Bothrops asper snake venom and
its metalloproteinase BaP–1
activate the complement system.
Role in leucocyte recruitment
Sandra H. P. Farsky1,CA, Lu õ´ s Roberto C. Gonçalves2,
Jose´ M. Gutie´rrez3, Adriana P. Correa1,
Alexandra Rucavado3, Philippe Gasque4 and
Denise V. Tambourgi1
Laboratories of 1Immunochemistry and
2Pathophysiology, Institute Butantan, Av. Vital Brazil,
1500 Sa˜o Paulo, 05503–900, SP, Brazil; 3Institute
Clodomiro Picado, Faculty of Microbiology,
University of Costa Rica, San Jose´, Costa Rica;
4Brain Inflammation and Immunity Group,
Department of Medical Biochemistry, University of
Wales College of Medicine, Cardiff, UK
CA Corresponding Author
Tel: 55 11 813 7222 ext. 2231
Fax: 55 11 815 1505
E-mail: farskyp@uol.com.br
TH E venom of the snake Bothrops asper, the most
important poisonous snake in Central America,
evokes an inflammatory response, the mechanisms
of which are not well characterized. The objectives of
this study were to investigate whether B. asper
venom and its purified toxins Ð phospholipases and
metalloproteinase Ð activate the complement system
and the contribution of the effect on leucocyte
recruitment. In vitro chemotaxis assays were per-
formed using Boyden’s chamber model to investigate
the ability of serum incubated with venom and its
purified toxins to induce neutrophil migration. The
complement consumption by the venom was eval-
uated using an in vitro haemolytic assay. The impor-
tance of complement activation by the venom on
neutrophil migration was investigated in vivo by
injecting the venom into the peritoneal cavity of
C5-deficient mice. Data obtained demonstrated that
serum incubated with crude venom and its purified
metalloproteinase BaPÐ1 are able to induce rat neu-
trophil chemotaxis, probably mediated by agent(s)
derived from the complement system. This hypoth-
esis was corroborated by the capacity of the venom to
activate this system in vitro. The involvement of C5a
in neutrophil chemotaxis induced by venom-acti-
vated serum was demonstrated by abolishing migra-
tion when neutrophils were pre-incubated with anti-
rat C5a receptor antibody. The relevance of the
complement system in in vivo leucocyte mobilization
was further demonstrated by the drastic decrease of
this response in C5-deficient mice. Pre-incubation of
serum with the soluble human recombinant comple-
ment receptor type 1 (sCR 1) did not prevent the
response induced by the venom, but abolished the
migration evoked by metalloproteinase-activated
serum. These data show the role of the complement
system in bothropic envenomation and the participa-
tion of metalloproteinase in the effect. Also, they
suggest that the venom may contain other compo-
nent(s) which can cause direct activation of C5a.
Key words: Bothrops asper snake venom, Leucocyte
infiltrate, Complement system, C5a, Metalloproteinase,
Inflammatory reaction
Introduction
The acute inflammatory response is characterized by
an increased passage of fluid and leucocytes, partic-
ularly neutrophils, out of the bloodstream into the
damaged tissue.1,2 Neutrophils are one of the central
inflammatory cells in the first line of host defence.
Their adhesion to endothelial cells and the sub-
sequent oriented transmigration are key events in
their recruitment during inflammation. Such events
are controlled by a dynamic interaction between
adhesion molecules (i.e. selectins) expressed on
leucocytes and the endothelial cells, as well as by a
direct action of secreted chemotactic mediators
which bind to the expressing seven transmembrane G
protein coupled receptors.2 –5
A local inflammatory reaction is characteristic of
envenomations by snakes of the genus Bothrops,
involving oedema formation and leucocyte mobiliza-
tion.6 –10 The mechanisms responsible for leucocyte
recruitment in these envenomations are not well
described. It has been shown that the accumulation
of leucocytes in animals injected with some Bothrops
sp. venoms is dependent on eicosanoid release7,9,10
and on chemotactic factors derived from the serum.10
The latter effect is probably due to complement
activation as venoms from crotaline snakes are known
to activate the complement system.11 The pathophy-
ISSN 0962-9351 print/ISSN 1466-1861 online/00/050213-09 © 2000 Taylor & Francis Ltd 213
DOI: 10.1080/09629350020025728
Research Paper
Mediators of Inflammation, 9, 213–221 (2000)
siology of envenomation by B. asper, the most
important poisonous snake in Central America,12
includes systemic alterations such as blood coagula-
tion disturbances,13 haemorrhage, cardiovascular
shock and renal failure,14 besides prominent local
tissue damage characterized by myonecrosis, haemor-
rhage and an acute inflammatory response.6,14 –17
Although, it has been demonstrated that oedema
evoked by the venom is multimediated,18 the mecha-
nisms involved in the local leucocyte accumulation
remain unknown.
The aims of the present study were to investigate
whether B. asper venom (BaV) is involved in comple-
ment activation and to test whether this activation
with the production of soluble complement anaphyla-
toxins leads to leucocyte recruitment. Our results
show that the venom is able to activate classical and
alternative complement system pathways, generating
C5a component. Serum treated with BaV was a
chemoattractant to neutrophils and we were able to
confirm, as expected, that C5a mediated this effect.
Indeed, an anti-rat C5a receptor (anti-ratC5aR) neu-
tralized the chemotaxis activity. We propose that the
metalloproteinase BaP–1 present in the venom is
involved in complement activation. It remains to be
tested whether complement activation can be attrib-
uted solely to this metalloproteinase or whether other
components of the venom have synergistic actions.
Materials and methods
Chemicals and reagents
Crystalline bovine serum albumin (fraction V), eosin Y,
lipopolysaccharide (LPS) from Escherichia coli (ser-
otype 026:B6), oyster glycogen type II, Sepharose–
polymyxin B columns were purchased from Sigma (St
Louis, MO, USA). Ester cellulose filters (0.22mm and
8.0mm) were from Millipore (Brazil). Liquemine was
acquired from Roche (Brazil).
Blood, sera, antibodies and other proteins
Normal sheep and rabbit blood, collected 1:1 in
Alsever’s old solution (pH 6.1, 114 mM citrate,
27 mM glucose, 72 mM NaCl), was obtained from the
animal facilities of Institute Butantan and stored at
4°C. Normal human or rat blood was obtained in-
house. Blood samples drawn to obtain sera were
collected without anticoagulant and allowed to clot
for 1 h at room temperature (RT). The normal human
serum (NHS) or normal rat serum (NRS) were stored
at –80°C. Anti-rat C5aR antiserum was produced in-
house (P. Gasque) by injecting a rabbit with a multiple
array C5a peptide (amino acids 12–35:
TYDYSDGTPNPDMPADGVYIPKME) corresponding
to the N-terminal of the receptor involved in the C5a
binding. The IgG fraction was obtained by affinity
purification on a protein A Sepharose column (Prosep
A, Bioprocessing, Princeton, NJ, USA). The rabbit anti-
C5aR stained specifically rat monocyte cell line NR83/
83 and rat C5aR-transfected CHO cells (P. Gasque,
unpublished observation). Soluble recombinant com-
plement receptor type 1 (sCR 1) was obtained from
Avant Immunotherapeutics (Needham, MA, USA).
Venom and purified toxins
Lyophilized crude BaV was a pool obtained from more
than 50 adult specimens collected in the Atlantic
region of Costa Rica and kept at the Serpentarium of
Instituto Clodomiro Picado, Costa Rica. The venom
was lyophilized and maintained at –20°C and dis-
solved at the moment of use. Prior to the experi-
ments, the venom solutions were filtered through a
cellulose ester membrane of 0.22mm average pore
size. Phospholipase A2 myotoxins II and III and
haemorrhagic metalloproteinase BaP–1 were purified
from crude venom as previously described.19 – 21 In
some experiments, BaV was loaded on to a Sephar-
ose–polymixin B column in order to remove any
traces of LPS which could be present in the venom.
Animals
Male Wistar rats weighing 170–190 g were obtained
from “Biote´rio Central do Instituto Butantan” and
male B10/A (H–2a) and A/J (H–2a ) isogenic mice
strains weighing 17–20 g were obtained from “Bio-
te´rio de Camundongos isogeˆnicos do Instituto de
Cieˆncias Biome´dicas da Universidade de Sa˜o Paulo
(Sa˜o Paulo, SP. Brazil)”. The animals were maintained
with free access to standard rat food pellets and water
ad libitum.
Haemolytic assay
In order to assess the capacity of BaV and related
toxins to activate the complement system in vitro,
samples of NHS were incubated with different con-
centrations of venom or toxin for 30 min at 37°C.
Control samples were incubated with phosphate-
buffered solution (PBS, pH 7.2, containing 10 mM Na
phosphate, 150 mM NaCl) alone. The haemolytic
assays were performed in 96-well microtitre plates.
Sheep erythrocytes (Es) were sensitized with rabbit
antibodies against Es (1:1000) for 30 min at 37°C.
Aliquots (100ml) of sensitized cells [2% suspension in
veronal-buffered saline (VBS++, pH 7.4, containing
10 mM Na barbitone, 0.15 mM CaCl2 and 0.5 mM
MgCl2 )] were incubated with dilutions of NHS
(100ml) as a source of complement for 60 min at
37°C. The plates were centrifuged to remove intact
cells and the absorbance of the supernatant was
S. H. P. Farsky et al.
214 Mediators of Inflammation · Vol 9 · 2000
measured at 414 nm. Es incubated without NHS were
used as the standard for lysis and water-lysed E were
used as the standard for 100% lysis. Alternative
pathway complement activation was measured using
serum diluted in VBS containing ethylene glycol
tetraacetic acid (EGTA)-Mg++ (VBS plus EGTA 0.1 M
MgCl2 and 0.1 M EGTA) and non-antibody sensitized
rabbit E (Er) as target cells.
Neutrophil preparations
Neutrophils were obtained from normal Wistar rats
4 h after the intraperitoneal 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 Hanks’ balanced
solution (HBSS, containing 138 mM NaCl, 5.36 mM
KCl, 1.25 mM Na2HPO4 . 12H20, 0.44 mM KH2PO4 ,
2.94 mM NaHCO3 , 0.13 mM CaCl2 , 0.1 mM MgSO4 ,
5.5 mM glucose) plus 1 U/ml heparin. Cell viability
was assessed by the eosin Y exclusion test. The final
cell suspensions contained 1% crystalline bovine
serum albumin in HBSS.
Neutrophil chemotaxis assay
The migration assay was performed as described by
Boyden22 and Zigmond and Hirsch23 using a multi-
well chemotaxis chamber. In brief, aliquots of cell
suspensions containing 1.5 ´ 106 neutrophils were
added to the upper compartment of the chamber
separated from the chemotactic agent(s) in the lower
compartment by an ester cellulose filter (8mm
average pore size). The chemotactic agent was substi-
tuted for HBSS for assessment of random migration.
The experiments were performed to evaluate: (1) the
capacity of BaV to directly induce neutrophil chem-
otaxis, (2) the neutrophil chemotaxis in response to
serum pre-treated with BaV (see below), (3) the
capacity of myotoxins II or III and metalloproteinase
BaP–1 to generate chemotactic factors derived from
serum, (4) the participation of C5a/C5aR in the
chemotaxis event. Activated serum was obtained by
incubation with E. coli LPS (65mg/ml) for 30 min at
37°C. Varying concentrations of LPS-activated serum
were prepared using HBSS as the diluent.
The chemotaxis chambers were incubated in
humidified air at 37°C for 60 min, followed by
removal of the filters for fixation and staining of the
cells. Neutrophil migration within the filter was
determined under light microscopy by the “leading
front” method,23 in which the distance was measured
from the top of the filter to the farthest plane of focus
still containing two cells observed with the ´ 40
objective. Five fields were counted and averaged for
each experiment, which was performed in
duplicate.
Determination of the number and specific cell
type of leucocytes of peritoneal exudate
following BaV injection
To evaluate leucocyte recruitment induced by BaV,
10mg of venom dissolved in 1 ml of sterile saline was
injected into the peritoneal cavity of B10/A (H–2a;
C5-sufficient) and A/J (H–2a; C5-deficient) isogenic
mice strains under light ether anaesthesia. Match
controls of each group of animals received 1 ml of
sterile saline by the same route. After 4 h, the animals
were re-anaesthetized with ether, the abdominal
cavity was opened, and the exudate was withdrawn
after washing the peritoneum with 3 ml of PBS
containing 5 U/ml heparin. Total leucocyte counts
were performed on Neubauer chambers and differ-
ential counts on smears stained with panchromatic
dye.
Analysis of data
Means and standard error of the mean (SEM) of all
data are presented and compared by Student’s t-test or
analysis of variance. When appropriate, the data were
analysed by the Newman–Keuls test.24
Results
Activation and consumption of the haemolytic
activity of the complement system following
treatment with BaV
The effect of BaV on the activity of the complement
system was determined in vitro, using NHS as a
Complement system and snake venom
Mediators of Inflammation · Vol 9 · 2000 215
FIG. 1. Consumption of the haemolytic activity of the
complement system following treatment with Bothrops
asper venom (BaV). Samples of normal human serum (NHS)
were incubated with different concentrations of venom for
30 min at 37°C. Control samples were incubated with phos-
phate-buffered solution (PBS) alone. Classical pathway (CP)
activation of the complement system was determined by
incubating aliquots of EA with dilutions of NHS in veronal-
buffered saline (VBS) for 60 min at 37°C. The plates were
centrifuged to remove intact cells and the absorbance of the
supernatant was measured at 414 nm. Alternative pathway
(AP) activation of the complement system was measured by
using serum diluted in VBS containing EGTA-Mg++ and
rabbit E as target cells. Values are mean ± standard error (SE)
of three experiments each performed in duplicate.
source of complement, with different doses of venom
for 30 min at 37°C. The residual complement activity
was determined by haemolytic assays performed in
conditions for measuring classical or alternative
pathway activation. Figure 1 shows that BaV induces
a dose-dependent reduction in haemolytic activity as
measured by both classical and alternative
pathways.
Leucocyte chemotaxis assay
To evaluate the capacity of BaV per se to induce
neutrophil migration, varying doses of venom (1, 10,
50 or 100mg/ml) were dissolved in HBSS and placed
in the bottom compartment of Boyden’s chamber. The
upper compartment was filled with neutrophils
suspended in HBSS. The observed neutrophil migra-
tion was equivalent in all doses used, being similar to
that observed in random migration (HBSS) (data not
shown), indicating that BaV has no direct neutrophil
transmigration activity.
In order to investigate the influence of BaV on
neutrophil-oriented migration capacity, neutrophils
were incubated with venom (1 ´ 107 cells suspended
in 1, 10, 50 or 100mg BaV/ml in HBSS) for 1 h at 37°C,
centrifuged and immediately resuspended in HBSS for
testing. These venom doses did not affect cell viability.
The results indicated that pre-incubation with BaV
does not alter neutrophil chemotactic response to
LPS-activated serum. The migration obtained with
cells pre-incubated with different doses of BaV in
response to HBSS, 1 or 10% of LPS-activated serum
were similar and equivalent to that observed with
cells pre-incubated with HBSS (data not shown).
To investigate the ability of BaV to generate
complement chemotactic factors following comple-
ment activation, varying doses of venom were incu-
bated with serum for 30 min at 37°C, diluted (v/v) in
HBSS and tested in Boyden’s chamber. The migration
observed with serum treated with 1 or 10mg BaV/ml
was similar to that observed with non-treated serum
(data not shown). However, higher doses of venom
(50 or 100mg/ml) were able to induce neutrophil
migration equivalent to that observed with LPS-
activated serum (Fig. 2A). This effect is not dependent
on bacterial LPS components present in the venom, as
equivalent migration was obtained with polymyxin-
treated BaV (serum + BaV = 120 ± 9.22mm, serum +
polymyxin-treated BaV = 109 ± 8.65mm). Heating the
serum for 1 h at 56°C prior to the addition of venom
completely abolished the ability to induce neutrophil
chemotaxis (Fig. 2B).
Participation of C5a component on the
neutrophil chemotaxis induced by the venom
In order to evaluate the possible involvement of the
complement-derived chemotactic fragment C5a in
the neutrophil chemotaxis induced by BaV, rat
neutrophils were pre-incubated for 15 min with
S. H. P. Farsky et al.
216 Mediators of Inflammation · Vol 9 · 2000
FIG. 2. Chemotactic responses of neutrophils to Bothrops asper venom (BaV) and to lipopolysaccharide (LPS)-treated serum.
Neutrophils were placed in the top compartment of the chamber and allowed to migrate for 1 h. (A) BaV and LPS were
incubated with serum for 30 min at 37°C, diluted in Hanks’ balanced solution (HBSS) and used as chemotactic agents. (B)
Heating the serum for 1 h at 56°C prior to the addition of BaV or LPS prevents its ability to induce neutrophil migration. Values
are mean ± standard error of the mean (SEM) of four experiments each performed in duplicate. *p < 0.01 versus values in the
group using non-activated serum.
different dilutions of anti-rat C5a receptor serum and
tested in chemotaxis assays. Figure 3 shows that the
migration of neutrophils was inhibited by anti-rat
C5aR antibody, in a dose-dependent manner. The anti-
C5aR antibody was similar in blocking neutrophil
chemotaxis induced by LPS-activated serum.
Effect of venom phospholipases and
metalloproteinase on the generation of
chemotactic factors from serum
Chemotaxis assays were performed with serum pre-
incubated with different concentrations of phospholi-
pases A2 myotoxin II or III and metalloproteinase
BaP–1 at 37°C for 30 min. Data obtained showed that
only metalloproteinase-activated serum was able to
induce neutrophil chemotaxis. The magnitude of the
response induced by 5 or 10mg/ml was equivalent to
that induced by LPS-activated serum (Fig. 4). Pre-
heating the serum at 56°C for 1 h abolished the ability
of the metalloproteinase to induce neutrophil chem-
otaxis (data not shown). Serum incubated with
myotoxin II or III (doses of 1, 10, 50mg/ml) did not
induce in vitro neutrophil locomotion (data not
shown).
Analysis of the mechanism of C5a generation
in rat serum induced by BaV and
metalloproteinase BaP–1
In order to investigate the mechanism of comple-
ment activation induced by the venom and metal-
loproteinase BaP–1, normal serum was incubated
with different concentrations of sCR1 at 37°C for
5 min before venom or metalloproteinase addition.
The same experimental procedure was performed
on LPS-activated serum. The solutions were incu-
bated at 37°C for 30 min and used in chemotaxis
assays. The results obtained showed that sCR1 did
not inhibit the generation of chemotactic factors in
venom-treated serum (Fig. 5). In contrast, sCR1 was
able to reduce the neutrophil chemotaxis serum
activity induced by both metalloproteinase and LPS
(Fig. 5).
Complement system and snake venom
Mediators of Inflammation · Vol 9 · 2000 217
FIG. 3. Chemotactic responses of neutrophils incubated with different concentrations of anti-rat C5a receptor antibody before
testing with Bothrops asper venom (BaV)-treated and lipopolysaccharide (LPS)-treated serum. Neutrophils were incubated
with the antibody at 37°C for 15min, placed in the top compartment of the chamber and allowed to migrate for 1 h. BaV and
LPS were incubated with serum for 30 min at 37°C, diluted in Hanks’ balanced solution (HBSS) and used as chemotactic agents.
Values are mean ± standard error of the mean (SEM) of three experiments each performed in duplicate. *p < 0.01 versus values
obtained with incubated serum with HBSS in each group.
Local leucocyte mobilization induced by venom
in C5-deficient mice
To evaluate the participation of C5a component on
local leucocyte recruitment induced by BaV, 10mg of
venom was injected into the peritoneal cavity of
B10/A (H–2a, C5-sufficient) and A/J (H–2a,
C5-deficient) isogenic mouse strains and local leuco-
cyte mobilization was determined 4 h after envenoma-
tion. The leucocyte mobilization into the peritoneal
cavity of the C5-deficient animals was significantly
reduced (50%) in comparison with that obtained in
the C5-sufficient animals (Fig. 6A). Differential counts
showed no significant migration of polymorphonu-
clear cells in C5-deficient mouse strain (Fig. 6B).
Discussion
BaV induces pronounced local effects, such as myone-
crosis, haemorrhage and inflammatory reaction, char-
acterized by long-lasting oedema and leucocyte infil-
tration.6,18,25,26 The mediators involved in the
development of oedema by this venom have been
partially studied18 and the mechanisms involved in
leucocyte recruitment remain unknown. Our results
indicate that BaV activates the complement system
leading to the genesis of C5a, which is notably
involved in the chemotaxis of neutrophils during
envenomation. Metalloproteinases, such as BaP-1,
present in the venom may participate in the effect,
but other component(s) may be involved.
In vitro chemotaxis studies suggest that BaV
neither activates neutrophil-specific membrane recep-
tors nor alters the intrinsic mechanisms involved in
leucocyte locomotion. These conclusions are sup-
ported by the observations that venom per se is
unable to evoke an oriented cell locomotion and does
not modify the intrinsic ability of neutrophils to
migrate in response to a chemotactic factor (LPS-
activated serum). However, BaV was able to generate
chemotactic factors when incubated with serum.
Elimination of this activity by heating the serum at
56°C suggests that the serum factor(s) inducing cell
migration may be complement activation products.
Our observations of in vitro reduction of serum
haemolytic activity by the venom corroborate this
hypothesis.
It is well known that fragments generated during
complement activation, such as C5a and C5a des arg,
cause neutrophil activation, up-regulation of adhesion
molecules, emigration and chemotaxis.27 C5a is also a
chemotactic factor to monocytes, macrophages, eosi-
nophils and basophils.28 Our results clearly demon-
strate that C5a is involved in both in vitro and in vivo
leucocyte locomotion induced by BaV. This was
clearly evidenced by the use of a rabbit antibody
generated against the N-terminal domain of the C5aR
involved in binding activity. These observations were
positively correlated with the experiments using
C5-deficient mice. In both cases, the chemotaxis
response was significantly reduced.
The abolishment of local neutrophil recruitment
observed in C5-deficient mice implicates that the
complement system and particularly C5a act as an
important pathogenic component of the inflamma-
tory response following envenomation. Other inflam-
matory mediators, as yet uncharacterized, certainly
play additional roles in the leucocyte recruitment
induced by BaV injection. A role for lipoxygenase
pathway-derived products in leucocyte recruitment
after injections of other Bothrops sp. venoms has
already been described.7,9,10 Furthermore, our results
indicate that the complement system has a central
role in this phenomenon.
The presence of complement activators/inactiva-
tors has been demonstrated in a variety of animal
venoms.11,29– 31 Venoms of species classified in the
families Elapidae, Crotalidae and Viperidae contain
components with a broad range of action on the
complement system. Some act by cleaving directly a
S. H. P. Farsky et al.
218 Mediators of Inflammation · Vol 9 · 2000
FIG. 4. Chemotactic responses of neutrophils to serum
treated with Bothrops asper metalloproteinase BaP–1 or
with lipopolysaccharide (LPS). Neutrophils were placed in
the top compartment of the chamber and allowed to migrate
for 1 h. BaP–1 or LPS were incubated with serum for 30 min
at 37°C, diluted in Hanks’ balanced solution (HBSS) and used
as chemotactic agents. Values are mean ± standard error of
the mean (SEM) of four experiments each performed in
duplicate. *p < 0.01 versus values obtained in the group
using non-activated serum.
Complement system and snake venom
Mediators of Inflammation · Vol 9 · 2000 219
FIG. 5. Effect of soluble recombinant complement receptor type 1 (sCR1) on neutrophil chemotaxis induced by Bothrops asper
venom (BaV), metalloproteinase BaP–1 or lipopolysaccharide (LPS)-treated serum. sCR1 was added to serum 5 min prior to the
addition of BaV, BaP–1 or LPS. The solutions were incubated at 37°C for 30 min, diluted in Hanks’ balanced solution (HBSS) and
used as chemotactic agents. Neutrophils were placed in the top compartment of the chamber. Migration was determined 1 h
after incubation of the chamber at 37°C. Values are mean ± standard error of the mean (SEM) of three experiments each
performed in duplicate. *p < 0.001 versus values in the corresponding samples of serum without sCR1.
FIG. 6. Number of total (A) and differential (B) leucocytes migrated into the peritoneal cavity of male B10A (H–2a ) or A/J (H–2a )
mice 4 h after intraperitoneal injection of 10mg of Bothrops asper venom (BaV) dissolved in 1 ml of sterile saline. Both groups
of animals received venom or the equivalent volume of sterile saline. Values are mean ± standard error of the mean (SEM) of
five animals in each group. *p < 0.01 versus values obtained in control animals treated with BaV, **p < 0.001 versus values
obtained in control animals treated with saline and ***p < 0.001 versus values obtained in control animals treated with
BaV.
particular component, while others interact with
complement components with the resulting complex
being able to activate part of the complement
cascade.
The complement activation induced by BaV was
not attributed to the action of bacterial LPS. All venom
solutions were sterilized by filtration and the eventual
contamination by LPS was removed following poly-
myxin B column purification. Interestingly, venom-
induced complement activation was not affected by
incubation of the serum with sCR 1, whereas this
soluble complement inhibitor abrogated neutrophil
chemotaxis of LPS-activated serum.
Various tissue-damaging toxins have been isolated
from BaV. There are at least four myotoxic phospholi-
pases A2 which induce acute muscle damage by
directly affecting the integrity of the muscle cell
plasma membrane.17 These myotoxins also induce
oedema.17,32 In addition, five haemorrhagic metal-
loproteinases have been purified from this
venom.21,33,34 Besides affecting the integrity of capil-
lary blood vessels,35,36 metalloproteinases also induce
myonecrosis,36,37 skin damage38 and oedema.37
Our results indicate that metalloproteinase BaP–1 is
able to activate the complement system, as shown by
the decrease in the haemolytic activity of the serum
with the toxin (data not shown). In addition, neu-
trophil chemotaxis was evidenced when exposed to
serum treated with BaP–1. In contrast, the two
myotoxic phospholipases A2 did not activate the
complement system nor induce complement-depend-
ent chemotaxis. Interestingly, the mechanism of
complement activation by metalloproteinase seems to
be dependent on C3 convertase assembly, as the
addition of sCR 1 to BaP–1-treated serum prevented
the generation of chemotactic factors. In contrast,
complement activation by venom does not seem to be
dependent on C3 convertase formation. Hence, sCR1
was unable to inhibit the neutrophil chemotaxis in
response to venom-treated serum, suggesting that
specific venom component(s) may act on the comple-
ment system by cleaving directly C5 component to
generate a functional C5a anaphylatoxin fragment.
The precise mechanism by which the venom
induces complement activation and the identification
of other venom component(s) involved in the genera-
tion of C5a are the subjects of further study.
ACKNOWLEDGEMENTS: This project was supported by FAPESP grants, by
Vicerrector´õ a de Investigacio´n, Universidad de Costa Rica (projects
741–98–202) and the International Foundation for Science (project
F/2707–1). Dr P. Gasque is supported by the British Medical Research
Council. We thank Avant Immunotherapeutics, Inc. for providing sCR 1.
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Received 8 September 2000;
accepted 19 September 2000
Complement system and snake venom
Mediators of Inflammation · Vol 9 · 2000 221
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