Leukocyte response induced by Bothrops jararaca crude venom: In vivo and in vitro studies

Pergamon

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LEUKOCYTE RESPONSE INDUCED BY BOTHROPS JARARACA CRUDE VENOM: IN VZJ’O AND IN VZTRO STUDIES SANDRA H. P. FARSKY,’ JOSE WALBER, M. COSTA-CRUZ,YARA CURRY’ and CATARINA F. P. TEIXEIRA’* ‘Laboratory

of Pathophysiology, and ‘Laboratory of Pharmacology, Vital Brazil, 1500, SBo Paulo, Brazil

Institut

Butantan,

A\

(Received 29 March 1996; accepted 24 June 1996)

S. H. P. Farsky, J. W. M. Costa-Cruz, Y. Cury and C. F. P. Teixeira. Leukocyte response induced by Bothrops jararaca crude venom: in viva and in vitro studies. Toxicon 35, 185-193, 1997.-The effect of Bothrops jararaca crude venom (BjV) on the cellular component of inflammatory responses was investigated in vivo and in vitro. In vivo leukocyte accumulation and release of eicosanoids (thromboxane A2, TXA?, and leukotriene B4, LTB,) at the site of injection of the venom were assessed using the air pouch method in rats. Administration of BjV caused a significant cell accumulation, maximal values being obtained after 6-8 hr. Neutrophils were the predominant cell type in the inflammatory exudate. High concentrations of LTB, were detected 14 hr after the injection of the venom. TXA, concentrations were significantly increased only at the early stages of the response to the venom. In z-itro chemotaxis assays were performed and showed that the venom per se was not able to induce oriented neutrophil migration because varying concentrations of the venom dissolved in Hanks’ balanced salt solution (HBSS) evoked a response equivalent to that of HBSS alone. Furthermore, the venom did not affect cellular intrinsic mechanisms involved with neutrophil locomotion because previous incubation of the cells with BjV produced no effect. However. high concentrations of the venom were able to generate serum chemotactic factor(s). Incubation of serum with the venom evoked a neutrophil migration similar to that observed with serum activated by lipopolysaccharide from Escherichia coli. Participation of chemotactic factors derived from the complement system is suggested by data showing loss of this activity when serum was heated (56°C) before the addition of BjV. The present results suggest that leukocyte accumulation in the locality of a lesion induced by BjV is dependent on secretion or activation of endogenous components responsible for several steps in leukocyte recruitment instead of a direct effect of the venom on leukocytes. 0 1997 Elsevier Science Ltd. All rights reserved

*Author

to whom

correspondence

should

be addressed. 185

et al.

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INTRODUCTION

Snakes from the Bothrops genus are responsible for 90% of ophidic accidents in Brazil (Rosenfeld, 1971). Their venoms cause pronounced local effects in humans and animals, with haemorrhage, oedema, pain and necrosis (Rosenfeld, 1971; Gutierrez et al., 1984). Such effects are related to the combined action of proteases, haemorrhagic factors and phospholipases present in the venoms, as well as to the release of endogenous mediators generated by the venoms (Rothischild and Rothischild, 1979; Gutierrez and Lomonte, 1989). Among these endogenous substances, products of arachidonic acid metabolism and platelet activating factor (PAF) are involved in the oedematogenic and hyperalgesic responses induced by Bothrops juraraca venom (BjV) (Trebien and Calixto, 1989; Teixeira et al., 1994). In addition to oedema, there is a considerable local inflammatory cell response associated with bothropic envenomation as described for B. asper, B. erythromelas and B. alternatus venoms (Gutierrez et al., 1986; Flores et al., 1993). Leukocytes are relevant factors in inflammation. Any stage of the inflammatory reaction is characterized by a progressive infiltration of perivascular tissues by different subsets of leukocytes. Granulocytes migrate into the affected tissues from the early stages of the response. The recruitment of neutrophils is dependent on the generation of chemotactic factors as well as the expression of adhesion molecules, which can affect any particular type of leukocyte. At the local of injury, phagocytic cells release pro-inflammatory mediators and reactive oxygen species which contribute to the elimination of the injurious agent and lead to the resolution of the inflammatory reaction (see Weiss, 1989; Rampart, 1994, for reviews). Despite the fact that B. juraraca is responsible for most snake poisoning in Brazil, little is known about the cellular component of the local reaction induced by its venom. The present study was, therefore, undertaken to investigate the mechanisms through which BjV induces leukocyte accumulation in tissue. The effects of the venom on leukocyte chemotactic properties and on the release of active inflammatory mediators, such as leukotriene B, (LTB,) and thromboxane A, (TXA,), were assessed.

MATERIALS Venom Lyophilized crude Butantan, Brazil. Animals Male Wistar libilum

venom

rats weighing

AND

METHODS

of B. jararaca (BjV) was supplied

18G220

g were used. The animals

by the Laboratorio

were allowed

de Herpetologia,

a standard

Institute

diet and water

ad

Air pouch formation Rat skin air pouches were produced according to Edwards et al. (1981). A volume of 20 ml sterile air (using fluoropore filters, 0.22 pm) was insufflated into the subcutaneous tissue of the back trunk of the animals under ether anaesthesia. Three days later an additional volume of 10 ml sterile air was insufflated, and on day 6 1 ml of a solution of BjV in phosphate-buffered saline (PBS) containing 5, 10 or 25 pg/ml was injected into the pouch under ether anaesthesia and in aseptic conditions. Control animals received I ml of sterile PBS alone, by the same route. Collection and processing of exudales At varying intervals after the injection of the venom, the animals were reanaesthetized with ether and the inflammatory exudate was withdrawn after washing the cavities with 2 ml PBS containing 5 U/ml heparin.

Leukocyte

Response

to B. jararaca

Venom

IX7

Aliquots of the washes were used to determine total cell counts in Neubauer chambers. Differential leukocyte counts were performed on stained (0.05% crystal violet dissolved in 30% acetic acid) cell suspensions of the same aliquots. The remaining volume of fluid was centrifuged at 500 g for 6 min at - 4°C. Supernatants were stored at - 70°C and later used for the determination of eicosanoid concentration.

Eicosanoid

assays

Concentrations of TXBz (stable metabolite of TXA,) and LTB, in the pouch washes were measured by specific enzymatic immunoassay (EIA) as previously described (Pradelles et al., 1985) after extraction of eicosanoids on SepPak Cl8 columns eluted with ethanol. In brief, 100 ~1 aliquots of each extracted sample were incubated Nith the eicosanoids conjugated with acetylcholinesterase and the specific antiserum in 96-well microtitration plates. coated with anti-IgG immunoglobulins. After addition of the enzymatic substrate, the optical density of the samples was determined at 412 nm in a microplate reader and the concentration of the eicosanoids estimated from standard curves.

Leukocyte-chemotaxis

assay

The migratory assay was performed as described by Boyden (1962) and Zigmond and Hirsch (1973) using a multiwell chemotaxis chamber. In brief, aliquots of cell suspensions containing 1.5 x lo6 neutrophils were added to the upper compartment of the chamber separated from the chemotactic agent(s) in the lower compartment by a ester cellulose filter, 8 pm average pore size. Hanks’ balanced salt solution (HBSS) was substituted for the chemotactic agent for assessment of random migration. Neutrophils were obtained from normal rats 4 hr after the i.p. injection of 20 ml of a 1% oyster glycogen solution in PBS. The animals were anaesthetized with ether and the cells collected by rinsing the abdominal cavity with HBSS containing 1 U/ml heparin. Cell viability was confirmed by the eosin Y exclusion test. Final cell suspensions contained 0.1% crystalline bovine albumin in HBSS. The experiments were performed to evaluate: (I) the capacity of BjV per se to induce neutrophil migration: (2) the influence of BjV on neutrophil migration in response to activated serum (see below); and (3) the ability of BjV to generate chemotactic factors derived from serum. Activated serum was obtained by incubation with lipopolysaccharide from Escherichia coli (LPS; 65 pg/ml) for 1 hr at 37’C. Varying concentrations of LPS-activated serum were obtained with dilution in HBSS. The chemotaxis chambers were incubated in humidified air at 37’C for 120 min, followed by removal of rhe filters for fixation and staining of the cells. Neutrophil migration within the filter was determined under light microscopy by the ‘leading front’ method (Zigmond and Hirsch, 1973), in which the distance was measured from x 40 objective. the top of the filter to the farthest plane still containing two cells, using a Duplicates were always run for each variable. Five fields were counted and averaged for each filter.

Materials

Materials were obtained from the following suppliers: crystal violet (Sigma); crystalline bovine albumin (fraction V, Sigma); eosin Y (Sigma); ester cellulose filters (0.22 pm and 8.0 pm, Millipore); fluoropore fillers (0.22 pm); lipopolysaccharide from E. co/i (serotype 026:B6, Sigma); liquemine (Roche); oyster glycogen. type II (Sigma); and reagents for eicosanoids EIA (Cayman Chem. Co).

Statistical

anal_vsis

Means and S.E.M. of all data are presented and were compared by Student’s f-test or analysis of variance the data were analysed by the with significance probability levels of less than 0.01. When appropriate, Newman-Keuls test (Sokal and Rohlf, 1981).

RESULTS

Cellular influx into air pouches The number of inflammatory cells in the washes was determined 4 hr after the injection of varying concentrations of BjV (5, 10 and 25 pg/ml/rat) into the air pouches. A significant leukocyte influx into the pouches was induced by 10 and 25 ~g/ml!rat. whereas 5 pug/ml/rat did not cause a significant cell accumulation into the pouch (Table 1). Differential counts showed that the predominant cells were polymorphonuclear leukocytes (PMN), mainly neutrophils, whereas the population of mononuclear leukocytes (MN) remained practically unchanged in each group. Equivalent responses were

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et al.

observed with filtered BjV (ester cellulose filter, 0.22 pm pore size; data not shown). In subsequent studies the amount of BjV injected was 10 pg dissolved in 1 ml PBS because this concentration induced a significant cell influx but no haemorrhage, as assessed microscopically. Time course migration of leukocytes into the rat air pouch induced by BjV is shown in Fig. 1. Total leukocyte counts attained highest values between 6 and 8 hr after BjV injection, decreasing rapidly thereafter. Maximal PMN and MN cell migrations were observed 6-S hr and 8 hr after the injection of the venom, respectively. Evaluation of eicosanoid release

Injection of BjV (10 pg/ml) into the rat pouch was followed by the release of TXB, and LTB, between 1 and 4 hr. A peak increase in TXB, was detected 1 hr after BjV injection, whereas the maximum increase in LTB, was observed 4 hr afterwards (Fig. 2). Leukocyte-chemotaxis

assay

To evaluate the capacity of BjV to induce neutrophil migration, varying concentrations of the venom (10, 20, 50, 100 or 200 pg/ml), suspended in HBSS, were placed at the was filled with bottom compartment of the chamber. The upper compartment neutrophils suspended in HBSS. Neutrophil migration was equivalent with all concentrations used and was analogous to that observed in HBSS-induced random migration (24 f 2.0 pm). To investigate the influence of the BjV on neutrophil migration induced by LPS-activated serum, the cells were incubated with the venom (10 x 106cells suspended in 10 pg BjVjml HBSS) for 1 hr at 37°C then centrifuged and immediately ressuspended in HBSS for testing. The concentration of 10 pg BjVjml was used for incubation because higher amounts caused an alteration in cell viability. Preincubation with BjV did not alter the neutrophil chemotactic response to different concentrations of LPS-activated serum (5% or 10%). Values obtained (5% = 63 f 5.3 and 10% = 92 f 5.1 ,nm) were equivalent to those observed with cells preincubated with HBSS (5% = 71 + 7.0 and 10% = 95 + 5.2 pm). To investigate the ability of BjV to generate chemotactic factors derived from serum, varying concentrations of the venom were incubated with the serum for 1 hr at 37°C. Serum was diluted (v/v) in HBSS and placed at the bottom compartment of the chamber. Table

1. Leukocyte

accumulation

into the rat air pouch after injection venom

Cells x ltY/ml Polymorphonuclear

Group Total Control BjV (5 pg/rat) Control BjV (10 pg/rat) Control BjV (25 pg/rat)

1416.2 1586.1 1897.0 3301.2 1688.3 3858.0

f k k f + +

of varying

123.4 144.9 182.0 205.4* 123.0 180.7*

605.5 768.9 609,8 2628.5 1019.6 3099.4

+ + 4 + + +

72.5 128.7 71.9 162.5* 112.9 176.9*

concentrations

of B. jararaca

Mononuclear 810.7 817.2 780.0 672.0 668.7 758.6

& f k + k k

83.8 79.6 67.9 121.3 37.5 95.0

BjV in sterile PBS or sterile PBS alone (control) was injected into the rat air pouch in a final volume of 1.O ml. Total leukocytes and polymorphonuclear and mononuclear cell counts were determined in pouch washes harvested 4 hr after BjV or PBS injections. Values are mean k S.E.M. for seven animals in each group. *P < 0.05 compared with corresponding control group.

Leukocyte

Response

to

B.,jararaca Venom

MN

A ---II 1

4

6

6

12

24

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1

4

6

6

12

24

Time Fig. 1. Time course migration of leukocytes induced into the rat air pouch by BjV. Groups of seven animals were injected with 10 fig of BjV in sterile PBS (a) or sterile PBS alone (m) in a final volume of 1.0 ml. Total leukocytes, polymorphonuclear (PMN) and mononuclear (MN) cell counts were determined at varying intervals. Each point represents the mean f S.F.M. *P < 0.01compared with the control group.

The upper compartment was filled with neutrophils suspended in HBSS. The migration rate observed with serum preincubated with 10, 20 and 50 pg BjVjml was similar to that observed with normal serum (data not shown). However, higher doses of the venom (100 and 200 pg/ml) were capable of inducing neutrophil migration equivalent to that observed with LPS-activated serum (Fig. 3). Equivalent responses were observed with filtered venom (ester cellulose filter, 0.22 pm pore size; data not shown). Heating the serum for I hr at 56°C before the addition of the venom completely prevented its ability to induce neutrophil migration in this circumstance (Fig. 3). DISCUSSION

The present results indicate that BjV is capable of inducing a characteristic local inflammatory reaction, because its injection into the rat air pouch resulted in a marked leukocyte influx to the injured area. The effect was not due to contamination of the venom with bacterial endotoxins, as leukocyte migration was unchanged after filtration of the venom in sterilizing membranes. At the early stages of the reaction, the predominant cells migrating into the pouches were neutrophils. The cell population changed subsequently. with the appearance of mononuclear leukocytes. A similar pattern of cell infiltration is induced in rats by bothropic venoms from er_vthromelas and alternatus species (Flares et al., 1993). Neutrophils play a vital role in host defence. They are usually the first cell type to reach the site of injury and predominate numerically in a recent lesion. Neutrophil attraction and emigration are effected by chemical signals (chemoattractants) generated at the injured area. The initial event in this process is the adherence of neutrophils to endothelial cells owing to the intervention of adhesion molecules. Neutrophils then migrate across the endothelial layer. This is thought to occur through interendothelial tight junctions (see

190

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et al.

Albelda et al., 1994; Granger and Kubes, 1994, for reviews). Once in the extracellular tissue, neutrophils are orientated through pseudpod emission, responding to a concentration gradient of locally generated chemoattractants (chemotaxis). Following chemoattractant-receptor interaction, a polarized elongation of the cell is observed that is associated with specific topographical reorganization of plasma organellae controlled by cytoskeletal structures (Snyderman and Goetzl, 1981). Mediators of the inflammatory reaction appear to be involved in the BjV-induced leukocyte influx. Release of tissue mediators responsible for the leukocyte accumulation in an inflamed area is described. Among them, products of arachidonic acid metabolism

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Time Fig. 2. LTB, and TXB? concentrations in the rat air pouch after BjV injection. Groups of seven animals were injected with 10 pg of BjV in sterile PBS (hatched bars) or sterile PBS alone (open bars) in a final volume of 1.0 ml. LTB, and TXAz were quantitated by specific ELISA in pouch washes collected at 30 min, 1 and 4 hr after BjV or PBS injection. Each bar represents mean f S.E.M. *P i 0.05 compared with the control group.

Leukocyte

Response

to

B.jararaca

191

Venom

200’

5

10

20

5

10

20

% of Activated Serum Fig. 3. Chemotactic

responses

(micropore

filter system) of neutrophils to BjV and LPS-activated serum. Cells were placed at the top compartment of the chamber and allowed to migrate for 120 min. (A) LPS and BjV were incubated with serum for I hr at 37’C, diluted in HBSS and used as chemotactic agents. (B) Heating of sera for 1 hr at 56°C prior to the addition of BjV prevents its ability IO induce neutrophil migration. Values are means S.E.M. of five experiments, each done in duplicate. *P < 0.01compared with values in the group using non-activated serum.

formed by the action of the cycloxygenase and lipoxygenase enzymes are relevant. particularly TXA, and LTB, (Lewis and Austen, 1988). The present results suggest the contribution of lipoxygenase products in the BjV-induced leukocyte influx into the rat air pouch. Elevated concentrations of LTB, into the rat pouch were detected, and LTB, is one of the most potent chemotactic agents for PMN leukocytes. Interaction of LTB, with its receptor on the neutrophil surface results in chemotaxis, chemokinesis, aggregation and adhesion to endothelial cells (Bray, 1982; Hoover et al., 1984; Palmblad et al., 1990). Furthermore, LTB, elicits leukocyte migration in ciuo in experimental animals and humans (Camp et al., 1982; Bhattacheryee et al., 1981). Our data showing high concentrations of LTB, from 1 to 4 hr after injection of the venom suggest a role for it in both the initiation and propagation steps of neutrophil recruitment into the pouch. Reports showing that LTB, is released by neutrophils and macrophages during phagocytosis (Palmer and Salmon, 1983; Claesson et al., 1985) are in agreement with the later occurrence of LTB, in the pouch following venom injection. TXA, is also likely to be involved with leukocyte infiltration induced by BjV. The finding that TXA, concentrations are significantly increased only at the early stages following BjV injection suggests its participation in the initial events responsible for leukocyte migration and subsequent accumulation in the locality of the BjV injection. This hypothesis is corroborated by the observation that TXA, mediates leukocyte adhesion to the endothelium by inducing the expression of adhesion molecules (Wiles et ul., 1991). Taken together, the present results suggest that LTB, and TXA, may have a relevant pathogenic role in the development of local effects induced by BjV. However, the extent to which both factors mediate BjV-induced cell influx into the pouch remains to be established. In addition, the in vitro chemotaxis studies presented here suggest that the BjV neither activated neutrophil-specific membrane receptors nor altered the intrinsic mechanisms involved in leukocyte locomotion; first, because the venom per se is not able to evoke an

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orientated cell locomotion; and second, because it did not modify the intrinsic capability of neutrophils to migrate in response to a chemotactic factor (LPS-activated serum). However, BjV was able to generate serum chemotactic factors. Its incubation with rat serum induced neutrophil chemotaxis when in the presence of the serum. Destruction of this activity by heating the active serum to 56°C suggested that the serum factor(s) thus generated is related to the complement system. This hypothesis is consistent with previous observations showing that activation of the complement system occurs by intervention of bothropic venoms (Dias da Silva et al., 1995). It is well known that the component parts C5a and C5a Des Arg generated by activation of the complement system are chemotactic to neutrophils, monocytes, macrophages, eosinophils and basophils (Snyderman and Goetzel, 198 1). Nevertheless, these factors were generated by only high doses of BjV under our experimental conditions. The participation of serum chemotactic fibrinopeptides (fibrinopeptide B) eventually generated by the BjV (Santoro and Farsky, unpublished material) may be ruled out in the present circumstances, as indicated by the fact that heating the serum to temperatures lower than those necessary to inactivate the peptides, in turn inactivated the chemotactic activity. In conclusion, the present results show that in uivo BjV evokes leukocyte infiltration into the site of injury following envenomation. The effect is indirect, via release or generation of endogenous mediators. The venom per se is not able to influence leukocyte chemotactic properties. LTB, and TXA? may play important roles as mediators of the BjV-induced cell migration. However, the participation of complement components is also likely to occur. In view of the capacity of neutrophils to cause tissue damage in the host in conditions of uncontrolled or unwanted stimulation, the present observations suggest that leukocyte accumulation at the site of bothropic envenomation may be considered in the design of future therapeutic strategies to confront the significant local effect induced by this venom.

Acknowledgements-The authors wish to thank Prof. J. Garcia-Leme for assistance with the preparation of the manuscript and Lindonea dos Santos for valuable technical assistance. This work was supported in part by FAPESP and CNPq grants.

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