Lomonte

et al., 1994; Landucci et al., 2000). Similar observations

have been carried out with a basic PLA2 from Trimeresurus

mucrosquamatus venom (Wang and Teng, 1990).

A prominent leukocyte infiltrate, composed predomi-

nantly of neutrophils, was observed after injection of MT-III

and MT-II. Previous studies have documented polymorpho-

nuclear and mononuclear cellular infiltrate after injection of

myotoxic PLA2s from the venoms of B. asper (Lomonte

et al., 1993), B. nummifer (Gutierrez et al., 1989) and B.

jararacussu (Gutierrez et al., 1991) in mouse skeletal

muscle, and after intrapleural administration of similar

myotoxins from B. jararacussu and B. pirajai venoms

(de Castro et al., 2000). Asp-49 MT-III induced a prominent

infiltrate, whereas Lys-49 MT-II caused this effect only at

higher doses, in agreement with a previous study using

Fig. 5. Levels of IL-1, IL-6 and TNF-a in the peritoneal exudate after injection of MT-II and MT-III. Animals were injected i.p. with MT-II

(1.0 mg/kg), MT-III (1.0 mg/kg) or sterile saline alone (control) in a final volume of 1 mL. IL-1 and IL-6 were quantified by specific EIA, and

TNF-a was quantified by cytotoxic activity on L929 cells in peritoneal fluids collected at the indicated time intervals after PLA2s or saline

injection as described in Materials and Methods. Each bar represents mean GSEM of 4–5 animals. *P!0.05 when compared with the

corresponding control groups; #P!0.05 when compared with corresponding MT-II group.

J.P. Zuliani et al. / Toxicon 45 (2005) 335–346342

similar components from the venoms of B. jararacussu and

B. pirajai (de Castro et al., 2000). These observations

suggest that enzymatic activity is relevant for inflammatory

cell recruitment in catalytically-active PLA2s, a conclusion

supported by the observation that pBPB modification of

MT-III caused a parallel loss of enzymatic activity and

cellular infiltrate.

The involvement of a number of adhesion molecules in

the PLA2s-induced neutrophil influx into the peritoneal

cavity of mice was investigated by pre-treating animals with

J.P. Zuliani et al. / Toxicon 45 (2005) 335–346 343

mAb against L-selectin, LFA-1, CD18, ICAM-1 and

PECAM-1 adhesion molecules. Present results showed

that anti-mouse L-selectin mAb significantly reduced the

neutrophil migration induced by both MT-II and MT-III.

L-selectin is shed from the neutrophil surface under cell

activation and contributes to neutrophil rolling at the initial

phase of the adhesion cascade (Bevilacqua and Nelson,

1993). Thus, our data suggest that both PLA2s up regulate

the expression of a member of the selectin family of

adhesion molecules, which mediates the rolling event.

Rolling is not sufficient for leukocyte firm adhesion, which

requires activation of the CD18 integrins LFA-1 and

MAC-1 on neutrophils by chemoattractants (Lawrence and

Springer, 1991; Seo et al., 2001), resulting in binding to one

of the intercellular adhesion molecules at surfaces of

endothelial cells. The present data showing that anti-

mouse LFA-1 (CD11a) mAb markedly reduced the

neutrophil influx suggest the ability of both PLA2s to

up-regulate the expression of an integrin adhesion molecule,

relevant for firm adhesion of leukocytes. Our findings in

mice injected with anti-mouse CD18 mAb further indicate

the contribution of the b2 (CD18)-subunit of integrins to the

neutrophil influx induced by both MT-II and MT-III.

Interestingly, these three adhesion molecules are expressed

in the membrane of leukocytes and participate in the

processes of rolling, adherence and transmigration, respect-

ively (Zimmerman et al., 1992). On the other hand our data

showed that blocking ICAM-1 or PECAM-1 adhesion

molecules did not change the neutrophil emigration induced

by both PLA2s. Therefore, these two adhesion molecules,

constitutively expressed by endothelial cells (Thompson

et al., 2001), may not play a significant role in mediating the

PLA2s-induced neutrophil emigration or are redundant with

other adhesion molecules ligands for CD18 integrins. Thus,

these results suggest that the studied PLA2s do not display a

specific inflammatory stimulus for expression of these two

endothelium adhesion molecules. ICAM-1 and PECAM-1

are ligands for b2 integrin and leukocyte PECAM-1,

respectively. These interactions are relevant for the

transmigration event. Based on our data it is suggest that

endothelial adhesion molecules ligands for LFA-1

and CD18 different from ICAM-1 are involved in the

PLA2s-induced neutrophil emigration indicating that there

is an ICAM-1-independent but b2-integrin-dependent path-

way of adhesion in our model. Thus, the identity of

endothelial cell adhesion molecules involved in cell

recruitment in this model remains to be established. On

the other hand, since neither MT-II nor MT-III alter the

cellular intrinsic mechanisms involved in neutrophil

locomotion (Farsky et al., 2000), the observed cell

recruitment must depend on the release of chemotactic

mediators in the tissues as a consequence of the actions of

these toxins.

A number of inflammatory mediators, released in the

peritoneal cavity after injection of MTs, may be responsible

for the leukocyte recruitment. Our observations demonstrate

a marked increase in LTB4 and TXA2 in the peritoneal

cavity as a consequence of injection of MT-III, whereas no

such effect was detected after injection of MT-II, in

agreement with its lack of enzymatic activity. Our data are

in accordance with Gambero et al. (2002) in that high

concentration of Piratoxin-I, a Lys-49 PLA2, and Bothrop-

stoxin-II, an Asp-49 PLA2, induced in vitro neutrophil

chemotaxis and this effect was associated with LTB4. This

eicosanoid is one of the most potent chemotactic agents for

PMN leukocytes. Interaction of this eicosanoid with its

receptor on the neutrophil surface results in chemotaxis,

chemokinesis, aggregation and adhesion to endothelial cells

(Hoover et al., 1984; Palmblad et al., 1990). This mediator

induces the expression of LFA-1, MAC-1 and b2 integrin in

leukocytes and of ICAM-1 on the endothelial cell surface

(Seo et al., 2001; Zimmerman et al., 1992). Thus, LTB4 may

be relevant for the expression of LFA-1 and b2 integrin in

our experimental conditions, thereby contributing for

neutrophil influx induced by MT-III. TXA2 is another

important chemotactic agent involved in leukocyte adher-

ence phenomena by its ability to induce the expression of b2

integrins in neutrophils and VCAM-1 by endothelial cells

(Wiles et al., 1991; Ishizuka et al., 1996). The release of this

lipid mediator by MT-III strongly suggests that the catalytic

activity of this PLA2 plays a role in this mediation, probably

due to the direct hydrolysis of membrane phospholipids,

with the consequent release of arachidonic acid and the

increase in biosynthetic pathways involved in eicosanoid

production. In addition to this enzymatic effect, it might be

that MT-III is able to interact with cells, activating

intracellular class IV PLA2 and, probably, inducing the

expression of endogenous forms of secreted class IIA or

class V PLA2s, all of which have been shown to play a

relevant role in eicosanoid synthesis (Dennis, 2000).

Moreover, class IIA PLA2 up-regulates cyclooxygenase-2

and amplifies the production of eicosanoids by inflamma-

tory cells at low concentrations of TNF-a (Bidgood et al.,

2000). Thus, besides the role of directly supplying

arachidonic acid through the enzymatic activity of MT-III,

a complex interplay involving the activation and secretion

of endogenous PLA2s and other enzymes associated with

eicosanoid production is probably associated with the

observed increase in the levels of these lipid inflammatory

mediators in our model. However, the drastic difference

observed in the ability of these two PLA2s to stimulate the

production of LTB4 and TXA2 strongly argue for a key role

of enzymatic activity of MT-III in this effect.

Cytokines, such as IL-1, IL-6 and TNF-a, are also

relevant mediators for leukocyte migration and participate

in several inflammatory conditions. Our results showed that

both PLA2s induced increase in IL-1 and IL-6 in the

peritoneal fluid, MT-III exerting a stronger effect than

MT-II. IL-1 induces the expression of adhesion molecules

by endothelial cells and stimulates the release of both IL-6

and TNF-a (Stylianou and Saklatvala, 1998). Thus, our

results suggest that IL-1 may contribute for the leukocyte

J.P. Zuliani et al. / Toxicon 45 (2005) 335–346344

influx induced by both MT-II and MT-III. In addition, the

similarity observed in the time course of IL-1 and IL-6

increase in the peritoneal exudates may indicate a positive

regulatory role for IL-1 on the release of IL-6 induced by

both toxins. IL-6, an important mediator of inflammation

causes leukocytosis characterized by a rapid neutrophilia by

releasing of PMN leukocytes from the bone marrow

(Suwa et al., 2000; Snick, 1990). In addition, IL-6

up-regulates ICAM-1 expression by endothelial cells but

decreases the levels of L-selectin on circulating PMN

leukocytes contributing for firm adhesion, the next step of

cell migration (Suwa et al., 2002). Therefore, this cytokine

may have a role in the formation of cellular infiltrate in our

experimental model. In accordance with our results,

increases in serum levels of IL-6 have been described in

mice injected with MT-II (Lomonte et al., 1993) and with

the venom of B. asper (Petricevich et al., 2000). TNF-a is

also likely to be involved in leukocyte infiltration induced

by MT-II and MT-III, since these PLA2s caused a significant

increase of TNF-a levels in the peritoneal exudate. TNF-ainduces the expression of E-selectin, CD11b/CD18 and

ICAM-1 and triggers the release of several cytokines such as

IL-1 and IL-6 and eicosanoids. Thus, our results suggest that

TNF-a may have a role in the expression of CD18 and the

release of other cytokines following MT-II and MT-III

injection, thereby being relevant for neutrophil influx and

for increase of VP. It is interesting that TNF-a and IL-6, as

well as IL-1, may induce or potentiate the expression and

release of group IIA PLA2 (Crowl et al., 1991; Schalwijk

et al., 1991). Therefore, PLA2 and proinflammatory

cytokines can cooperate to promote inflammation by

enhancing each other’s secretion. The cellular sources of

eicosanoids and cytokines were not addressed in this study,

although it is suggested that peritoneal macrophages and

mast cells are likely to be involved. The effects of both

PLA2s on these cells are currently being investigated.

In conclusion, both Asp-49 and Lys-49 myotoxic PLA2s

induce a marked inflammatory reaction in the mouse

peritoneal cavity. Since basic myotoxic PLA2s comprise

approximately 30% of B. asper venom (Gutierrez and

Lomonte, 1995), these toxins must play a relevant role in the

proinflammatory activity that characterizes this venom. The

fact that MT-III elicited a stronger reaction than MT-II

argues in favor of a role of enzymatic phospholipid

hydrolysis in this phenomenon, either through the direct

release of arachidonic acid from plasma membranes or

through activation of intracellular processes in target cells,

probably in peritoneal macrophages. On the other hand, the

observation that enzymatically inactive MT-II also elicits

inflammatory events clearly indicates that molecular regions

distinct from the catalytic site in Lys-49 PLA2s can interact

with cellular membranes to induce cellular activation

leading to an inflammatory reaction, which is cytokine-

dependent. Interestingly, a PLA2-like protein devoid of

enzymatic activity, due a mutation in active site His, was

described in mouse and human tissues (Roault et al., 2003).

In addition, a PLA2-like protein, named otoconin-90, has

been cloned. This protein has two internal domains

homologous to PLA2, but lack key residues at the

Ca2C-binding loop and the active site, being probably

devoid of enzymatic activity (Wang et al., 1998). Therefore,

catalytically inactive PLA2 homologues are present in

mammalian tissues and may play relevant pharmacological

roles. Our present observations indicate that it would be

important to search for catalytically inactive PLA2s

homologues in inflammatory exudates, since they may be

present and might play a role in physiological and

pathological processes, as has been demonstrated for

catalytically inactive PLA2s homologues from snake

venoms.

Acknowledgements

The authors thank Dr Bruno Lomonte for his collabor-

ation in the isolation of myotoxins and to Maria Zelma da

Silva for technical assistance. This project was supported

by grants from FAPESP-Brazil (Grants 98/00162-9;

98/15657-3), CNPq (301199/91-4) and Vicerrectorıa de

Investigacion, Universidad de Costa Rica.

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