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doi:10.1016/j.tox

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(K. Conceic- ao).

Toxicon 49 (2007) 625–633

www.elsevier.com/locate/toxicon

Unusual profile of leukocyte recruitment in mice induced by askin secretion of the tree frog Phyllomedusa hypochondrialis

Katia Conceic- aoa,�, Fernanda Miriane Brunia,b, Alessandra Pareja-Santosa,b,Marta M. Antoniazzic, Carlos Jaredc, Monica Lopes-Ferreiraa,b,

Carla Limaa,b, Daniel C. Pimentaa

aLETA (Laboratorio Especial de Toxinologia Aplicada) Center for Applied Toxinology (CAT/CEPID),

Butantan Institute, Sao Paulo, SP, BrazilbLaboratory of Immunopathology, Butantan Institute, Sao Paulo, SP, BrazilcDepartment of Celullar Biology, Butantan Institute, Sao Paulo, SP, Brazil

Received 16 June 2006; received in revised form 24 October 2006; accepted 25 October 2006

Available online 29 October 2006

Abstract

Phyllomedusa hypochondrialis skin secretion can cause both systemic and local effects. In this study, we describe the

pattern of local acute inflammatory response after P. hypochondrialis skin secretion injection. The inflammatory reaction in

the mice footpad was analysed, including the leukocyte recruitment into local tissue from the peripheral blood, in a mouse

model of tissue injury. We also investigated the release of the cytokines IL-1, IL-6 and TNF-a, chemokines KC and MCP-1

and the eicosanoids LTB 4 and PGE2 in mice. The present findings support the ability of P. hypochondrialis skin secretion

to induce local inflammation. In addition, these skin secretion components play a role in the initial rolling and slowing of

recruited leukocytes and the transition from rolling to adhesion. Levels of the proinflammatory cytokine IL-6, chemokines

KC and MCP-1 as well as the eicosanoid PGE2 were significantly increased after injection of a skin secretion of P.

hypochondrialis (0.6 mg/30ml intraplantar), whereas no changes in other parameters were observed. Finally, the mechanisms

involved in the local inflammatory process induced by P. hypochondrialis skin secretion is one of the questions of relevance

related to the complex pathophysiology induced by this particular secretion and other toxins.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Phyllomedusa hypochondrialis; Amphibia; Anura; Skin secretion; Effect local; Inflammation

1. Introduction

Amphibian skin is a morphologically, biochemi-cally and physiologically complex organ whichfulfills a wide range of functions necessary for

front matter r 2006 Elsevier Ltd. All rights reserved

icon.2006.10.009

ng author. Tel.: +5511 3726 7222x2042;

1 6605.

ss: conceicaokatia@butantan.gov.br

amphibian survival, including respiration, waterregulation, anti-predator, antimicrobial defense,excretion, temperature control, and others (Clarke,1997). The skin of most amphibians contains severaltypes of secretory glands, either mucus or granular,distributed throughout the body region. Mucousglands provide the moist coating necessary forcutaneous respiration, whereas granular glandssynthesise and expel a variety of biologically activepeptides/molecules (Erspamer and Melchiorri,1983;

.

ARTICLE IN PRESSK. Conceic- ao et al. / Toxicon 49 (2007) 625–633626

Bevins and Zasloff, 1990). These compounds arethought to play various roles, either in the regula-tion of physiological functions of the skin or indefense against predators or microorganisms(Barthalmus, 1994; Barra and Simmaco, 1995). Inparticular, numerous antimicrobial peptides havebeen found in the skin secretions of many amphi-bian species that are responsible for the innateimmune defense, and most of them havea molecular weight less than �5 kDa (Simmacoet al., 1998; Apponyi et al., 2004; Bevier et al., 2004;Conlon et al., 2004a, b; King et al., 2005; Conceicaoet al., unpublished data).

The granular secretions of the subfamily ofPhyllomedusinae are known to contain largenumbers of pharmacologically active peptides, suchas sauvagine and dermorphin from Phyllomedusa

sauvagei (Erspamer et al., 1985; Montecucchi et al.,1981), and tryptophillin from Phyllomedusa rohdei

(Montecucchi, 1985). A recent study with P.

hypochondrialis crude skin secretion have beencarried out in our laboratory. Ours results showthat the skin secretion is composed of toxins ofproteinaceous nature with significant levels ofphospholipase A2 (PLA2) and creatine kinase(CK) activity. In mice, systemic effects wereobserved as erection of the hair, tachycardia andslobbering. The onset of local effects as such aspain, edema and necrosis is very rapid, and a reportof necrosis 48 h was observed (Conceic- ao et al.,unpublished data). Preliminary experimental obser-vations in mice indicate that P. hypochondrialis

crude skin secretion induces myofibrilar hypercon-traction of skeletal muscle cells with histologicalfeatures distinct from those characterising myone-crosis caused by myotoxins isolated from snakevenoms visualised by intravital microscopy.

In the view of these facts, the present study wascarried out in order to describe the pattern of localacute inflammatory response after P. hypochondrialis

skin secretion injection, including the leukocyte recruit-ment into local tissue from the peripheral blood, in amice model of tissue injury. A better understanding ofthe inflammatory events could lead us to reveal the roleof the proteins found in this skin secretion.

2. Materials and methods

2.1. Animals

Seven to eight week-old male Swiss mice (n ¼ 6)obtained from a colony at Butantan Institute (Sao

Paulo, Brazil) were maintained at the animal housefacilities of the Laboratory of Immunopathology,under specific pathogen-free conditions. All theprocedures involving mice were in accordance withthe guidelines provided by the Brazilian College ofAnimal Experimentation.

2.2. Skin secretions

Specimens (n ¼ 12) of Phyllomedusa hypochon-

drialis of both sexes were collected from Angicos inthe state of Rio Grande do Norte, Brazil. The treefrogs were kept alive in the biotherium of theDepartment of Cellular Biology of Butantan In-stitute, Sao Paulo, Brazil. Glandular secretions ofthe frogs were obtained from adult specimens, thatwere submerged in a beaker containing deionisedwater and manually and gently compressed. Thesesolutions were frozen, lyophilised prior to analysisand stored at �20 1C. Protein content was deter-mined by the method of Bradford (1976) usingbovine serum albumin (Sigma Chemical Co., StLouis, MO) as a standard protein.

2.3. Estimation of edema-inducing activity

Edematogenic activity of the skin secretion wasassayed according to the methods of Lima et al.(2003). Samples of 30 ml containing 0.6 mg of proteinskin secretion were injected by intraplantar injection(i.pl.) in the right footpad of mice. Local edema wasquantified by measuring the thickness of injectedpaws with a paquimeter (Mytutoyo Sul Americana,SP, Brazil) at 2 and 24 h after injection. Miceinjected with 30 ml of sterile PBS acted as thecontrol-group. The results were expressed by thedifference between experimental and control foot-pad thickness. Each data point represents mean7SEM of three independent experiments.

2.4. Microcirculatory alterations

The dynamic of alterations in the microcircula-tory network were determined using intravitalmicroscopy by transillumination of mice cremastermuscle after topical application of the skin secretion(0.6 mg of protein dissolved in 20 ml of sterile saline).Administration of the same amount of sterile salinewas used as control. In three independent experi-ments (n ¼ 4) mice were anaesthetised with pento-barbital sodium (Hypnols Cristalia; 50mg/kg,intraperitoneal route) and the cremaster muscle

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was exposed for microscopic examination in situ asdescribed by Baez (1973) and Lomonte et al. (1994).The animals were maintained on a special boardthermostatically controlled at 37 1C, which includeda transparent platform on which the tissue to betransilluminated was placed. After the stabilisationof the microcirculator, the number of roller cells andadherent leukocytes in the postcapillary venuleswere counted 10min after skin secretion injection.In some experiments, paired arterioles with dia-meters of 15–25 mm and lengths 4150 mm were seenclose to the venule in the field of view. Arteriolardiameter and velocity were also measured in theseexperiments as described for the venules. Thenumber of adherent leukocytes was determinedoff-line during playback of videotaped images. Aleukocyte was considered adherent to venularendothelium if it remained stationary for 30 s orlonger. Adherent leukocytes were quantified as thenumber per 100-mm length of venule. A rollingleukocyte was defined as a white cell that movedslower than the stream of flowing erythrocytes. Thenumber of rolling leukocytes was quantified as thenumber of white cells that passed a fixed point(25 mm window) on the television monitor (Shige-matsu et al., 1999). The study of the microvascularsystem of the transilluminated tissue was accom-plished with an optical microscope (Axiolab, Carl-Zeiss, Oberkochen, DE) coupled to a photographiccamera (Coolpix 990-Nikon) using an � 10/025longitudinal distance objective/numeric apertureand 1.6 optovar.

2.5. Induction of a local inflammatory reaction

The induction of an inflammatory reaction by theskin secretion of P. hypochondrialis was assayedaccording to the methods of Lima et al. (2003).Samples of 0.6 mg of protein in 30 ml of PBS wasinjected in the intraplantar region of the right hindfootpad. Animals injected with 30 ml PBS wereconsidered as a control group. Two or 24 h afterinjection, animals were sacrificed and the right pawswere amputated, the tissue was disrupted withscissors and homogenised with a glass piston in200 ml of PBS to reach a 1ml of cell suspension(Lima et al., 2003).

2.6. Cell harvesting and counting

Leukocyte migration was assessed 2 or 24 h afterskin secretion of P. hypochondrialis or PBS admin-

istration in the footpad. The samples were immedi-ately centrifuged at 3000 rpm, at 4 1C, for 20min.The supernatants were stored at �20 1C for futuredeterminations. The cell pellets were resuspended in1ml of PBS+0.1% BSA for cell counts. Total cellcounts were performed in a hemocytometer anddifferential leukocyte counts in cytocentrifuge pre-parations stained with Wright–Giemsa. Cells weredifferentially counted by microscopy, evaluating300 cells per slide. The results represent themean7SEM per millilitre of cell suspension ofthree independent experiments.

2.7. Quantification of cytokines and chemokines

Cytokines and chemokines were measured in thesupernatant of the footpad cell suspension by aspecific two-site sandwich ELISA, using the OpteIAfor IL-1b (Interleukin-1 beta), TNF-a (Tumornecrosis factor-alpha), and IL-6 (Interleukin–6),KC (Chemokine family with homology to humanIL-8), and MCP-1 (Monocyte chemoattractantprotein-1) according to the manufacturer’s instruc-tions (B&D Pharmingen, Oxford, UK). Binding ofbiotinylated monoclonal antibodies was detectedusing streptavidin-biotinylated horseradish perox-idase complex and 3,30,5,50-tetramethylbenzidine(B&D Pharmingen, Oxford, UK). Samples werequantified by comparison with standard curves ofrecombinant mice cytokines and chemokines. Theresults were expressed as the arithmetic mean7SEM for triplicate samples.

2.8. Eicosanoid assays

Concentrations of LTB4 (Leukotriene B4) andPGE2 (Prostaglandin E2) were measured in thehomogenate of tissue footpad at 2 h after i.pl.injections of skin secretions of P. hypochondrialis orsterile PBS, by a specific enzymatic immunoassay(EIA) using a commercial kit (Cayman Chemicals,MI, USA). The absorbances of the samples wererecorded at 412 nm in a microplate reader, andconcentrations of the eicosanoids were estimatedfrom standard curves.

2.9. Statistical analysis

A one way analysis of variance (ANOVA)followed by a Dunnett’s test was used to determinethe levels of difference between all groups. Differ-ences were considered statistically significant at

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po0.05. The SPSS statistical package (Release 8.0,Standard Version, 1997) was employed.

3. Results

3.1. Effect of skin secretion of P. hypochondrialis on

mice footpad edema

A dose of 0.6 mg/footpad was used to determinethe edematogenic response induced by P. hypochon-

drialis skin secretion. Fig. 1 shows that intraplantarinjection of 0.6 mg induced a significant edema atboth 2 and 24 h post-injection.

3.2. Leukocyte-endothelial interactions

We analysed the dynamic of alterations in themicrocirculatory network using intravital micro-scopy by transillumination of mice cremaster muscleafter topical application of sample (0.6 mg of proteindissolved in 20 ml of sterile saline). The contributionof skin secretion to leukocyte interactions wasexamined by determining the number of rollingand adhered cells in postcapillary venules. Tenminutes following skin secretion application therolling of leukocyte increased four-fold in animals

*

0

0.5

1.0

1.5

2.0

2.5

Ede

ma (

mm

)

Control

2 h 24 h

P. hypo Control P. hypo

*

Fig. 1. Estimation of edema-inducing activity. Samples of 30 mlcontaining 0.6mg of protein of P.hypochondrialis skin secretion

were injected (i.pl.) in the right footpad of mice. Local edema was

quantified at 2 and 24 h after injection. The control group

received an equal volume of sterile PBS were considered as

control-group. The results were expressed by the difference

between experimental and control footpad thickness. Each point

represents mean 7SEM. *po0.05 compared with control-group

of three independent experiments.

and remained elevated throughout the experimentalperiod (Fig. 2). Topical application of skin secretioninduced a large increase in the number of adherentleukocytes, reaching a maximum seven-fold increaseover control-animals at 20min post-application(Fig. 3). No changes in rolling leukocyte velocityor numbers of adhered cells were seen in controlanimals receiving PBS.

3.3. Modulation of leukocyte migration

Leukocyte recruitment to the site of injury afterP. hypochondrialis skin secretion injection wasevaluated in mice. Fig. 4 shows the increment incell recruitment into footpad tissue of the group ofanimals receiving the skin secretion at 2 h, char-acterized by the higher recruitment of macrophages.Therefore the skin secretion did not induce sig-nificant leukocyte influx in 24 h, but the number ofneutrophils was significant (approximately12.5� 105 neutrophils) in this period.

3.4. Release of acute phase cytokines and eicosanoids

In the view of the local (intraplantar) relationshipbetween vessel permeability and protein extravasa-tion, footpad homogenates were tested for thepresence of TNF-a, IL-1b and IL-6 cytokines asshown in Fig. 5. Only significantly increased levelsof IL-6 compared with control-group were observedat 2 h, returning to the normal levels in 24 h. Asshown in Fig. 6, elevated levels of KC and MCP-1were observed only in 2 h. Interestingly, the skinsecretion of P. hypochondrialis provoked a libera-tion of PGE2 in supernatant of footpad. Nosignificantly raised levels of LCB4 were detected(Fig. 7).

4. Discussion

In this work we describe the pattern of local acuteinflammatory response after P. hypochondrialis skinsecretion injection, including the leukocyte recruit-ment into local tissue from the peripheral blood in amice model of tissue injury.

In models of inflammation induced by thevenoms of various species, protein extravasation,edema formation and leukocyte recruitment havealso been observed (Farsky et al., 1997; Costa et al.,1997; Lima et al., 2003). We have also shown thatthe skin secretion of P. hypochondrialis is a potentedematogenic factor. This acute increase in plasma

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10

20

30

40

50

Num

ber

of R

olli

ng L

eukocyte

s

(per

min

)

Time (min)

Baseline 10 20 30

*

*

*

Ten minutes after topical application of the skin secretion of P. hypochondrialis

0

Fig. 2. Number of leukocytes rolling. Rolling leukocytes in postcapillary venules of the mice cremaster muscle after topical application of

P. hypochondrialis skin secretion (0.6 mg of protein dissolved in 20 ml of sterile saline) or sterile saline (20ml, control). Determinations were

performed 10, 20 and 30min after skin secretion and values averaged of three independent experiments. Results were obtained in recorded

images on optical microscope (Axiolab, Carl-Zeiss) coupled to a photographic camera (Coolpix 990-Nikon) using an � 10/025

longitudinal distance objective/numeric aperture and 1.6 optovar.

0

5

10

15

20

Adhere

nt Leukocyte

s

(per

100 μ

m)

Time (min)

Baseline 10 20 30

*

*

*

Twenty minutes after topical application of the skin secretion of P. hypochondrialis

Fig. 3. Adhered leukocytes in postcapillary venules of cremaster muscle after topical application of skin secretion. Adhered leukocytes was

observed after application of P. hypochondrialis skin secretion (0.6mg of protein dissolved in 20 ml of sterile saline) or sterile saline (20ml,control). Determinations were performed 10, 20 and 30min after skin secretion injection and values averaged of three independent

experiments. Results were obtained in recorded images on optical microscope (Axiolab, Carl-Zeiss) coupled to a photographic camera

(Coolpix 990-Nikon) using an � 10/025 longitudinal distance objective/numeric aperture and 1.6 optovar.

K. Conceic- ao et al. / Toxicon 49 (2007) 625–633 629

extravasation after skin secretion injection suggestsearly release of edematogenic mediators. In agree-ment with this we also observed the liberation ofprostaglandin E2 in tissue injured by skin secretionsof P. hypochondrialis. This mediator, a potentvasodilator, is enhanced in inflammatory reactions,causing edema, erythema and pain (Higgs et al.,1984; Funk 2001).

Prostaglandin E2 (PGE2), an arachidonic acid(AA) metabolite generated by the cyclooxygenase

enzymes COX-1 and COX-2 in macrophages,fibroblasts, and mast cells, is recognised as a potent,proinflammatory mediator, which exerts its biolo-gical activities by interaction with one or acombination of subtype receptors designated EP1,EP2, EP3, and EP4, variously induced by proin-flammatory signals (Coleman et al., 1994). In someinflammatory conditions, a direct relationship isdetected between inflammatory disease activity andlevels of PGE2 in tissues. COX-2 inhibitors, which

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0

50

100

150

*

Tota

l celln

um

ber

(x 1

05)

50

0

25

Neutr

ophils

(x 1

05)

75

0

25

50

*

Macro

phages (

x 1

05)

Control

2 h

P. hypo Control

24 h

P. hypo

*

Fig. 4. Effect of P. hypochondrialis skin secretion on leukocyte

recruitment. Samples of 0.6 mg of protein in 30 ml of PBS was

injected in the intraplantar region of the right hind footpad.

Animals injected with 30 ml PBS were considered as control-

group. Two or 24 h after injection, animals were sacrificed and

the right paws were amputated, the tissue was processed for cell

count. The results represent the mean7SEM. po0.05 of three

independent experiments compared with control-group.

0

500

1000

1500

IL6 (

ng/m

L)

*

0

100

200

300

IL-1

β (p

g/m

L)

0

250

500

750

TN

F -

α (ρ

g/m

L)

Control

2 h 24 h

P. hypo Control P. hypo

Fig. 5. Quantification of cytokines in homogenates of footpad

from mice injected with P. hypochondrilais skin secretion.

Samples (0.6mg protein in 30 ml of PBS) was injected in the

intraplantar region of the right hind footpad (skin secretion-

group). Mice only injected with PBS were considered as control-

group. After 2 and 24 h, animals were sacrificed and the right

paws were amputated, and homogenised for ELISA cytokine

determinations. Each bar represents mean7SEM. po0.05 for

triplicate samples compared with control-group.

K. Conceic- ao et al. / Toxicon 49 (2007) 625–633630

can reduce PGE2 production, are effective atreducing inflammation and inflammatory diseaseprogression (Osiri and Moreland, 1999).

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0

1000

2000

KC

(ρg

/mL)

0

250

500

750

MC

P -

1 (

ρg/m

L)

Control

2 h

Control P. hypoP. hypo

*

*

24 h

Fig. 6. Quantification of chemokines in homogenates of footpad

from mice injected with P. hypochondrilais skin secretion.

Samples (0.6mg protein in 30ml of PBS) was injected in the

intraplantar region of the right hind footpad (skin secretion-

group). Mice only injected with PBS were considered as control-

group. After 2 and 24 h, animals were sacrificed and the right

paws were amputated, and homogenised for ELISA chemokine

determinations. Each bar represents mean7SEM. po0.05 for

triplicate samples compared with control-group.

Control P. hypochondrialis

*

0

250

500

750

PG

E2 (n

g/m

L)

0

50

100

150

200

LC

T B

4 (

ng

/mL

)

Control P. hypochondrialis

Fig. 7. Quantification of eicosanoid in homogenates of footpad

from mice injected with P. hypochondrialis skin secretion.

Concentrations of LTB4 and PGE2 were measured in the

homogenate of tisuue footpad at 2 h after injections of skin

secretion of P. hypochondrialis or sterile PBS, by a specific

enzymatic immunoassay (EIA). The absorbances of the samples

were recorded at 412 nm in a microplate reader, and concentra-

tions of the eicosanoids were estimated from standard curves.

Each bar represents mean7SEM. po0.05 of three independent

experiments compared with control-group.

K. Conceic- ao et al. / Toxicon 49 (2007) 625–633 631

The early control of local macrophage migrationfollowed by PMN migration could, in this modelalso be regulated by mast cells through theliberation of histamine. Mast cell-derived histaminehas been shown to up-regulate endothelial cellP-selectin and blocking either histamine or P-selectin after mast cell activation inhibited endothe-lial leukocyte capture and rolling (Gaboury et al.,1995). Then the edema induced by the skinsecretions of P. hypochondrialis can be reduced bythe treatment of mice with COX or histamineinhibitors.

Increase leukocyte adhesion is a marker of aninflamed and dysfuncional endothelium (Ulbrichet al., 2003). The current study showed that skinsecretions of P. hypochondrialis induce increasedleukocyte rolling followed by a gradual increase infirmly adherent cells to the endothelium in intravitalexperiments. The selectin family of adhesion mole-cules is associated with the initial phase of leukocyterecruitment characterised by leukocyte rolling (Ley,1996). P-selectin is more critical in the initial rollingand slowing of recruited leukocytes (Robinsonet al., 1999), whereas E-selectin is more important

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in leukocyte arrest or the transition from slowrolling to firm adhesion, as postulated by Smith etal. (2004). In addition, -1 and -2 integrin adhesionmolecules (VCAM-1, ICAM-1) are critical for firmattachment of activated leukocytes in the recruit-ment cascade (Ley, 1996). Our result support thehypothesis that in the skin secretions of P.

hypochondrialis are present components with activ-ity in endothelium, up-regulating the expressionof import adhesion molecules for leukocyte re-cruitment.

Neutrophils are recruited rapidly into sites ofacute infection and dominate the initial influx ofleukocytes (Issekutz and Movat, 1980). Later ininflammation, leukocytes of the monocyte/macro-phage lineage replace the neutrophil as thepredominant leukocyte, suggesting a bimodal re-cruitment pattern involving a switch from neutro-phils to monocytes. Recruited neutrophils arethought to mediate this switch by releasing solublefactors into the early inflammatory site that initiatemonocyte recruitment (Ryan and Majno, 1977;Doherty et al., 1988). The macrophage plays acentral role in the inflammatory response, releasingcytokines that control key events in the initiation,resolution, and repair processes of inflammation(reviewed in Adams and Hamilton, 1992). Ingeneral, the major chemokine responsible forrecruiting monocytes to inflammatory sites is thechemokine MCP-1, and neutrophils are instrumen-tal in mediating a chemokine switch promotingmonocyte chemoattraction (Kaplanski et al., 2003).Interestingly, we showed here an unusual profile ofleukocyte recruitment with a predominance ofmacrophages followed by neutrophilic influx intofootpads of mice, and elevated levels of MCP-1 andKC protein at 2 h. These results corroborate theview that elicited neutrophils are not the only cellresponsible for stimulating the MCP-1 synthesis orrelease, and resident macrophages and also me-sothelial cells contribute to the MCP-1 levelsproviding an immediate source of MCP-1 forrecruitment of monocytes from the circulation(Henderson et al., 2003).

It is interesting that recent studies aboutPGE2 modulation of MCP-1 activities have de-scribed a more vigorous monocyte chemotacticresponse to MCP-1 in the presence of PGE2 (Panzerand Uguccioni, 2004), suggesting that PGE2 mayexert additional stimulatory activities on mono-cytes. The inflammation induced by skin secretionsof P. hypochondrialis was characterised by the

presence of PGE2 and MCP-1 in the footpads ofmice.

Finally, we detected high levels of IL-6 in thefootpad injured by P. hypochondrialis skin secretion.IL-6 is a pleiotropic cytokine that is commonlyproduced at local tissue sites and released into thecirculation in almost all situations of homeostaticperturbation typically including endotoxemia, en-dotoxic lung, trauma, and acute inflammation(Kishimoto et al., 1992; Zitnik and Elias, 1993).Circulating IL-6, together with other alarm cyto-kines such as TNF-a and IL-1, is known to berequired for the induction of acute phase reactionscomposed of fever, corticorsterone release, andhepatic production of acute phase proteins (Bau-mann and Gauldie 1994; Xing et al., 1995, 1998).

In conclusion, we have demonstrated that themurine chemokines, MCP-1 and KC protein andthe cytokine IL-6 are expressed in footpad afterinjection of crude skin secretion of P. hypochon-

drialis; that these mediators induce local macro-phage followed by PMN migration; and that theearly control of leukocyte migration could, in thismodel be regulated by mast cells through theliberation of PGE2 and histamine. And finally, wecan suggest the MCP-1 is produced in situ by tissueresident macrophages and or mesothelial cellsindependently of a simultaneous infiltration ofneutrophils. Current work is in progress in ourlaboratory targeting the biochemical and molecularcharacterisation of several toxins present in the skinsecretion of P. hypochondrialis.

Acknowledgements

We wish to thank the Laboratorio de Fisiopato-logia of Instituto Butantan for the use of theinstrument for intravital microscopy. The tree frogswere collected according to the Brazilian Environ-mental Agency (IBAMA-Instituto Brasileiro do MeioAmbiente e dos Recursos Naturais Renovaveis)under the license no. 02027.023238/03-91. Supportedby funds provided by FAPESP, CAT/CEPID,CAPES and CNPq (Grant #303516/2005-4).

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