Delayed local inflammatory response induced by Thalassophryne nattereri venom is related to...

ORIG INAL ART ICLE

Delayed local inflammatory response induced by Thalassophrynenattereri venom is related to extracellular matrix degradation

Alessandra Pareja-Santos*, Tania Cristina Saraiva*, Erica Pereira Costa�, Marinilce Fagundes Santos�,

Telma Tenorio Zorn�, Valdenia Maria Oliveira Souza�, Monica Lopes-Ferreira* and Carla Lima*

*Special Laboratory of Applied Toxinology (CAT ⁄ CEPID), Butantan Institute, Sao Paulo;�Department of Developmental and Cell

Biology, Institute of Biomedical Sciences, University of Sao Paulo;�Laboratory of Immunopathology Keizo Asami, Federal

University of Pernambuco, Recife; Brazil

Thalassophryne nattereri fish envenomation is commonly

reported among fishermen and bathers in the Brazilian

north and north-east coast, estimated at hundreds of acci-

dents per year (Haddad et al. 2003). On Ceara State sea-

side, 16 cases of envenomation by T. nattereri were

notified, from 1992 to 2002 (Faco et al. 2005). According

INTERNATIONAL

JOURNAL OF

EXPERIMENTAL

PATHOLOGY

Received for publication:

29 January 2008

Accepted for publication:

15 June 2008

Correspondence:

Dr Carla Lima

Special Laboratory of Applied

Toxinology (CAT ⁄ CEPID)

Butantan Institute

Av. Vital Brazil, 1500, Butantan

05503-900 Sao Paulo

Brazil

Tel. ⁄ Fax: +55 11 3676 1392

E-mail: [email protected]

Summary

Symptoms evoked by Thalassophryne nattereri fish envenomation include local

oedema, severe pain and intense necrosis with strikingly inefficient healing, contin-

uing for several weeks or months. Investigations carried out in our laboratory

showed that, in the venom-induced acute inflammation, thrombosis in venules and

constrictions in arterioles were highly visible, in contrast to a notable lack of inflam-

matory cell. Nevertheless, the reason that the venom toxins favour delayed local

inflammatory response is poorly defined. In this study, we analysed the movement of

leucocytes after T. nattereri venom injection in the intraplantar region of Swiss mice,

the production of pro-inflammatory mediators and the venom potential to elicit

matrix metalloproteinase production and extracellular matrix degradation. Total

absence of mononuclear and neutrophil influx was observed until 14 days, but the

venom stimulates pro-inflammatory mediator secretion. Matrix metalloproteinases

(MMP)-2 and MMP-9 were detected in greater quantities, accompanied by tissue

degradation of collagenous fibre. An influx of mononuclear cells was noted very late

and at this time the levels of IL-6, IL-1b and MMP-2 remained high. Additionally,

the action of venom on the cytoskeletal organization was assessed in vitro. Swift

F-actin disruption and subsequent loss of focal adhesion was noted. Collectively

these findings show that the altered specific interaction cell-matrix during the

inflammatory process creates an inadequate environment for infiltration of inflam-

matory cells.

Keywords

cytoskeleton, extracellular matrix, fish venom, metalloproteinase, Thalassophryne

nattereri

Int. J. Exp. Path. (2009), 90, 34–43

doi: 10.1111/j.1365-2613.2008.00603.x

34� 2009 The Authors

Journal compilation � 2009 Blackwell Publishing Ltd

to Fonseca and Lopes-Ferreira (2000), the palm of the

hands or soles of the feet are the areas most commonly

affected in the victims when pierced by spines connected

to the fish’s venom glands. Envenoming symptoms are

readily evident, including local oedema, erythema and

severe pain followed by intense necrosis and a markedly

inefficient healing response. This problem of the inefficient

healing is very important for the evolution and treatment

of the accident, which is devoid of specific drug treatment

(Lopes-Ferreira et al., 2000; Haddad et al. 2003). Accord-

ing to this, the symptoms advance, taking weeks or even

months for complete recovery (Fonseca & Lopes-Ferreira

2000).

Investigations carried out in our laboratory have shown

that the dramatic symptoms of T. nattereri envenomation

are related to kallikrein–kinin cascade, identified as the

major mechanism involved in the nociception as well as

the oedematous response to venom, as only the

administration of novel tissue kallikrein inhibitor

(phenylacetyl-Phe-Ser-Arg-N-(2,4-dinitrophenyl)-ethylenedia-

mine – TKI), and not PKSI-527 and SBTI, specific and

non-specific plasma kallikrein inhibitors, respectively,

reduced these clinical manifestations (Lopes-Ferreira et al.

2004).

Thalassophryne nattereri induces direct damage to skele-

tal muscle plasma membrane together with thrombosis

and other alterations in the microvasculature of mice,

without inducing haemorrhage (Lopes-Ferreira et al. 1998,

2001). Analyses of the cremaster muscle showed that the

venom elicits a peculiar alteration of the microcirculation

with intense vascular congestion, thrombosis in venules

and focal transient constrictions in arterioles (Lopes-

Ferreira et al. 2002). In contrast to other models of

myonecrosis, in which the microvasculature is not affected,

phagocytosis of necrotic material is well advanced by 24 h

and no remnants of necrotic muscle cells are observed

after 1 week (Harris et al., 1975; Gutierrez et al., 1991;

Morini et al., 1998. Curiously, low numbers of phagocytic

cells during the first 24 h after) T. nattereri injection, and

the presence of necrotic material which had not been

cleared out 7 days after envenomation was described

(Lopes-Ferreira et al. 2001; Lima et al. 2003). Neverthe-

less, the reason that the venom toxins favour delayed local

inflammatory response is ill defined.

This study was carried out to describe leucocyte

movement into mice tissue after the injection of T. natter-

eri venom in order to investigate the role of inflammatory

mediators, metalloproteinases and cell adhesion to

extracellular matrix (ECM) components present in this

envenomation.

Material and methods

Animals

Male Swiss mice weighing 18–20 g were obtained from a

colony at the Butantan Institute, Sao Paulo, Brazil. Animals

were housed in a laminar flow holding unit (Gelman Sci-

ences, Sydney, Australia) in autoclaved cages on autoclaved

bedding, in an air-conditioned room in a 12-h ⁄ 12-h light ⁄ -dark cycle. Irradiated food and acidified water were pro-

vided ad libitum. All the procedures involving animals were

in accordance with the guidelines provided by the Brazilian

College of Animal Experimentation (276 ⁄ 06).

Venom

Venom was obtained from specimens of adult T. nattereri

fish collected in the Brazilian state of Alagoas. Venom was

collected by pressing the spines at their base, and was either

used immediately or frozen at )80 �C for later usage. Pro-

tein content was determined by using the colorimetric

method of Bradford (1976) using bovine serum albumin

(Sigma Chemicals, St Louis, MO, USA) as standard protein.

Induction of local inflammatory reaction

Leucocyte migration was assessed in the footpad of mice

according to Lima et al. (2003). Thalassophryne nattereri

venom (10 lg of protein in 30 ll of sterile saline) was

injected in the intraplantar region of the right hind footpad

(venom group). Animals injected with 30 ll of sterile saline

were considered as controls. Six, 24 and 48 h or 7, 14, and

21 days after injection, animals were killed and the right

paws were amputated; the tissue was dismembered with scis-

sors and homogenized with a glass piston in phosphate-buf-

fered saline (PBS). Samples were immediately centrifuged at

3000 rpm (1000 g) and 4 �C for 20 min. The supernatants

were stored at )20 �C for cytokine, chemokine and matrix

metalloproteinase (MMP) determination.

Cell harvesting and counting

The cell pellets were resuspended in PBS containing 0.1%

newborn calf serum (Sigma) for cell counts using a haemo-

cytometer and cytocentrifuge (Cytospin II; Shandon,

Cheshire, UK) slides were prepared, air dried, fixed in

methanol, and stained (Hema 3; Scientific Products,

Chicago, IL, USA). For differential cell counts, 300 leuco-

cytes were enumerated and identified as mononuclear cells

or polymorphonuclear neutrophils, on the basis of staining

Local inflammatory response and matrix degradation 35

� 2009 The Authors

Journal compilation � 2009 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 90, 34–43

and morphologic characteristics using a conventional light

microscope (Axio Imager A1; Carl Zeiss, Meditec, Dublin,

CA, USA).

Quantification of cytokines and chemokines

Cytokines and chemokines were measured in footpad tissue

homogenates by a specific two-site sandwich ELISA, using

the OpEIA Kit (BD Pharmingen, San Diego, CA, USA).

Detection limits were 7.8 pg ⁄ ml for IL-1b, IL-6, TNF-a and

MCP-1 or 15.62 pg ⁄ ml for keratinocyte-derived chemokine

or cytokine-induced neutrophil chemoattractant (KC).

Determination of MMP-2 and MMP-9 levels using

ELISA

Matrix metalloproteinase levels in footpad homogenate sam-

ples were analysed using mouse ELISA kits following manu-

facturer’s instructions (R&D Systems, Minneapolis, MN,

USA). Detection limit was 0.75 or 0.65 ng ⁄ ml for MMP-2

and MMP-9 respectively.

Determination of MMPs in footpad after venom injection

using gelatin zymography

Enzymatic activity in footpad homogenates of mice injected

or not with T. nattereri venom was analysed using zymo-

graphy gel containing 1 mg ⁄ ml gelatin, in the presence of

sodium dodecyl sulphate (SDS), under non-reducing condi-

tions as previously described (Hibbs et al. 1985; Kleiner &

Stetler-Stevenson 1994). After electrophoresis, the gels were

stained with Coomassie Brilliant blue and destained using

acetic acid in methanol and H2O (1:3:6), both for 2 h, to

visualize bands with gelatinolytic activity. Gels were then

photographed by means of a digital camera (Nikon Coolpix

S1; Nikon Instruments Inc, Melville, NY, USA). The molec-

ular mass (kDa) of the gelatinases was estimated against

markers of known recombinant proteins (R&D Systems).

Footpad total collagen measurement

Total collagen content was determined using the Sircol colla-

gen assay as previously described (Muro et al. 2008; Saito

et al. 2008). Briefly, frozen right footpad of each animal was

homogenized in 500 ll of radio-immunoprecipitation assay

buffer containing protease inhibitors. Homogenates were

centrifuged at 13,000 g for 10 min at 4 �C. Sirius red

reagent (50 ll) was added to each footpad homogenate

(50 ll) and mixed for 30 min. The collagen–dye complex

was precipitated by centrifugation at 16,000 g for 5 min,

washed with ethanol, and dissolved in 0.5 M NaOH.

Finally, absorbance was measured at 540 nm and compared

with a calibration curve generated using the collagen

standard provided by the manufacturer (Biocolor Ltd,

Carrickfergus, UK).

Tissue staining

Mice were killed and injected footpads were removed and

immediately fixed in 10% buffered formalin. The tissue was

then processed and embedded in paraffin. Five-micrometre

tissue sections were prepared and stained using haematoxy-

lin and eosin (H&E) methods. The stained, paraffin-embed-

ded sections were examined and qualitatively evaluated

using light photomicroscopy for inflammatory and architec-

tural changes to evaluate general morphology. All slides

were examined with light microscopy at a magnification of

·40 (Axio Imager A1; Carl Zeiss) calibrated with a reference

micrometre slide. For each group of six mice, four stained

footpad sections from each mouse were analysed.

Stimulation of epithelial cells with Thalassophryne

nattereri venom

The IEC-6 cells (ATCC # CRL 1592) were cultured in Dul-

becco’s Modified Eagle’s Medium with 5% FBS plus 10 lg

of insulin, 100 units of penicillin, 100 lg of streptomycin

sulphate and 0.29 mg of l-glutamine ⁄ ml (DMEM-FBS)

according to Quaroni et al. (1979). Approximately 2 · 105

cells were plated per 35-mm dish (each containing a glass

cover slip) and incubated with T. nattereri venom at a con-

centration of 5 lg ⁄ ml to the experimental dishes. Cells were

incubated for 1, 5 or 60 min at 37 �C, 5% CO2. Polylysine

(Sigma)-coated slides were used as a control for the effects

of non-specific adhesion.

F-actin staining

After culture, cells were fixed and permeabilized with 2%

formaldehyde plus 0.2% Triton X-100 in PEM buffer

(10 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6.8) for

10 min at RT and postfixed with 95% ethanol for 5 min at

)20 �C. In fibroblasts and most adherent cells, focal adhe-

sion involves interactions between adhesion molecules and

cytoskeletonal proteins, such as actin (Richardson & Parsons

1996). F-actin was detected using rhodamine–phalloidin

staining according to manufacturer’s instructions. Samples

were imaged with a Nikon PCM 2000 Laser Scanning

Confocal microscope (Nikon Instruments Inc, Melville, NY,

USA).

36 A. Pareja-Santos et al.



Statistical analysis

All values were expressed as mean ± SEM. Parametric data

were evaluated using an analysis of variance, followed by the

Bonferroni test. Non-parametric data were assessed using the

Mann–Whitney test. Differences were considered statistically

significant at P < 0.05. The spss statistical package (Release

13.0, Evaluation Version, 2004; SPSS Inc, Chicago, Illinois,

USA) was employed. Experiments were repeated at least

three times.

Results

Impaired early leucocyte migration induced by

Thalassophryne nattereri venom

Rapid accumulation of neutrophils to a site of inflammation

is a defining early event of innate immunity. Thalassophryne

nattereri venom does not induce an increase in neutrophils

in footpad during the first 24 h, but, by contrast, caused a

marked decrease in mononuclear cell recruitment at 48 h

(Figure 1c,d). A drastic reduction in neutrophil number com-

pared with control mice provoked by the venom was

observed at 14 and 21 days after injection (Figure 1b). Sig-

nificant influx of leucocytes to the footpad was only

observed 21 days after venom injection with augmented

number of mononuclear cells (Figure 1a,c,d). Thus, T. nat-

tereri venom causes inappropriate early polymorphonuclear

(PMN) recruitment in the footpad of mice.

Pro-inflammatory mediators elicited by Thalassophryne

nattereri venom

The induction of cytokines and chemokines involved in neu-

trophil recruitment was evaluated in footpad homogenates

of mice. IL-1b was augmented in the paw 48 h and 21 days

after venom administration (Figure 2a). IL-6 and TNF-a

were only observed after 21 days or 48 h, respectively, and

the latter reached baseline levels at 21 days (Figure 2b,c).

MCP-1 and KC, critical mediators of monocyte and neutro-

phil trafficking were detected after 48 h, but were not

detected in footpads of mice after 21 day from venom stimu-

lus (Figure 3).

Thalassophryne nattereri venom modulates MMPs

secretion

It is known that several pro-inflammatory cytokines, such as

TNF-a (Gan et al. 2001; Miyajima et al. 2001), IL-8 (or KC)

and IL-1b (Gan et al. 2001; Gooz et al. 2003), are capable of

inducing MMP secretion. As shown in Figure 4, control mice

constitutively secreted both MMPs, reaching significant levels

after 48 h of venom injection. MMPs returned to baseline

level at day 21. To verify whether T. nattereri induced MMP

activation, a gelatin zymogram was performed using footpad

homogenates. Comparisons with rMMP standards (left lines 1

and 2) indicated that venom induced active forms of MMP-9

and MMP-2 after 48 h. However after 21 days, MMP-9 was

observed in latent form, and latent and intermediary forms of

100

150 (a) (b)

(c) (d)

*

21d 0

50 *

To

tal c

ells

(x

105 )

T. nattereri venom

Cont 6h 24h 48h 7d 14d

50

Neu

tro

ph

ils (

x 10

5 )

0

25

* *

T. nattereri venom

21d Cont 6h 24h 48h 7d 14d

Lym

ph

ocy

tes

(x 1

05 )

20

30

* *

0

10 *

T. nattereri venom

21d Cont 6h 24h 48h 7d 14d

Mac

rop

hag

es (

x 10

5 )

100

150

*

0

50

*

T. nattereri venom

21d Cont 6h 24h 48h 7d 14d

Figure 1 Impaired early leucocyte

migration induced by Thalassophryne

nattereri venom. Several times after an

injection of 10 lg of protein venom,

footpad tissues were processed for total

leucocyte (·105, a), neutrophil (b),

lymphocyte (c) and macrophage

(d) counts. The results represent the

mean ± SEM of six animals ⁄ group.

*P < 0.05 compared with the control

group.




MMP-2 were produced (Figure 5). These observations corre-

late well with MMP determination as measured using an

ELISA.

Breakdown of the collagenous network induced by

Thalassophryne nattereri venom

Control animals showed normal tissue architecture without

leucocyte infiltration (Figure 6a). Forty-eight hours after

venom injection, the footpad of mice demonstrated no infiltra-

tion of leucocytes (Figure 6b). The ECM is a complex struc-

tural entity composed of fibrillar proteins and proteoglycans,

which provide support for surrounding cells within most

mammalian tissues and organs. In Figure 6c, an intense

1000 (a)

(b)

(c)

*

500 *

IL-1

β in

fo

otp

ad (

pg

/ml)

21d 0

T. nattereri venom

Control 48h

IL-6

in f

oo

tpad

(p

g/m

l)

150

50

100

150

*

0

50

T. nattereri venom

21dControl 48h

TN

F-α

in f

oo

tpad

(p

g/m

l)

200

300

*

0

100

T. nattereri venom

21dControl 48h

Figure 2 Analysis of IL-1b, IL-6 and TNF-a levels in footpad of

mice injected with Thalassophryne nattereri venom. Forty-eight

hours or 21 days after venom injection, footpad homogenates

were collected for IL-1b (a), IL-6 (b) and TNF-a (c) determina-

tion using an ELISA. The results represent the mean ± SEM of

six animals per group. *P < 0.05 compared with the control

group.

20

30 (a)

(b)

*

10

KC

in f

oo

tpad

(p

g/m

l)

21d 0

T. nattereri venom

Control 48h

MC

P-1

in f

oo

tpad

(p

g/m

l)

500

*

0

250

T. nattereri venom

21d Control 48h

Figure 3 KC and MCP-1 levels induced by Thalassophryne

nattereri venom. Forty-eight hours and 21 days after venom

injection, footpad homogenates were collected for KC (a) and

MCP-1 (b) determination using an ELISA. The results represent

the mean ± SEM of six animals per group. *P < 0.05 compared

with the control group.




MM

P-9

(n

g/m

l)

2.5

5.0

7.5

*

Control 21d

0.0

2

T. nattereri venom

48h

5.0(a)

(b)

*

0.0

2.5

MM

P-2

(n

g/m

l)

T. nattereri venom

Control 21d48h

Figure 4 MMP-2 and MMP-9 production in footpad after Thal-

assophryne nattereri venom injection. After 48 h and 21 days

after venom injection, footpad homogenates were collected for

MMP-2 (a) and MMP-9 (b) determination using an ELISA. The

results represent the mean ± SEM of six animals per group.

*P < 0.05 compared with the control group.

MMP-2 Cont TnV Cont TnVMMP-9

Latent MMP-2Intermediary MMP-2

Active MMP-2

Latent MMP-9

Active MMP-9

48 21

Figure 5 Thalassophryne nattereri venom modulates MMPs

secretion. After 48 h and 21 days after venom injection, footpad

homogenates were collected for MMPs determination using

gelatin zymography.

50

75

(a)

(b)

*

Control Tnv0

25

Co

llag

en (

µg

/Fo

otp

ad)

(c)

(d)

Figure 6 Pathological analysis and detection of total collagen

content in the footpad of mice injected with venom. H&E

staining of footpad tissue of venom-injected mice shows no

leucocytes in all interstitium (b) and few leucocyte infiltration

in control mice (a). Footpad total collagen content (Sircol assay)

was diminished 48 h or 21 days after Thalassophryne nattereri

venom injection (c).




rupture can be seen in the major ECM protein, such as colla-

gen fibres, accompanied by a significant decrease in the

amounts of total collagen content in the footpad after 48 h

(Figure 6d).

Thalassophryne nattereri venom alters cell adhesion by

acting in cytoskeletal organization

In vitro assays have demonstrated venom cytotoxic effect,

but intracellular events that precede cell death have not

yet been verified. Here, the effects of venom in the

cytoskeletal organization were evaluated using immuno-

fluorescent confocal microscopy. For this, epithelial cells

were cultured with 5 lg ⁄ ml of venom by 1, 5 and

60 min. Epithelial cells without venom stimulation spread

and formed focal adhesions when allowed to attach to

plastic (Figure 7a). Growth of the cells on polylysine-

coated slides (used as a control for the effects of non-spe-

cific adherences) had no effect on cell morphology or actin

polymerization. Within a few minutes, a decrease in F-

actin fibres stress could be noted in these cells, indicating

an impaired actin polymerization (Figure 7b). Accompany-

ing this first alteration, we observed loss of stress fibres

and the formation of lamellipodia and fillopodia

(Figure 7c,e,d).

Discussion

Symptoms evoked by T. nattereri fish venom include local

oedema, erythema, severe pain and intense necrosis with

strikingly inefficient healing extended for weeks or

months. In this study, we demonstrated that T. nattereri

venom alters ECM structure through induction and activa-

tion of MMPs and alters total collagen content during the

healing phase. In addition, the venom acts altering

cytoskeleton organization and pseudopodia formation of

epithelial cells.

Lima et al. (2003) recently reported that T. nattereri

venom induce upregulation of mRNA for IL-1b, IL-6 and

TNF-a genes as well as the release of these soluble cyto-

kines. Moreover, absence of leucocytes infiltration in the

intraplantar region of mice within a few hours after

venom application was also reported. Here, our results

confirm the pro-inflammatory effect of venom and provide

evidence of the induction of a chronic injury, as we

observed only a later recruitment of mononuclear cells

accompanied by the production of cytokines, chemokines

and MMPs. Really, the healing of the injuries caused by

toadfish of the Thalassophryne genus is slow (Sosa-Rosales

et al., 2005), in contrast to the other injuries caused by

venomous fish such as stingrays and catfish. Both venoms

are described to induce a large increase in the number of

rolling and adherent leucocytes in endothelium of the cre-

master muscle of mice (Magalhaes et al. 2006; Junqueira

et al. 2007).

Indeed, a notable activity of MMP-2 and MMP-9 and

an intense degradation of collagen fibres were observed.

Our findings thus support and expand on these earlier

investigations indicating a role for T. nattereri venom in

regulating the inflammatory response not only by

cytokines but also chemoattractants, as well as MMPs

which are upregulated in the footpad upon venom

stimulation.

The MMPs are considered an important enzyme for the

degradation of connective tissues in both physiological and

pathological situations. MMP production is stimulated by

cytokines, growth factors and cell–cell contact. The MMPs

participate in the first phase of the wound-healing process, by

removing devitalized tissue, and are therefore believed to play

an important role in normal wound healing and remodelling

(Yager & Nwomeh 1999). It is possible that during the

inflammatory process induced by T. nattereri venom, host-

derived enzymes, such as MMPs, break down the exposed

collagenous network leading to ECM disorganization,

preventing KC and MCP-1 functional binding and the

formation of chemotactic gradient in anchored components of

(a) (b)

(c) (d)

Figure 7 Effects of Thalassophryne nattereri venom on F-actin

arrangement in migrating epithelial cells. Migrating cells were

treated with 5 lg ⁄ ml venom for different periods of time. (a)

control migrating cells; (b–d) treatment with T. nattereri venom

for 1, 5 and 60 min respectively. Actin polymerization and the

formation of processes, such as filopodia, lamellipodia and

stress fibres, decreased.




the matrix, such as syndecan-1 and proteoglycans respectively

(Parks et al. 2004).

This scenario indicates an ambiguous role of the venom in

the inflammatory process, where it displays a potent stimu-

lus ability in contrast to an inefficient capacity for support-

ing infiltrated cells in inflamed tissue due to MMP

upregulation. Furthermore, we did not rule out the possibil-

ity that enzymatic toxins in the venom can act directly as

degrading factors of ECM. Indeed, previous characterization

of proteins in the T. nattereri venom showed that Natterins

family presented intense kininogenase activity, although not

sharing the molecular motif with MMPs or other already

known protease present in databanks (Magalhaes et al.

2005).

Interestingly, after 21 days when the level of active

MMP-2 was minimal and inactive MMP-9 was present,

mononuclear cell influx was replenished, indicating a par-

tial recovery of the specific cell matrix component and tis-

sue healing. This is, however, unlikely, as neutrophil

numbers are dramatically reduced. The total absence of

TNF-a, a potent signal for neutrophil influx and activation

could explain this phenomenon (Paul & Ruddle 1988;

Flynn et al. 1995). In addition, IL-1b has been associated

with impairment of normal homeostasis of cartilage ECM

through inhibition of proteoglycans and collagen synthesis

(Fernandes et al. 2002). In our model, elevated levels of

IL-1b were detected after venom application remained

high until 3 weeks.

Collectively, these results reinforce the view that the

most efficient therapy for controlling T. nattereri venom

poisoning can be based on neutralizing the toxins and

inducing polymorphonuclear recruitment in the lesion. A

vital role for neutrophil in injury and regenerative process

was recently described by Teixeira et al. (2003) showing

that mice treated by intraperitoneal injections of antisera

to neutrophils and monocytes and injected with snake

venom show a deficient regenerative response suggesting

the importance of neutrophils for normal muscle repair.

Another interesting observation from this study was that

animals depleted of neutrophils and monocytes also

showed more tissue debris in the injured muscles, raising

the possibility at least that the impaired capacity to

remove tissue debris by phagocytes could slow the regen-

erative process.

Inflammatory responses constitute a potential link

between acute injury and chronic remodelling and wound

healing. Some studies clearly demonstrate the essential

function of neutrophils in clearing antigens and toxins for

effective injury resolution (Savill et al. 1989; Teixeira

et al. 2003; Toumi et al. 2006). After entering tissues,

granulocytes promote the switch of arachidonic acid-

derived prostaglandins and leucotrienes to lipoxins, resolv-

ins and protectins which initiate the termination sequence,

leading to neutrophil clearance and release of anti-inflam-

matory and reparative cytokines, such as transforming

growth factor-b1, essential for later initiation of ECM

deposition and remodelling (Serhan & Savill 2005). We

can speculate that for the efficient clearance of the toxins,

polymorphonuclear recruitment would be an essential step

for resolving inflammation and initiation of healing.

The secondary aim of this study was to explore the effect

of venom on focal adhesion. Focal adhesion is now regarded

as an important route of signal transduction in cell growth

and migration (Valitutti et al. 1995), and is influenced by

ECM proteins including collagen, fibronectin and laminin

(Juliano & Haskill 1993). Additionally, disruption of actin

has been associated with MMP-2 production (Sanka et al.

2007) fostering the extreme degradation of ECM. Venom-

induced ECM degradation was associated with a loss of

stress fibres and the formation of lamellipodia and fillopodia

by epithelial cells, which are critically involved in the regula-

tion of cell migration. This well-established in vitro assay

could mimic the early alteration induced by the venom in

endothelial cells previously described (Lopes-Ferreira et al.

2002) and shows that the actin cytoskeleton signals play

important roles in the communication between different cell

types in inflammatory sites.

Collectively these findings show that the altered availabil-

ity of the specific cell matrix component during the early

inflammatory process induced by T. nattereri venom create

an inadequate environment for infiltration or survival of

inflammatory cells, damaging the healing phase and inflam-

mation resolution. Understanding the mechanism used by T.

nattereri venom to reduce the early neutrophilic influx to

injured tissue is essential for establishing an adequate ther-

apy for uncommon inflammatory response observed in

envenomation.

Acknowledgements

This work was supported by the Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo (FAPESP) and CNPq.

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